Technical

Manual
18TH EDITION
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Technical
Manual
18TH EDITION

E di te d b y

Mark K. Fung, MD, PhD

Fletcher Allen Health Care
Burlington, VT

Brenda J. Grossman, MD, MPH

Washington University School of Medicine
St. Louis, MO

Christopher D. Hillyer, MD
New York Blood Center
New York, NY

Connie M. Westhoff, PhD, SBB

New York Blood Center
New York, NY
Mention of specific products or equipment by contributors to this AABB publication does not represent
an endorsement of such products by the AABB Press nor does it indicate a preference for those
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imply or guarantee that the materials meet federal, state, or other applicable requirements. It is
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AABB authors are requested to comply with a conflict of interest policy that includes disclosure of
relationships with commercial firms. A copy of the policy is located at http://www.aabb.org.

Efforts are made to have publications of the AABB consistent in regard to acceptable practices.
However, for several reasons, they may not be. First, as new developments in the practice of blood
banking occur, changes may be recommended to the Standards for Blood Banks and Transfusion
Services. It is not possible, however, to revise each publication at the time such a change is adopted.
Thus, it is essential that the most recent edition of the Standards be consulted as a reference in regard to
current acceptable practices. Second, the views expressed in this publication represent the opinions of
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Copyright © 2014 by AABB. All rights reserved. No part of this book may be reproduced or transmitted
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AABB ISBN No. 978-1-56395-888-5

8101 Glenbrook Road Printed in the United States
Bethesda, Maryland 20814-2749

Cataloging-in-Publication Data

Technical manual / editor, Mark K. Fung—18th ed.

p. ; cm.
Including bibliographic references and index.
ISBN 978-1-56395-888-5
1. Blood Banks—Handbooks, manuals, etc. I. Fung, Mark K. II. AABB.
[DNLM: 1. Blood Banks-laboratory manuals. 2. Blood Transfusion-
laboratory manuals. WH 25 T2548 2014]
RM172.T43 2014
615’.39—dc23
DNLM/DLC
 Technical Manual
Authors
Colleen A. Aronson, MT(ASCP)SBB
Aleksandar M. Babic, MD, PhD
Robert A. Bray, PhD
Laura Cooling, MD, MS
Brian R. Curtis, PhD, D(ABMLI),
MT(ASCP)SBB
Robertson D. Davenport, MD
Meghan Delaney, DO, MPH
Gregory A. Denomme, PhD, FCSMLS(D)
Katharine A. Downes, MD
Larry J. Dumont, MBA, PhD
Deborah F. Dumont, MT(ASCP)SBB
Nancy M. Dunbar, MD
Anne F. Eder, MD, PhD
William FitzGerald, LTC USA (Ret)
Susan A. Galel, MD
Howard M. Gebel, PhD
Mary Ghiglione, RN, MSN, MHA
Janis R. Hamilton, MS, MT(ASCP)SBB
Jeanne E. Hendrickson, MD
Eapen K. Jacob, MD
Shweta Jain, MD
Yameena Jawed, MD
Betsy W. Jett, MT(ASCP), CQA(ASQ)CQM/OE
Brian Johnstone, PhD
Cassandra D. Josephson, MD
Melanie S. Kennedy, MD
Scott A. Koepsell, MD, PhD
Mickey B. C. Koh, MD, PhD
Patricia M. Kopko, MD
Regina M. Leger, MSQA, MT(ASCP)SBB, CMQ/
OE(ASQ)
Christine Lomas-Francis, MSc, FIBMS
Keith March, MD, PhD
Kim Maynard, BSN, RN, OCN
Catherine A. Mazzei, MD
David H. McKenna Jr, MD
Erin Meyer, DO, MPH
Tania L. Motschman, MS, MT(ASCP)SBB,
CQA(ASQ)
Maria D.L.A. Muniz, MD
Theresa Nester, MD
Mona Papari, MD
Jessica Poisson, MD
Marilyn S. Pollack, PhD
Mark A. Popovsky, MD
Kathleen E. Puca, MD, MT(ASCP)SBB
Glenn Ramsey, MD
Donna M. Regan, MT(ASCP)SBB
Rita A. Reik, MD
Edward R. Samuel, PhD, MSc
Scott Scrape, MD
Ira A. Shulman, MD
James W. Smith, MD, PhD
Steven L. Spitalnik, MD
Simon Stanworth, FRCP, FRCPath, DPhil
Jill R. Storry, PhD, FIBMS
Garnet Suck, PhD, MSc
Ruth D Sylvester, MS, MT(ASCP)SBB
Sreedhar Thirumala, PhD
Alan Tinmouth, MD, FRCPC, MSc
Christopher A. Tormey, MD
Lance D. Trainor, MD
Wendy Trivisonno
Phyllis S. Walker, MS, MT(ASCP)SBB
Connie M. Westhoff, PhD, SBB
Theresa Wiegmann, JD
Susan L. Wilkinson, EdD, MS, MT(ASCP)SBB
James C. Zimring, MD, PhD
 Acknowledgments
H E 1 8 T H E D I T I O N O F the Technical
T Manual was the work of many dedicated
individuals. In addition to the chapter authors,
I would like to thank my three associate edi-
tors: Brenda Grossman, Chris Hillyer, and
Connie Westhoff. Their efforts and long hours
in revising and rewriting chapters during the
review process made my job immeasurably
easier.
We would also like to acknowledge the
members of the following committees and
program units for their expert review of chap-
ters, methods, and appendices for the 18th
edition of the Technical Manual.
REVIEW IN G GROUPS
AABB Representative to ASFA
AATB Representative
Circular of Information Task Force
Clinical Transfusion Medicine Committee
Cord Blood Subsection of the Cellular Thera-
pies Section
Donor Center Accreditation Program Unit
Donor History Task Force
Immunohematology Reference Laboratories
Accreditation Program Unit
Immunohematology Reference Laboratories
Standards Program Unit
Information Systems Committee
Molecular Testing Laboratories Accreditation
Program Unit
Molecular Testing Laboratories Standards Pro-
gram Unit
Novel Therapies and CT Product Development
Subsection of the Cellular Therapies Section
Patient Blood Management Advisory Group
Perioperative Accreditation Program Unit
Perioperative Standards Program Unit
Product Collection and Clinical Practices
Subsection of the Cellular Therapies Section
Product Manufacturing and Testing Subsec-
tion of the Cellular Therapies Section
Quality Operations Subsection of the Cellular
Therapies Section
Quality Systems Accreditation Subcommittee
Regulatory Affairs Subsection of the Cellular
Therapies Section
Relationship Testing Accreditation Program
Unit
Relationship Testing Standards Program Unit
Transfusion-Transmitted Disease Committee
Finally, we would like to thank the editors,
authors, and program unit members of the
17th and earlier editions of the Technical Man-
ual for selected tables, figures, methods, and
written sections of the chapters that are valu-
able inclusions in the new edition.
Mark K. Fung, MD, PhD
Editor in Chief
 Preface
T I S W I T H T R E M E N D O U S pleasure that
I we introduce you to the 18th edition of the
AABB Technical Manual. As with all previous
editions, this revision is based on the solid
foundation of knowledge gathered by past
contributors to whom we are indebted. I
would like to especially acknowledge the tre-
mendous contributions of Drs. Hillyer and
Grossman who have helped guide previous
editions. With the 18th edition they both con-
clude their tenures as Associate Editors. Along
the same lines, I want to welcome Dr. Westhoff
who has joined me in this new challenge of
providing an up-to-date comprehensive
resource of information in the field of transfu-
sion medicine and cellular therapies.
This edition of the Technical Manual will
be most notable for several innovations. First,
the cellular therapy content has been
expanded and reorganized to include many
novel therapies that are moving from the
research setting into the clinical realm. In
addition to updates on the sources of stem
cells and the transfusion support of stem cell
transplantation, chapters focus on the quality
and regulatory issues associated with cord
banking, novel stem cell therapies using non-
hematopoietic stem cells, and tissue engineer-
ing. The new scope will be of great value to the
increasing number of professionals who now
include some aspect of cellular therapy in
their daily responsibilities.
In a similar manner, the content on
patient blood management (PBM) reflects the
growing scope of what is considered PBM. The
traditional topics covered as part of discus-
sions on blood utilization review and periop-
erative blood recovery are augmented by
detailed content on anemia management,
optimization of coagulation, and a host of
adjunctive therapies that can reduce the need
for transfusion. As health-care economics join
better patient care in prompting continued
adoption of PBM, readers will find that the
new emphasis is highly relevant to their needs.
Other content receiving special attention
in this edition is that involving molecular test-
ing. An increasing number of organizations
seek the more detailed test results possible
through investments in molecular technology.
Those in the workforce today need (or soon
will need) a solid understanding of the both
the theory and practice of molecular testing
systems, which is found in this volume.
Perhaps the most obvious upgrade in the
new edition is the relocation of the methods
from the printed pages to electronic storage
medium found inside the back cover. By mov-
ing the methods into the digital format, we are
able for the first time to give Technical Manual
users methods that are already set up as stan-
dard operating procedures (SOPs)—in a tem-
plate that reflects how procedures would
actually be used in real-life. Users are invited
to upload the methods to their facility net-
works and customize them for integration into
their existing SOPs.
In addition to these particular innova-
tions, all chapters have been revised. Some of
the chapter authors have added substantial
updates in great detail to assist the reader,
whether working at the bench or the bedside.
They have embraced the task of explaining the
issues that face all of us in the ever-changing
world of transfusion medicine and cellular
therapies. We welcome your comments and
feedback on the effectiveness of our labors.
Mark K. Fung, MD, PhD
Editor in Chief
ix
 Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

QUALI TY ISSUES

1. Quality Management Systems: Theory and Practice . . . . . . . . . . 1

Tania L. Motschman, MS, MT(ASCP)SBB, CQA(ASQ);
Betsy W. Jett, MT(ASCP), CQA(ASQ)CQM/OE; and
Susan L. Wilkinson, EdD, MS, MT(ASCP)SBB
Concepts in Quality. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2
Practical Application of Quality Management Principles. . . . . . . . . . . . . . . . . . .4
Key Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Appendix 1-1. Glossary of Commonly Used Quality Terms . . . . . . . . . . . . . . . 33
Appendix 1-2. Code of Federal Regulations Quality-Related References . . . 35
Appendix 1-3. Suggested Quality Control Performance Intervals for
Equipment and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

2. Facilities, Work Environment, and Safety. . . . . . . . . . . . . . . . . . . 39

Betsy W. Jett, MT(ASCP), CQA(ASQ)CQM/OE;
Susan L. Wilkinson, EdD, MS, MT(ASCP)SBB; and
Tania L. Motschman, MS, MT(ASCP)SBB, CQA(ASQ)
Facilities. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Safety Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Fire Prevention. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Electrical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Biosafety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Chemical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Radiation Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Shipping Hazardous Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
General Waste Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Key Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

xi
xii 䡲 AA BB TECHNICAL MANUAL

Appendix 2-1. Safety Regulations and Recommendations Applicable to

Health-Care Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Appendix 2-2. General Guidelines for Safe Work Practices, Personal
Protective Equipment, and Engineering Controls . . . . . . . . . . . . . . . . . . . . . 70
Appendix 2-3. Biosafety Level 2 Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Appendix 2-4. Sample List of Hazardous Chemicals that May Be
Encountered in a Blood Bank . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
Appendix 2-5. Chemical Categories and How to Work Safely with Them . . . 76
Appendix 2-6. Incidental Spill Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Appendix 2-7. Managing Hazardous Chemical Spills. . . . . . . . . . . . . . . . . . . . . 81

3. Regulatory Issues in Blood Banking . . . . . . . . . . . . . . . . . . . . . . . . 83

Glenn Ramsey, MD
Biological Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
Licensure and Registration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
FDA Inspections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Blood-Related Devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Hematopoietic Progenitor Cells as Tissues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Managing Recalls and Withdrawals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Medical Laboratory Laws and Regulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Hospital Regulations and Accreditation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Key Points. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

4. Disaster Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Ruth D. Sylvester, MS, MT(ASCP)SBB; William FitzGerald, LTC USA (Ret);
Wendy Trivisonno; and Theresa Wiegmann, JD
Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Business Operations Planning. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Regulatory Considerations in Emergencies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Testing the Disaster Plan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Summary of Lessons Learned from Recent Disasters . . . . . . . . . . . . . . . . . . . 112
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
Key Points. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113

BLOOD DONATI ON AND COLLECTION

5. Allogeneic and Autologous Blood Donor Selection . . . . . . . . . 117

Anne F. Eder, MD, PhD, and Maria D.L.A. Muniz, MD
Overview of Blood Donor Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Selection of Allogeneic Blood Donors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
Blood-Center-Defined Donor Eligibility Criteria . . . . . . . . . . . . . . . . . . . . . . . 127
 Table of Contents 䡲 xiii

Abbreviated DHQ for Frequent Donors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

Recipient-Specific “Designated” or “Directed” Blood Donation . . . . . . . . . 130
Key Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132

6. Whole-Blood Collection and Component Processing . . . . . . 135

Larry J. Dumont, MBA, PhD; Mona Papari, MD;
Colleen A. Aronson, MT(ASCP)SBB; and
Deborah F. Dumont, MT(ASCP)SBB
WB Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Blood Component Preparation and Processing . . . . . . . . . . . . . . . . . . . . . . . . 146
Descriptions of Major Blood Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
Blood Component Modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
Quarantine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Labeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
QC of Blood Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
Key Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

7. Blood Component Collection by Apheresis . . . . . . . . . . . . . . . 167

James W. Smith, MD, PhD
Component Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
Instruments and Systems for Donor Apheresis Collections. . . . . . . . . . . . . . 173
Key Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176

8. Infectious Disease Screening. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179

Susan A. Galel, MD
Historical Overview of Blood Donor Screening . . . . . . . . . . . . . . . . . . . . . . . . 179
Donor Screening Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
Residual Infectious Risks of Transfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
Screening for Specific Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
Pathogen Reduction Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Key Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206

9. Hospital Storage, Monitoring, Pretransfusion Processing,

Distribution, and Inventory Management of
Blood Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Nancy M. Dunbar, MD
Blood and Blood Component Storage and Monitoring . . . . . . . . . . . . . . . . . 213
Pretransfusion Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
Inventory Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
xiv 䡲 AA BB TECHNICAL MANUAL

Key Points. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229

BL OOD GROUPS

10. Molecular Biology and Immunology in Transfusion

Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
James C. Zimring, MD, PhD, and Steven L. Spitalnik, MD
Nucleic Acid Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
Protein Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
Basic Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
Key Points. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253

11. Blood Group Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255

Christine Lomas-Francis, MSc, FIBMS
Basic Principles of Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
Inheritance of Genetic Traits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
Population Genetics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
Relationship Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276
Blood Group Gene Mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
Chimerism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
Blood Group Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
Blood Group Genomics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
Key Points. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288

12. ABO, H, and Lewis Blood Groups and Structurally

Related Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
Laura Cooling, MD, MS
ABO System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
The H System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
The Lewis System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304
I and i Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
P Blood Groups/GLOB Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
Key Points. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313

13. The Rh System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317

Gregory A. Denomme, PhD, FCSMLS(D), and
Connie M. Westhoff, PhD, SBB
Characterization of Rh . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317
Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
 Table of Contents 䡲 xv

Rh Genes and Rh Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319

Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
Rh Genotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330
Rhnull Syndrome and RhAG Blood Group System . . . . . . . . . . . . . . . . . . . . . . 331
Rh Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
Technical Considerations for Rh Typing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
Key Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334

14. Other Blood Group Systems and Antigens . . . . . . . . . . . . . . . . 337

Jill R. Storry, PhD, FIBMS
The MNS System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337
M (MNS1), N (MNS2), S (MNS3), and s (MNS4) . . . . . . . . . . . . . . . . . . . . . . . . 341
The Lutheran System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344
The Kell and Kx Systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
The Duffy System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349
The Kidd System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
The Diego System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352
The Yt System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
The Xg System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
The Scianna System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
The Dombrock System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
The Colton System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355
The Landsteiner-Wiener System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355
The Chido/Rodgers System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356
The Gerbich System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
The Cromer System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
The Knops System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358
The Indian System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358
The Ok System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
The Raph System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
The John Milton Hagen System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
The GILL System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
The RHAG System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360
The FORS System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360
The Jr System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360
The Lan System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360
The Vel System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361
Antigens That Do Not Belong to a Blood Group System . . . . . . . . . . . . . . . . 361
Erythroid Phenotypes Caused by Mutations in Transcription
Factor Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362
Key Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
xvi 䡲 AA BB TECHNICAL MANUAL

ANTIGEN AND ANTIBODY TESTING

15. Pretransfusion Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367

Katharine A. Downes, MD, and Ira A. Shulman, MD
Requests for Transfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
Identification of Recipients and Labeling of Blood Specimens . . . . . . . . . . 368
Specimen Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370
Serologic Testing Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
Pretransfusion Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372
Tubeless Methods for Pretransfusion Testing . . . . . . . . . . . . . . . . . . . . . . . . . . 377
Comparison of Current Testing Results with Previous Records . . . . . . . . . . 378
Donor RBC Unit Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378
Donor RBC Unit Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378
Compatibility Testing or Crossmatch (Serologic or Computer/
Electronic) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
Interpretation of Antibody Screening and Crossmatch Results . . . . . . . . . . 381
Pretransfusion Orders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381
Availability of Compatible Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
Labeling of Blood and Blood Components with the Recipient’s
Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
Special Clinical Situations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385
Key Points. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387

16. Identification of Antibodies to Red Cell Antigens . . . . . . . . . . . 391

Phyllis S. Walker, MS, MT(ASCP)SBB, and
Janis R. Hamilton, MS, MT(ASCP)SBB
Significance of Alloantibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392
Preanalytical Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392
Analytical Phase of Antibody Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . 393
Postanalytical Considerations: Selecting Blood for Transfusion . . . . . . . . . 419
Key Points. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422
Suggested Readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424

17. The Positive Direct Antiglobulin Test and Immune-

Mediated Hemolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425
Regina M. Leger, MSQA, MT(ASCP)SBB, CMQ/OE(ASQ)
The DAT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425
Autoimmune Hemolytic Anemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430
Drug-Induced Immune Hemolytic Anemia . . . . . . . . . . . . . . . . . . . . . . . . . . . 440
Key Points. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445
Appendix 17-1. Drugs Associated with Immune Hemolytic Anemia . . . . . . 448
 Table of Contents 䡲 xvii

18. Platelet and Granulocyte Antigens and Antibodies . . . . . . . . . 453

Brian R. Curtis, PhD, D(ABMLI), MT(ASCP)SBB
Platelet Antigens and Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453
Granulocyte Antigens and Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 466
Key Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 470

19. The HLA System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 475

Robert A. Bray, PhD; Marilyn S. Pollack, PhD; and Howard M. Gebel, PhD
Genetics of the MHC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 476
Biochemistry, Tissue Distribution, and Structure . . . . . . . . . . . . . . . . . . . . . 480
Identification of HLA Antigens and Alleles . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484
Crossmatching and Detection of HLA Antibodies . . . . . . . . . . . . . . . . . . . . . 487
The HLA System and Transfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 488
HLA Testing and Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491
Other Clinically Significant Aspects of HLA . . . . . . . . . . . . . . . . . . . . . . . . . . . 493
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494
Key Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495

CLINICAL CONSIDERATI ONS I N

TRANSFUSION PRACTICE

20. Hemotherapy Decisions and Their Outcomes . . . . . . . . . . . . . 499

Theresa Nester, MD; Shweta Jain, MD; and Jessica Poisson, MD
Red Cell Transfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499
Platelet Transfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 507
Plasma Transfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517
Cryoprecipitate Transfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 522
Granulocyte Transfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 523
Plasma-Derivative Transfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 526
Key Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 532
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533

21 . Administration of Blood Components . . . . . . . . . . . . . . . . . . . 545

Kim Maynard, BSN, RN, OCN
Events and Considerations Before Dispensing Components . . . . . . . . . . . 545
Blood Component Transportation and Dispensing . . . . . . . . . . . . . . . . . . . . 549
Events and Considerations Before Component Administration . . . . . . . . . 550
Manual Administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 552
Unique Transfusion Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 555
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 556
Key Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 556
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557
xviii 䡲 AABB TECHNICAL MANUAL

22. Perinatal Issues in Transfusion Practice . . . . . . . . . . . . . . . . . . 561

Melanie S. Kennedy, MD; Meghan Delaney, DO, MPH; and
Scott Scrape, MD
HDFN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561
RhIG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 565
ABO Hemolytic Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 566
Immune Thrombocytopenia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 567
Key Points. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 568
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 569

23. Neonatal and Pediatric Transfusion Practice . . . . . . . . . . . . . . 571

Cassandra D. Josephson, MD, and Erin Meyer, DO, MPH
Transfusion in Infants Younger Than 4 Months . . . . . . . . . . . . . . . . . . . . . . . 571
Transfusion in Infants Older Than 4 Months and Children . . . . . . . . . . . . . 586
Prevention of Adverse Effects of Transfusion in Neonates,
Older Infants, and Children . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589
Key Points. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592

24. Patient Blood Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599

Mary Ghiglione, RN, MSN, MHA, and
Kathleen E. Puca, MD, MT(ASCP)SBB
Definition and Scope of Patient Blood Management . . . . . . . . . . . . . . . . . . . 599
The Rationale for Patient Blood Management . . . . . . . . . . . . . . . . . . . . . . . . . 600
Basic Elements of a PBM Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 603
Postoperative Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 608
Blood Utilization Review and Changing Physician Behavior . . . . . . . . . . . . . 610
Program Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 612
Key Points. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613
Appendix 24-1. Pharmacologic Therapies for Supporting Patient
Blood Management. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 620
Appendix 24-2. Responsibilities for Activity Levels 1, 2, and 3 PBM
Programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 629

25. Transfusion Support for Hematopoietic Stem Cell

Transplant Recipients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 631
Christopher A. Tormey, MD, and Jeanne E. Hendrickson, MD
Implications of ABO- and Non-ABO-Antigen-Incompatible
Red Blood Cell Transplantation for Transfusion . . . . . . . . . . . . . . . . . . . . . 632
Blood Component Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633
Patients with Neutropenia and Infection that Is Unresponsive to
Antimicrobial Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 638
 Table of Contents 䡲 xix

Special Processing of Blood Components for HSCT Recipients . . . . . . . . . 638

Special Considerations for Transfusions in Pediatric HSCT Recipients . . . 639
Information Portability for HSCT Recipients . . . . . . . . . . . . . . . . . . . . . . . . . 640
Key Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 640
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 641

26. Therapeutic Apheresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 645

Robertson D. Davenport, MD
Principles and Modalities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 645
Indications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 647
Anticoagulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 658
Adverse Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 658
Vascular Access . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 660
Patient Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 660
Key Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 661
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 662

27. Noninfectious Complications of Blood Transfusion . . . . . . . . 665

Catherine A. Mazzei, MD; Mark A. Popovsky, MD; and
Patricia M. Kopko, MD
Hemovigilance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 665
Recognition and Evaluation of a Suspected Transfusion Reaction . . . . . . 666
Delayed Transfusion Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 686
Fatality Reporting Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 691
Key Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 691
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 692

28. Approaches to Blood Utilization Auditing . . . . . . . . . . . . . . . . . 697

Alan Tinmouth, MD, FRCPC, MSc, and
Simon Stanworth, FRCP, FRCPath, DPhil
Rationale for Monitoring Blood Utilization . . . . . . . . . . . . . . . . . . . . . . . . . . . 698
Types of Transfusion Audits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 699
Interventions to Change Transfusion Practice . . . . . . . . . . . . . . . . . . . . . . . . . 704
Effectiveness of Monitoring and Interventions to Change
Transfusion Practice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 704
Selecting an Audit Process to Monitor Transfusions . . . . . . . . . . . . . . . . . . . 705
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 705
Key Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 707
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 708
Appendix 28-1. Transfusion Order Form in Use at St. Vincent
Indianapolis Hospital Since 2001 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 711
xx 䡲 AABB T ECHNICAL M ANUAL

TRA NSPLA NTA TION

29. The Collection and Processing of Hematopoietic

Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713
Scott A. Koepsell, MD, PhD; Eapen K. Jacob, MD; and
David H. McKenna Jr, MD
Clinical Utility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713
Determination of Graft Source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 717
Collection/Sources of HSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 718
Processing of HSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 720
Specialized Cell-Processing Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 721
Cryopreservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 722
Shipping and Transport of HSC Cellular Products . . . . . . . . . . . . . . . . . . . . . . 723
Patient Care . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 724
Other Regulatory Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 725
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 725
Key Points. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 725
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 726

30. Umbilical Cord Blood Banking . . . . . . . . . . . . . . . . . . . . . . . . . . . 729

Aleksandar M. Babic, MD, PhD, and Donna M. Regan, MT(ASCP)SBB
Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 729
Donor-Related Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 730
UCB Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 733
UCB Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 734
Shipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 737
Receipt of UCB for Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 738
Thawing and Washing of UCB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 740
Infusion of UCB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 741
Economic Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 742
Regulations and Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 743
Key Points. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 747
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 747

31. Tissue-Derived Non-Hematopoietic Stem Cell Sources

for Use in Cell-Based Therapies . . . . . . . . . . . . . . . . . . . . . . . . 753
Yameena Jawed, MD; Brian Johnstone, PhD;
Sreedhar Thirumala, PhD; and Keith March, MD, PhD
MSC Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 754
Properties of Clinical Relevance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 758
Isolation and Expansion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 758
Standardization of Methods for Isolation and Expansion of
Clinical Product . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 760
Cell Product Banking and Management of Supply Chain and End Use . . . 761
Therapeutic Applications of MSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 762
 Table of Contents 䡲 xxi

Current Research and Development: Focus on Cell Culture and

Handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 764
Conclusions and Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 765
Key Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 766
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 766

32. Human Allografts and the Hospital Transfusion Service . . . . 773

Lance D. Trainor, MD, and Rita A. Reik, MD
Tissue Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 773
Regulations and Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 777
Hospital Tissue Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 778
Transfusion Service Support for Organ Transplantation . . . . . . . . . . . . . . . . 783
Key Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 784

33. Blood and Marrow-Derived Nonhematopoietic Stem Cell

Sources and Immune Cells for Clinical Applications . . . . . 785
Mickey B. C. Koh, MD, PhD; Edward R. Samuel, PhD, MSc; and
Garnet Suck, PhD, MSc
Immune Cells for Clinical Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786
Induced Pluripotent Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 795
Regulatory and Oversight Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797
Key Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 798

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 803

METHO DS

Methods Introduction

1. General Laboratory Methods—Introduction

Method 1-1. Shipping Hazardous Materials
Method 1-2. Monitoring Temperature During Shipment of Blood
Method 1-3. Treating Incompletely Clotted Specimens
Method 1-4. Solution Preparation Procedure
Method 1-5. Serum Dilution Procedure
Method 1-6. Dilution of Percentage Solutions Procedure
Method 1-7. Preparing a 3% Red Cell Suspension
xxii 䡲 AA BB T ECHNICAL MANUAL

Method 1-8. Preparing and Using Phosphate Buffer

Method 1-9. Reading and Grading Tube Agglutination

2. Red Cell Typing Methods—Introduction

Method 2-1. Determining ABO Group of Red Cells—Slide Test
Method 2-2. Determining ABO Group of Red Cells and Serum—Tube Test
Method 2-3. Determining ABO Group of Red Cells and Serum—Microplate Test
Method 2-4. Initial Investigation of ABO Grouping Discrepancies Procedure
Method 2-5. Detecting Weak A and B Antigens and Antibodies by
Cold Temperature Enhancement
Method 2-6. Confirming Weak A and B Antigens Using Enzyme-Treated
Red Cells
Method 2-7. Confirming Weak A or B Subgroup by Adsorption and Elution
Method 2-8. Testing Saliva for A, B, H, Lea, and Leb Antigens
Method 2-9. Confirming Anti-A1 in an A2 or Weak A Subgroup
Method 2-10. Resolving ABO Discrepancies Caused by Unexpected
Alloantibodies
Method 2-11. Determining Serum Group Without Centrifugation
Method 2-12. Determining Rh (D) Type—Slide Test
Method 2-13. Determining Rh (D) Type—Tube Test
Method 2-14. Determining Rh (D) Type—Microplate Test
Method 2-15. Testing for Weak D
Method 2-16. Preparing and Using Lectins
Method 2-17. Removing Autoantibody by Warm Saline Washes
Method 2-18. Using Sulfhydryl Reagents to Disperse Autoagglutination
Method 2-19. Using Gentle Heat Elution to Test Red Cells with a Positive DAT
Method 2-20. Dissociating IgG by Chloroquine for Antigen Testing of Red Cells
with a Positive DAT
Method 2-21. Using Acid Glycine/EDTA to Remove Antibodies from Red Cells
Method 2-22. Separating Transfused from Autologous Red Cells by Simple
Centrifugation
Method 2-23. Separating Transfused from Autologous Red Cells in Patients with
Hemoglobin S Disease

3. Antibody Detection, Identification, and Compatibility Testing—

Introduction
Method 3-1. Using Immediate-Spin Compatibility Testing to Demonstrate ABO
Incompatibility
Method 3-2. Saline Indirect Antiglobulin Test Procedure
Method 3-3. Albumin or LISS-Additive Indirect Antiglobulin Test Procedure
Method 3-4. LISS-Suspension Indirect Antiglobulin Test Procedure
Method 3-5. PEG Indirect Antiglobulin Test Procedure
Method 3-6. Prewarming Procedure
Method 3-7. Detecting Antibodies in the Presence of Rouleaux—Saline
Replacement
Method 3-8. Preparing Ficin Enzyme Stock, 1% w/v
Method 3-9. Preparing Papain Enzyme Stock, 1% w/v
Method 3-10. Standardizing Enzyme Procedures
 Table of Contents 䡲 xxiii

Method 3-11. Evaluating Enzyme-Treated Red Cells

Method 3-12. One-Stage Enzyme Procedure
Method 3-13. Two-Stage Enzyme Procedure
Method 3-14. Performing a Direct Antiglobulin Test
Method 3-15. Antibody Titration Procedure
Method 3-16. Using Sulfhydryl Reagents to Distinguish IgM from IgG Antibodies
Method 3-17. Using Plasma Inhibition to Distinguish Anti-Ch and -Rg from
Other Antibodies with Similar Characteristics
Method 3-18. Treating Red Cells Using DTT or AET
Method 3-19. Neutralizing Anti-Sda with Urine
Method 3-20. Adsorption Procedure
Method 3-21. Using the American Rare Donor Program

4. Investigation of a Positive DAT Result—Introduction

Method 4-1. Cold-Acid Elution Procedure
Method 4-2. Glycine-HCl/EDTA Elution Procedure
Method 4-3. Heat Elution Procedure
Method 4-4. Lui Freeze-Thaw Elution Procedure
Method 4-5. Cold Autoadsorption Procedure
Method 4-6. Determining the Specificity of Cold-Reactive Autoagglutinins
Method 4-7. Cold Agglutinin Titer Procedure
Method 4-8. Adsorbing Warm-Reactive Autoantibodies Using Autologous Red
Cells
Method 4-9. Adsorbing Warm-Reactive Autoantibodies Using Allogeneic Red
Cells
Method 4-10. Polyethylene Glycol Adsorption Procedure
Method 4-11. Performing the Donath-Landsteiner Test
Method 4-12. Detecting Drug Antibodies by Testing Drug-Treated Red Cells
Method 4-13. Detecting Drug Antibodies by Testing in the Presence of Drug

5. Hemolytic Disease of the Fetus and Newborn—Introduction

Method 5-1. Testing for Fetomaternal Hemorrhage—The Rosette Test
Method 5-2. Testing for Fetomaternal Hemorrhage—Modified
Kleihauer-Betke Test
Method 5-3. Using Antibody Titration Studies to Assist in Early
Detection of Hemolytic Disease of the Fetus and Newborn

6. Blood Collection, Component Preparation, and Storage—

Introduction
Method 6-1. Screening Donors for Anemia—Copper Sulfate Method
Method 6-2. Preparing the Donor’s Arm for Blood Collection
Method 6-3. Collecting Blood and Samples for Processing and Compatibility
Tests
Method 6-4. Preparing Red Blood Cells from Whole Blood
Method 6-5. Preparing Prestorage Red Blood Cells Leukocytes Reduced from
Whole Blood
Method 6-6. Using High-Concentration Glycerol to Cryopreserve Red Cells—
Meryman Method
xxiv 䡲 AA BB T ECHNICAL MANUAL

Method 6-7. Using High-Concentration Glycerol to Cryopreserve Red Cells—

Valeri Method
Method 6-8. Checking the Adequacy of Deglycerolization of Red Blood Cells
Method 6-9. Preparing Fresh Frozen Plasma from Whole Blood
Method 6-10. Preparing Cryoprecipitated AHF from Whole Blood
Method 6-11. Thawing and Pooling Cryoprecipitated AHF
Method 6-12. Preparing Platelets from Whole Blood
Method 6-13. Removing Plasma from Platelets (Volume Reduction)

7. Transplantation of Cells and Tissue—Introduction

Method 7-1. Infusing Cryopreserved Hematopoietic Cells
Method 7-2. Processing Umbilical Cord Blood
Method 7-3. Investigating Adverse Events and Infections Following Tissue
Allograft Use

8. Quality Control Methods—Introduction

Method 8-1. Validating Copper Sulfate Solution
Method 8-2. Calibrating Liquid-in-Glass Laboratory Thermometers
Method 8-3. Calibrating Electronic Oral Thermometers
Method 8-4. Testing Refrigerator Alarms
Method 8-5. Testing Freezer Alarms
Method 8-6. Calibrating Centrifuges for Platelet Separation
Method 8-7. Calibrating a Serologic Centrifuge for Immediate Agglutination
Method 8-8. Calibrating a Serologic Centrifuge for Washing and Antiglobulin
Testing
Method 8-9. Testing Automatic Cell Washers
Method 8-10. Monitoring Cell Counts of Apheresis Components
Method 8-11. Counting Residual White Cells in Leukocyte-Reduced Blood and
Components—Manual Method

A P P E N DI C E S

Appendix 1. Normal Values in Adults

Appendix 2. Selected Normal Values in Children
Appendix 3. Typical Normal Values in Tests of Hemostasis and Coagulation
(Adults)
Appendix 4. Coagulation Factor Values in Platelet Concentrates
Appendix 5. Approximate Normal Values for Red Cell, Plasma, and Blood
Volumes
Appendix 6. Blood Group Antigens Assigned to Systems
Appendix 7. Examples of Gene, Antigen, and Phenotype Symbols in
Conventional and International Society of Blood Transfusion Terminology
Appendix 8. Examples of Correct and Incorrect Terminology
Appendix 9. Distribution of ABO/Rh Phenotypes by Race or Ethnicity
Appendix 10. Example of a Maximum Surgical Blood Order Schedule
Appendix 11. Directory of Organizations
 Abbreviations
AATB American Association of Tissue Banks cDNA complementary deoxyribonucleic acid
ACD acid-citrate-dextrose CDRH Center for Devices and Radiological
ACE angiotensin-converting enzyme Health
ACOG American College of Obstetricians and CFR Code of Federal Regulations
Gynecologists CFU colony-forming unit
ADP adenosine diphosphate CGD chronic granulomatous disease
AET 2-aminoethylisothiouronium cGMP current good manufacturing practice
AHF antihemophilic factor cGTP current good tissue practice
AHG antihuman globulin cGy centiGray
AHTR acute hemolytic transfusion reaction CI confidence interval
AIDS acquired immune deficiency syndrome CIDP chronic inflammatory demyelinating
AIHA autoimmune hemolytic anemia polyneuropathy
ALDH aldehyde dehydrogenase CJD Creutzfeldt-Jakob disease
ALT alanine aminotransferase CLIA Clinical Laboratory Improvement
Amendments
AML acute myelogenous leukemia
CLSI Clinical and Laboratory Standards
AMR antibody-mediated rejection Institute
ANH acute normovolemic hemodilution CML chronic myelogenous leukemia
AORN Association of periOperative Registered CMS Centers for Medicare and Medicaid
Nurses Services
APC antigen-presenting cell CMV cytomegalovirus
aPTT activated partial thromboplastin time CNS central nervous system
ARDP American Rare Donor Program CP2D citrate-phosphate-dextrose-dextrose
AS additive solution CPD citrate-phosphate-dextrose
ASFA American Society for Apheresis CPDA-1 citrate-phosphate-dextrose-adenine-1
ASHI American Society for Histocompatibility CR complement receptor
and Immunogenetics
CREG cross-reactive group
ATP adenosine triphosphate
CRYO cryoprecipitate (Cryoprecipitated AHF)
BCR B-cell receptor
C/T crossmatch/transfusion
BLA biologics license application
CV coefficient of variation
BPD biological product deviation
DAF decay-accelerating factor
BSA bovine serum albumin or body surface
area DAT direct antiglobulin test
BSC biological safety cabinet DDAVP deamino-D-arginine vasopressin
BSL-1 Biosafety Level 1 DHQ donor history questionnaire
CAP College of American Pathologists DHTR delayed hemolytic transfusion reaction
CAS cold agglutinin syndrome DIC disseminated intravascular coagulation
CBER Center for Biologics Evaluation and DMSO dimethylsulfoxide
Research DNA deoxyribonucleic acid
CCI corrected count increment DOT (US) Department of Transportation
CD clusters of differentiation 2,3-DPG 2,3-diphosphoglycerate
CDC Centers for Disease Control and DRG diagnosis-related group
Prevention
DSTR delayed serologic transfusion reaction HDFN hemolytic disease of the fetus and
DTT dithiothreitol newborn
EACA epsilon aminocaproic acid HES hydroxyethyl starch
EBAA Eye Bank Association of America HHS (US) Department of Health and Human
Services
ECMO extracorporeal membrane oxygenation
HIT heparin-induced thrombocytopenia
ECV extracorporeal volume
HIV human immunodeficiency virus
EDTA ethylenediaminetetraacetic acid
HNA human neutrophil antigen
EIA enzyme immunoassay
HPA human platelet antigen
ELBW extremely low birthweight
HPC hematopoietic progenitor cell
ELISA enzyme-linked immunosorbent assay
HPC(A) HPCs from apheresis (HPC, Apheresis)
EMAs emergency management agencies
HPC(C) HPCs from cord blood (HPC, Cord
EPO erythropoietin
Blood)
FACT Foundation for the Accreditation of
HPC(M) HPCs from marrow (HPC, Marrow)
Cellular Therapy
HSC hematopoietic stem cell
FcR Fc gamma receptor
HSCT hematopoietic stem cell transplantation
FDA Food and Drug Administration
HTLV-I human T-cell lymphotropic virus, type I
FFP Fresh Frozen Plasma
HTR hemolytic transfusion reaction
FMH fetomaternal hemorrhage
HUS hemolytic uremic syndrome
FNAIT fetal/neonatal alloimmune
thrombocytopenia IAT indirect antiglobulin test
FNHTR febrile nonhemolytic transfusion IATA International Air Transport Association
reaction ICAM-1 intercellular adhesion molecule-1
FTA-ABS fluorescent treponemal antibody ID indentification or individual donation
absorption test Ig immunoglobulin
G-CSF granulocyte colony-stimulating factor IL-1 interleukin-1 alpha
GalNAc N-acetylgalactosamine IL-1 interleukin-1 beta
GM-CSF granulocyte-macrophage colony- IL-2 interleukin-2
stimulating factor
IM intramuscular
GMP good manufacturing practice
IND investigational new drug
GPIa glycoprotein Ia
INR international normalized ratio
GPA glycophorin A
iPSCs induced pluripotent stem cells
GPB glycophorin B
IRL immunohematology reference
GPC glycophorin C laboratory
GPD glycophorin D IS immediate spin
GTP good tissue practice ISBT International Society of Blood
GVHD graft-vs-host disease Transfusion
Gy Gray ISO International Organization for
HAV hepatitis A virus Standardization
HAZMAT hazardous material ITP immune thrombocytopenia
Hb hemoglobin IU international unit
HBc hepatitis B core antigen IV intravenous
HBsAg hepatitis B surface antigen IVIG intravenous immune globulin
HBV hepatitis B virus LDH lactate dehydrogenase
Hct hematocrit LDL low-density lipoprotein
HCT/Ps human cells, tissues, and cellular and LISS low-ionic-strength saline
tissue-based products LN2 liquid nitrogen
HCV hepatitis C virus LR leukocyte-reduced
MAC membrane attack complex QC quality control
2-ME 2-mercaptoethanol QSE Quality System Essential
MF mixed field RBCs Red Blood Cells (blood donor unit)
MHC major histocompatibility complex RFLP restriction fragment length
MNC mononuclear cell polymorphism
MoAb monoclonal antibody rFVIIa recombinant Factor VIIa
MPHA mixed passive hemagglutination assay Rh Rhesus factor
mRNA messenger ribonucleic acid RHAG Rh-associated glycoprotein
MSC mesenchymal stem cell RhIG Rh Immune Globulin
MSDS material safety data sheet RIBA recombinant immunoblot assay
MSM male who has sex with another male RIPA radioimmunoprecipitation assay
NAIT neonatal alloimmune thrombocytopenia RNA ribonucleic acid
NAN neonatal alloimmune neutropenia RPR rapid plasma reagin (serologic test for
syphilis)
NAT nucleic acid testing
RT room temperature or reverse
NHLBI National Heart, Lung, and Blood transcriptase
Institute
SCF stem cell factor
NIH National Institutes of Health
SD standard deviation or solvent/detergent
NIPA nonimmunologic protein adsorption
SNP single nucleotide polymorphism
NK natural killer
SOP standard operating procedure
NMDP National Marrow Donor Program
SPRCA solid-phase red cell adherence
NRC Nuclear Regulatory Commission
TA transfusion-associated
NRF National Response Framework
TACO transfusion-associated circulatory
OSHA Occupational Safety and Health overload
Administration
TCR T-cell receptor
p probability
TMA transcription-mediated amplification
PAD preoperative autologous (blood)
donation TNCs total nucleated cells
PBM patient blood management TNF- tumor necrosis factor alpha
PBS phosphate-buffered saline TPE therapeutic plasma exchange
PCH paroxysmal cold hemoglobinuria TPO thrombopoietin
PCR polymerase chain reaction TRALI transfusion-related acute lung injury
PEG polyethylene glycol TSE transmissible spongiform
encephalopathy
PF24 Plasma Frozen Within 24 Hours After
TTP thrombotic thrombocytopenic purpura
Phlebotomy
UCB umbilical cord blood
PF24 RT24 Plasma Frozen Within 24 Hours After
Phlebotomy Held at Room Temperature UDP uridine diphosphate
Up to 24 Hours after Phlebotomy UNOS United Network for Organ Sharing
PPE personal protective equipment USC United States Code
PRA panel-reactive antibody vCJD variant Creutzfeldt-Jakob disease
PRCA pure red cell aplasia VLBW very low birthweight
PRP platelet-rich plasma vWD von Willebrand disease
PRT pathogen reduction technology vWF von Willebrand factor
PT prothrombin time or proficiency testing WAIHA warm autoimmune hemolytic anemia
PTP posttransfusion purpura WB whole blood or Western blot
PTT partial thromboplastin time WBC white blood cell
PVC polyvinyl chloride WHO World Health Organization
QA quality assessment or quality assurance WNV West Nile virus
 C h a p t e r 1

Quality Management Systems:

Theory and Practice

Tania L. Motschman, MS, MT(ASCP)SBB, CQA(ASQ);

Betsy W. Jett, MT(ASCP), CQA(ASQ)CQM/OE; and
Susan L. Wilkinson, EdD, MS, MT(ASCP)SBB

A PRIMARY GOAL of transfusion istration (FDA), especially in the current good

medicine, cellular therapies, and clinical
diagnostics is to promote high standards of
quality in all aspects of patient care and relat-
ed products and services. This commitment to
quality is reflected in standards of practice set
forth by the AABB. AABB standards use a quali-
ty management system as the framework for
quality. A quality management system in-
cludes the organizational structure, responsi-
bilities, policies, processes, procedures, and
resources established by executive manage-
ment to achieve and maintain quality. (A glos-
sary of quality terms used in this chapter is in-
cluded in Appendix 1-1.)
The establishment of a formal quality as-
surance program is required under the Centers
for Medicare and Medicaid Services (CMS)
Clinical Laboratory Improvement Amend-
ments (CLIA)1 and the Food and Drug Admin-
manufacturing practice (cGMP) and current
good tissue practice (cGTP) regulations.2-5 The
FDA regulations in the Code of Federal Regula-
tions (CFR) Title 21, Part 211.22 require an in-
dependent quality control (QC) or quality as-
surance unit that has responsibility for the
overall quality of the facility’s finished product
and authority to control the processes that
may affect this product.3 (See frequently used
CFR quality-related citations in Appendix 1-2.)
Professional and accrediting organizations
such as the AABB, 6,7 College of American Pa-
thologists (CAP), 8 The Joint Commission, 9,10
Clinical and Laboratory Standards Institute
(CLSI),11 and Foundation for the Accreditation
of Cellular Therapy (FACT),12 have also estab-
lished requirements and guidelines to address
quality issues. The International Organization
for Standardization (ISO) quality manage-

Tania L. Motschman, MS, MT(ASCP)SBB, CQA(ASQ), Quality Director, Esoteric Business Unit, Laboratory
Corporation of America, Burlington, North Carolina; Betsy W. Jett, MT(ASCP), CQA(ASQ)CQM/OE, Vice Presi-
dent for Quality and Regulatory Affairs, New York Blood Center, New York, New York; and Susan L. Wilkinson,
EdD, MS, MT(ASCP)SBB, Interim Department Head, Analytical and Diagnostic Sciences, College of Allied
Health Sciences, and Associate Professor Emerita, University of Cincinnati, Cincinnati, Ohio
The authors have disclosed no conflicts of interest.

1
2 䡲 AABB TECHNICAL MANUAL
ment standards (ISO 9001) are generic to any
industry and describe the important mini-
mum elements of a quality management sys-
tem.13 The ISO 15189 standards are specific to
laboratory medicine.14 In addition, the Health
Care Criteria for Performance Excellence pub-
lished by the Baldrige Performance Excellence
Program15 provides an excellent framework for
implementing quality on an organizational
level.
The AABB has defined the minimum ele-
ments that must be addressed in its quality
system essentials (QSEs). 16 The AABB QSEs
were developed to be compatible with ISO
9001 standards, the FDA Guideline for Quality
Assurance in Blood Establishments,5 and other
FDA quality system approaches.17,18
CO NCE P T S I N QUA LI T Y
Quality Control, Quality Assurance,
and Quality Management
The purpose of QC is to provide feedback to
operational staff about the state of a process
that is in progress. QC tells staff whether to
continue (everything is acceptable) or to stop
until a problem has been resolved (something
is found to be out of control).
Historically, transfusion services and do-
nor centers have used many QC measures as
standard practices in their operations. Exam-
ples include reagent QC; product QC; clerical
checks; visual inspections; and measure-
ments, such as temperature readings on refrig-
erators and volume or cell counts on finished
blood components.
Quality assurance activities are not tied to
the actual performance of a process. Rather,
they include activities, such as the develop-
ment of documents like standard operating
procedures (SOPs), to ensure consistent and
correct performance of processes, training of
personnel, and qualification of materials and
equipment. Quality assurance activities also
include retrospective reviews and analyses of
operational performance data to determine
whether the overall process is in a state of con-
trol and to detect shifts or trends that require
attention. Quality assurance provides infor-
mation to process managers regarding levels
of performance that can be used in setting pri-
orities for process improvement. Examples of
quality assurance activities in transfusion
medicine and cellular therapies include record
reviews, monitoring of quality indicators, and
internal assessments.
Quality management considers interrelat-
ed processes in the context of the organization
and its relations with customers and suppliers.
It addresses the leadership role of executive
management in creating a commitment to
quality throughout the organization, the un-
derstanding of suppliers and customers as
partners in quality, the management of human
and other resources, and quality planning.
The quality systems approach described
in this chapter encompasses all of these activi-
ties. It ensures application of quality principles
throughout the organization and reflects the
changing focus of quality efforts from detec-
tion to prevention.
Juran’s Quality Trilogy
Juran’s Quality Trilogy is one example of a
quality management approach. This model
centers around three fundamental processes
for managing quality in any organization:
planning, control, and improvement.19(p2.5)
The planning process for a new product
or service includes activities to identify re-
quirements, develop product and process
specifications that meet those requirements,
and design the process. During the planning
phase, the facility must perform the following
steps:
1. Establish quality goals for the project.
2. Identify the customers.
3. Determine customer needs and expecta-
tions.
4. Develop product and service specifications
to meet customer, operational, regulatory,
and accreditation requirements.
5. Develop operational processes for produc-
tion and delivery, including written proce-
dures and resources requirements.
6. Develop process controls and validate the
process in the operational setting.
 CHAPTER 1 Quality Management Systems: Theory and Practice
The results of the planning process are re-
ferred to as “design output.”13
Once the plan is implemented, the con-
trol process provides a feedback loop for oper-
ations that includes the following:
1. Evaluation of performance.
2. Comparison of performance to goals.
3. Action to correct any discrepancy between
the two.
The control process addresses inputs,
production, and delivery of products and ser-
vices to meet specifications. Process controls
should allow staff to recognize when things are
going wrong and to either make appropriate
adjustments to ensure a product’s quality or
stop the process.
An important goal in quality manage-
ment is to establish a set of controls that en-
sure process and product quality but are not
excessive. Controls that do not add value
should be eliminated to conserve limited
resources and allow staff to focus attention on
those controls that are critical to the opera-
tion.
Statistical tools, such as process capabili-
ty measurement and control charts, allow a fa-
cility to evaluate process performance during
the planning stage and in operations. These
tools help determine if a process is stable
(ie, in statistical control) and if it is capable of
meeting product and ser vice specifica-
tions.19(p22.19)
Quality improvement is intended to en-
able an organization to attain higher levels of
performance by creating new or better fea-
tures that add value or by removing deficien-
cies in the process, product, or service. Oppor-
t u n i t i e s t o i m p r ove m a y b e re l a t e d t o
deficiencies in the initial planning process;
unforeseen factors discovered on implementa-
tion; shifts in customer needs; or changes in
starting materials, environmental factors, or
other variables that affect the process. Im-
provements must be based on data-driven
analysis; an ongoing measurement and assess-
ment program is fundamental to that process.
䡲 3
Process Approach
In its most generic form, a process includes all
of the resources and activities that transform
an input into an output. An understanding of
how to manage and control processes in trans-
fusion medicine, cellular therapies, and clini-
cal diagnostic activities is based on this simple
equation:
INPUT  PROCESS  OUTPUT
For example, a key process for donor cen-
ters is donor selection. The “input” includes
the individual who presents for donation and
all of the resources required for that donor’s
health screening. Through a series of activities
(a process), including the verification of the
donor’s identity, a deferral status review, a
mini-physical exam, and a health history
questionnaire, an individual is deemed an “eli-
gible donor.” The “output” is either an eligible
donor who can continue to the next process
(blood collection) or an ineligible donor who is
deferred. When the selection process results in
a deferred donor, the resources (inputs) asso-
ciated with that process do not continue
through the process but contribute to the cost
of quality. One way that donor centers at-
tempt to minimize this cost is to educate po-
tential donors before screening so that those
who are not eligible do not enter the selection
process.
Strategies for managing a process should
address all of its components, including its in-
terrelated activities, inputs, outputs, and re-
sources. Supplier qualification, formal agree-
ments, supply verification, and inventory
control are strategies for ensuring that the
inputs to a process meet specifications.
Personnel training and competence assess-
ment, equipment maintenance and control,
management of documents and records, and
implementation of appropriate in-process
controls provide assurance that the process
will operate as intended. End-product testing
and inspection, customer feedback, and
outcome measurement provide data to evalu-
ate product quality and improve the process.
These output measurements and quality
4 䡲 AABB TECHNICAL MANUAL
indicators are used to evaluate the effective-
ness of the process and process controls.
To manage a system of processes effec-
tively, the facility must understand how its
processes interact and what cause-and-effect
relationships exist between them. In the donor
selection scenario, the consequences of ac-
cepting a donor who is not eligible reach into
almost every other process in the facility. For
example, if a donor with a history of high-risk
behavior is not identified as such during the
selection process, the donated unit(s) may re-
turn positive test results for one of the viral
marker assays, triggering follow-up testing,
look-back investigations, and donor deferral
and notification procedures. Components
must be quarantined and their discard docu-
mented. Personnel involved in collecting and
processing the unit(s) are at risk of exposure to
infectious agents. Part of quality planning is to
identify these relationships so that quick and
appropriate corrective action can be taken
when process controls fail.
It is important to remember that opera-
tional processes include not only product
manufacture or service creation, but also the
delivery of a product or service. Delivery gen-
erally involves interaction with the customer.
The quality of that transaction is critical to
customer satisfaction and should not be over-
looked in the design and ongoing assessment
of the quality management system.
Service vs Production
Quality management principles apply equally
to a broad spectrum of activities, from those
related to processing and production to those
involving interactions between individuals in
delivering a service. However, different strate-
gies may be appropriate when there are differ-
ing expectations related to customer satisfac-
tion. Although the emphasis in a production
process is on minimizing variation to create a
product that consistently meets specifications,
service processes require a certain degree of
flexibility to address customer needs and cir-
cumstances at the time of the transaction. In
production, personnel need to know how to
maintain uniformity in day-to-day operations.
In service, personnel need to be able to adapt
a service in a way that meets customer ex-
pectations but does not compromise quality.
To do this, personnel must have sufficient
knowledge and understanding of interrelated
processes to use independent judgment ap-
propriately, or they must have ready access to
higher-level decision makers.
When one designs quality management
systems for production processes, it is useful
to think of the process as the driver, with peo-
ple providing the oversight and support need-
ed to keep it running smoothly and effectively.
In service, people are the focus; the underlying
process provides a foundation that enables
staff to deliver safe and effective services that
meet customers’ needs in almost any situa-
tion.
Quality Management as an Evolving
Science
The principles and tools used in quality man-
agement today will change as research pro-
vides new knowledge of organizational behav-
ior, technology provides new solutions, and
the transfusion medicine and cellular thera-
pies fields present new challenges. Periodic as-
sessments of the quality management system
will help identify practices that are no longer
effective or that could be improved through
the use of new technology or new tools.
PRACTICAL APP L ICATION OF
QUAL I TY MAN AGE ME NT
PR IN CI PL E S
The remainder of this chapter addresses the
elements of a quality management system and
practical application of quality management
principles to the transfusion medicine, cellular
therapies, and clinical diagnostics environ-
ments. These basic elements include the fol-
lowing:
䡲 Organization and leadership.
䡲 Customer focus.
䡲 Facilities, work environment, and safety.
䡲 Human resources.
䡲 Suppliers and materials management.
 CHAPTER 1 Quality Management Systems: Theory and Practice
䡲 Equipment management.
䡲 Process management.
䡲 Documents and records.
䡲 Information management.
䡲 Management of nonconforming events.
䡲 Monitoring and assessment.
䡲 Process improvement.
Organization and Leadership
The facility should be organized in a manner
that promotes effective implementation and
management of its operational and quality
management system. The structure of the or-
ganization must be documented, and the roles
and responsibilities for the provision of tests,
products, and services must be clearly defined.
These provisions should include a description
of the relationships and avenues of communi-
cation between organizational units and those
responsible for key quality functions. Each fa-
cility may define its structure in any format
that suits its operations. Organizational trees
or charts that show the structure and relation-
ships are helpful.
The facility should define in writing the
authority and responsibilities of executive
management to establish and maintain the
quality management system. These responsi-
bilities include the following:
䡲 Establishing a quality policy and associated
quality goals and quality objectives.
䡲 Providing adequate facilities as well as hu-
man, equipment, and material resources to
carry out the operations of the facility and
the quality management system.
䡲 Ensuring appropriate design and effective
implementation of new or modified pro-
cesses and procedures.
䡲 Participating in the review and approval of
quality and technical policies, processes,
and procedures.
䡲 Enforcing adherence to operational and
quality policies, processes, and procedures.
䡲 Overseeing operations and regulatory and
accreditation compliance.
䡲 Periodically reviewing and assessing quality
management system effectiveness.
䡲 5
䡲 Identifying designees and defining their re-
sponsibilities when assisting executive
management in carrying out these duties.
Executive management support for the
quality management system goals, objectives,
and policies is critical to the program’s success.
Executive management needs to clearly com-
municate its commitment to quality goals and
create an organizational culture that embraces
quality principles.
The individual designated to oversee the
facility’s quality functions should report di-
rectly to executive management. In addition to
having the responsibility to coordinate, moni-
tor, and facilitate quality system activities, this
person should have the authority to recom-
mend and initiate corrective action when ap-
propriate.5 The designated individual need not
perform all of the quality functions personally.
Ideally, this person should be independent of
the facility’s operational functions. In small fa-
cilities, this independence may not always be
possible and carrying out this function may re-
quire some creativity. Depending on the orga-
nization’s size and scope, the designated over-
sight person may work in the transfusion
service, have laboratory-wide responsibilities,
supervise other workers (eg, a quality unit), or
be part of an organization-wide quality unit
(eg, hospital quality or risk management). In-
dividuals with dual quality and operational
responsibilities should not provide quality
oversight for operational work that they have
performed.
Quality oversight functions may include
the following5:
䡲 Review and approval of SOPs and training
plans.
䡲 Review and approval of validation plans
and results.
䡲 Review and approval of document control
and record-keeping systems.
䡲 Audit of operational functions.
䡲 Development of criteria for evaluating sys-
tems.
䡲 Review and approval of suppliers.
䡲 Review and approval of product and service
specifications (eg, the requirements to be
6 䡲 AABB TECHNICAL MANUAL
met in the manufacture, distribution, or ad-
ministration of blood components, cellular
therapy products, tissues, and derivatives).
䡲 Review of reports of adverse reactions, de-
viations in the manufacturing process,
nonconforming products and services, and
customer complaints.
䡲 Participation in decisions to determine
whether blood components, cellular thera-
py products, tissues, derivatives, and ser-
vices are suitable for use, distribution, or
recall.
䡲 Review and approval of corrective action
plans.
䡲 Surveillance of problems (eg, event or inci-
dent reports, Form FDA 483 observations,
or customer complaints) and the effective-
ness of corrective actions implemented to
solve those problems.
䡲 Use of data resources to identify trends and
potential problems before a situation wors-
ens and patients and/or products are af-
fected.
䡲 Preparation of periodic (as specified by the
organization) reports of quality issues,
trends, findings, and corrective and pre-
ventive actions.
Quality oversight functions may be
shared among existing staff, departments, and
facilities or, in some instances, may be per-
formed by an outside firm under a contract.
The goal is to provide an independent evalua-
tion of the facility’s quality activities to the ex-
tent possible. Policies, processes, and proce-
dures should exist to define the roles and
responsibilities of all individuals in the devel-
opment and maintenance of these quality
goals. Quality management system policies
and processes should be applicable across the
entire facility. A blood bank, tissue bank, trans-
fusion service, or cellular therapy product ser-
vice need not develop its own quality policies
if it is part of a larger entity whose quality
management system addresses all of the mini-
mum requirements. The quality management
system should address all matters related to
compliance with federal, state, and local regu-
lations and accreditation standards that are
applicable to the organization.
Customer Focus
A primary focus for any organization interest-
ed in quality is serving the needs of its custom-
ers. Customers have a variety of needs and ex-
pectations. The most appropriate way to
ensure that these needs and expectations are
met is for the facility and its customers to de-
fine them in an agreement, a contract, or an-
other document. Additional information on
agreements can be found in the “Suppliers and
Materials Management” section.
When planning for new or changed prod-
ucts or services, the facility should take the
customer’s needs and expectations into ac-
count. If these changes are determined to be
critical to the quality or effectiveness of the
products and services provided by the facility,
they should be incorporated into the product
or service specifications as customer require-
ments. The facility must have a process to
manage needs and expectations that are not
met. For example, for a facility that has agreed
to deliver leukocyte-reduced components dai-
ly to one of its customers, processing compo-
nents in a manner that ensures adequate leu-
kocyte removal is critical to this product’s
quality. Such an expectation should be incor-
porated into the product specifications. Daily
delivery of products is a customer need and
expectation, but it is not critical to the quality
of the manufactured product. The facility
should have a process to manage this agreed-
on expectation and ensure that the product
delivery mechanism meets this customer
need. If this need cannot be met, the facility
should have a process to address this failure.
Once agreements have been made be-
tween the facility and its customers, there
should be a means to obtain feedback from
the customer to ensure that the facility is
meeting the customer’s expectations. Mecha-
nisms for obtaining such feedback proactively
include satisfaction surveys and periodic re-
views of agreements. Reactive feedback is ob-
tained through customer complaints. A review
of event data may also indicate failures to meet
customer needs and expectations. Data ob-
tained through these mechanisms should be
evaluated, and appropriate follow-up actions
 CHAPTER 1 Quality Management Systems: Theory and Practice
must be taken. One such action could be to
change the agreement. Inadequately address-
ing customer concerns or failing to meet ex-
pectations may result in loss of the customer.
Facilities, Work Environment, and
Safety
The facility should provide a safe workplace
with adequate environmental controls and
emergency procedures to ensure the safety of
patients, donors, staff, and visitors. Space allo-
cation, building utilities, and the communica-
tion infrastructure should adequately support
the facility’s activities. The facility should be
kept clean and well maintained so that the
products and services provided are not com-
promised. Procedures should be in place to
address the following:
䡲 General safety.
䡲 Disaster preparedness, response, and re-
covery.
䡲 Biological safety (eg, protection from blood-
borne pathogens).
䡲 Chemical safety.
䡲 Fire safety.
䡲 Radiation safety, if applicable.
䡲 Discard of biologic and other hazardous
substances.
cGMP regulations require quality plan-
ning and control of the physical work environ-
ment, including the following:
䡲 Adequate space and ventilation.
䡲 Sanitation and trash disposal.
䡲 Equipment for controlling air quality and
pressure, humidity, and temperature.
䡲 Water systems.
䡲 Toilet and hand-washing facilities.
An evaluation of the infrastructure and its
limitations before implementation of proce-
dures or installation of equipment will help
ensure maximum efficiency and safety. A more
thorough discussion of facilities and safety can
be found in Chapter 2.
䡲 7
Human Resources
This element of the quality management sys-
tem is aimed at management of personnel, in-
cluding selection, orientation, training, com-
petence assessment, and staffing.
Selection
Each facility should have a process to provide
adequate numbers of qualified personnel to
perform, verify, and manage all activities in the
facility. Qualification requirements for person-
nel are determined on the basis of job respon-
sibilities. The selection process should consid-
er the applicant’s qualifications for a particular
position as determined by his or her educa-
tion, training, experience, certifications, and
licensure. For laboratory testing staff, the stan-
dards for personnel qualifications should be
compatible with the regulatory requirements
established under CLIA.1 Job descriptions are
required for all personnel involved in process-
es and procedures that affect the quality of
tests, products, and services. Effective job de-
scriptions clearly define the qualifications and
responsibilities of the positions as well as their
reporting relationships.
Orientation, Training, and Competence
Assessment
Once hired, employees should be oriented to
their position and the organization’s policies
and procedures and be trained in their new
duties. The orientation program should cover
facility-specific requirements and policies that
address issues such as safety, quality, informa-
tion systems, security, and confidentiality. The
job-related portion of the orientation program
should cover operational issues specific to the
work area. Training should be provided for
each procedure for which employees are re-
sponsible. The ultimate result of the orienta-
tion and training program is to deem new em-
ployees competent to independently perform
the duties and responsibilities defined in their
job descriptions. Time frames should be estab-
lished to accomplish this goal.
Before the introduction of a new test or
service, existing personnel should be trained
8 䡲 AABB TECHNICAL MANUAL
to perform their newly assigned duties and
must be deemed competent in these duties.
During orientation and training, employees
should be given the opportunity to ask ques-
tions and seek additional help or clarification.
All aspects of the training should be docu-
mented, and the facility trainer or designated
facility management representative and em-
ployees should mutually agree on how em-
ployees’ competence will be determined.
FDA cGMP training is required for staff
involved in the manufacture of blood and
blood products, including staff involved in col-
lection, testing, processing, preservation, stor-
age, distribution, and transport. 3 Likewise,
cGTP training is required for personnel in-
volved in similar activities for human cells, tis-
sues, and cellular and tissue-based products
(HCT/Ps).4 Such training should provide staff
with an understanding of the regulatory basis
for the facility’s policies and procedures as well
as the specific application of the cGMP and
cGTP requirements as described in the facili-
ty’s own written operating procedures. This
training should be provided periodically to en-
sure that personnel remain familiar with regu-
latory requirements.
To ensure that skills are maintained, the
facility should have regularly scheduled com-
petence evaluations of all staff members
whose activities affect the quality of laboratory
testing, manufacture of products, or provision
of products or services.1,5-10 Competence as-
sessment of management personnel should
also be considered.
Depending on the nature of the job du-
ties, competence assessments may include
written evaluations; direct observations of ac-
tivities; reviews of work records or reports, in-
formation system records, and QC records;
testing of unknown samples; and evaluations
of employees’ problem-solving skills.5 For all
testing personnel, CMS requires that each of
the following methods be used, when applica-
ble, for each test system annually1:
䡲 Direct observations of
– Routine patient test performance (in-
cluding patient preparation, if applica-
ble), specimen handling, processing, and
testing.
– Performance of instrument maintenance
and function checks.
䡲 Monitoring of the recording and reporting
of test results.
䡲 Review of intermediate test results or work-
sheets, QC records, proficiency testing re-
sults, and preventive maintenance records.
䡲 Assessment of
– Test performance by testing previously
analyzed specimens, internal blind test-
ing samples, or external proficiency test-
ing samples.
– Problem-solving skills.
A formal competency plan that includes a
schedule of assessments, a defined minimum
for acceptable performance, and remedial
measures is one way to ensure appropriate
and consistent competence assessments. As-
sessments need not be targeted to each indi-
vidual test or procedure performed by the em-
ployee; instead, they can be grouped together
to assess similar techniques or methods. How-
ever, any test with unique aspects, problems,
or procedures should be assessed separately to
ensure that staff maintain their competency to
report test results promptly, accurately, and
proficiently.1 Written tests can be used effec-
tively to evaluate problem-solving skills and
cover many topics by asking one or more ques-
tions for each area to be assessed. CMS re-
quires that employees who perform testing
be assessed semiannually during the first
year that patient specimens are tested and an-
nually thereafter.1 Initial training verification
activities may serve as the first of these com-
petence assessments.
The quality oversight personnel should
assist in the development, review, and approv-
al of training programs, including the criteria
for retraining. 5 Quality oversight personnel
also monitor the effectiveness of training pro-
grams and competence evaluations, and they
recommend changes as needed. In addition,
The Joint Commission requires analyses of ag-
gregate competence assessment data to iden-
tify staff learning needs.9
 CHAPTER 1 Quality Management Systems: Theory and Practice
Staffing
Management should have a staffing plan that
describes the number and qualifications of
personnel needed to perform the facility’s
functions safely and effectively. There should
be an adequate number of staff to perform the
operational activities and support quality
management system activities. Organizations
should assess staffing effectiveness by evaluat-
ing human resource indicators (eg, overtime,
staff injuries, and staff satisfaction) in con-
junction with operational performance indi-
cators (eg, adverse events and patient com-
plaints). The results of this evaluation should
feed into the facility’s human resource plan-
ning process along with projections based on
new or changing operational needs.
Suppliers and Materials Management
Materials, supplies, and services used as in-
puts to a process are considered critical if they
affect the quality of products and services be-
ing produced. Examples of critical supplies are
blood components, blood bags, test kits, and
reagents. Examples of critical services are
infectious disease testing, blood component
irradiation, transportation, equipment cali-
bration, and preventive maintenance. The
suppliers of these materials and services may
be internal (eg, other departments within the
same organization) or external (outside ven-
dors). Supplies and services used in the collec-
tion, testing, processing, preservation, storage,
distribution, transport, and administration of
blood components, cellular therapy products,
tissues, and derivatives that have the potential
to affect quality should be qualified before use
and obtained from suppliers who can meet the
facility’s requirements. Three important ele-
ments of supplier and materials management
are 1) supplier qualification; 2) agreements;
and 3) receipt, inspection, and testing of in-
coming supplies.
Supplier Qualification
Critical supplies and services must be quali-
fied on the basis of defined requirements. Sim-
ilarly, suppliers should be qualified to ensure
䡲 9
that they are reliable sources of materials. The
facility should clearly define requirements or
expectations for its suppliers and share this in-
formation with staff and suppliers. Suppliers’
ability to consistently meet specifications for a
supply or service should be evaluated along
with performance relative to availability, deliv-
ery, and support. The following are examples
of factors that could be considered to qualify
suppliers:
䡲 Licensure, certification, or accreditation.
䡲 Supply or product requirements.
䡲 Supplier-relevant quality documents.
䡲 Results of audits or inspections.
䡲 Quality summary reports.
䡲 Customer complaints.
䡲 Experience with that supplier.
䡲 Cost of materials or services.
䡲 Delivery arrangements.
䡲 Financial security, market position, and
customer satisfaction.
䡲 Support after sales.
A list of approved suppliers should be
maintained that includes both primary suppli-
ers and suitable alternatives for contingency
planning. Critical supplies and services should
be purchased only from suppliers that have
been qualified. Once suppliers are qualified,
periodic evaluations of supplier performance
help ensure suppliers’ continued ability to
meet requirements. Tracking suppliers’ ability
to meet expectations gives the facility valuable
information about the stability of each suppli-
er’s processes and commitment to quality.
Documented failures of supplies or suppliers
to meet defined requirements should result in
immediate action by the facility, including no-
tification of the supplier, quality oversight per-
sonnel, and management with contracting
authority, if applicable. Supplies may need to
be replaced or quarantined until all quality is-
sues are resolved.
Agreements
Contracts and other agreements define expec-
tations and reflect the concurrence of the par-
ties involved. Periodic reviews of agreements
10 䡲 AABB TECHNICAL MANUAL
ensure that the expectations of all parties con-
tinue to be met. Changes should be mutually
agreed upon and incorporated as needed.
Transfusion and cellular therapy services
should maintain written contracts or agree-
ments with outside suppliers of critical mate-
rials and services, such as blood components,
cellular therapy products, irradiation, compat-
ibility testing, or infectious disease marker
testing. An outside supplier may be another
department within the same facility that is
managed independently, or it may be another
facility (eg, contract manufacturer). The con-
tracting facility assumes responsibility for the
manufacture of the product; ensuring the safe-
ty, purity, and potency of the product; and en-
suring that the contract manufacturer com-
plies with all applicable product standards and
regulations. Both the contracting facility and
the contractor are legally responsible for the
work performed by the contractor.
It is important for the transfusion or cel-
lular therapy service to participate in the eval-
uation and selection of suppliers. The service
should review contracts and agreements to en-
sure that all important aspects for their critical
materials and services are addressed. Exam-
ples of issues that could be addressed in an
agreement or contract include the responsibil-
ity for a product or blood sample during ship-
ment; the supplier’s responsibility to promptly
notify the facility when changes have been
made that could affect the safety of blood
components, cellular therapy products, tis-
sues, derivatives, or services for patients; and
the supplier’s responsibility to notify the facili-
ty when information is discovered indicating
that a product may not be considered safe,
such as during look-back procedures.
Receipt, Inspection, and Testing of
Incoming Supplies
Before acceptance and use, critical materials
such as reagents, blood components, cellular
therapy products, tissues, and derivatives
should be inspected and tested (if necessary)
to ensure that they meet specifications for
their intended use. It is essential that supplies
used in the collection, processing, preserva-
tion, testing, storage, distribution, transport,
and administration of blood, components, tis-
sues, and cellular therapy products also meet
FDA requirements.
The facility must define acceptance crite-
ria for critical supplies (see 21 CFR 210.3)3 and
must develop procedures to control and pre-
vent the inadvertent use of materials that do
not meet specifications. Corrective action may
include returning the material to the vendor or
destroying it. Receipt and inspection records
enable the facility to trace materials that have
been used in a particular process and provide
information for ongoing supplier qualifica-
tion.
Equipment Management
Equipment that must operate within defined
specifications to ensure the quality of blood
components, cellular therapy products, tis-
sues, derivatives, and services is referred to as
“critical equipment” in the quality manage-
ment system. Critical equipment may include
instruments, measuring devices, and comput-
er systems (hardware and software).
Activities designed to ensure that equip-
ment performs as intended include qualifica-
tion, calibration, maintenance, and monitor-
ing. Calibration, functional and safety checks,
and preventive maintenance should be sched-
uled and performed according to the manu-
facturer’s recommendations and regulatory re-
quirements of the FDA 2-4 and CMS.1 Written
procedures for equipment use and control
should comply with the manufacturer’s rec-
ommendations unless an alternative method
has been validated by the facility and, in some
instances, approved by the appropriate regula-
tory and accrediting agencies.
When one selects new equipment, it is
important to consider not only the perfor-
mance of the equipment as it will be used in
the facility but also any issues regarding ongo-
ing service and support by the supplier. There
should be a written plan for installation, oper-
ational, and performance qualifications.6 The
plan should provide for 1) installation accord-
ing to the manufacturer’s specifications, 2)
verification of the equipment’s functionality
 CHAPTER 1 Quality Management Systems: Theory and Practice
before use by ensuring that the criteria estab-
lished by the manufacturer for its intended use
are met, and 3) assurance that the equipment
performs as expected in the facility’s process-
es. After the equipment is installed, any prob-
lems and follow-up actions taken should be
documented. Recalibration and requalifica-
tion may be necessary if repairs are made that
affect the equipment’s critical operating func-
tions. Recalibration and requalification should
also be considered when existing equipment is
relocated.
The facility must develop a mechanism to
uniquely identify and track all critical equip-
ment, including equipment software versions,
if applicable. The unique identifier assigned
by the manufacturer may be used, or a unique
identification code may be applied by the
transfusion or cellular therapy service or
assigned through a laboratory-wide or organi-
zation-wide identification system. Maintain-
ing a list of all critical equipment helps in the
control function of scheduling and performing
functional and safety checks, calibrations, pre-
ventive maintenance, and repair. The equip-
ment list can be used to ensure that all appro-
priate actions have been performed and
recorded. Evaluating and trending equipment
calibration, maintenance, and repair data help
the facility assess equipment functionality,
manage defective equipment, and identify
equipment needing replacement. When
equipment is found to be operating outside
acceptable parameters, the potential effects
on the quality of products or test results must
be evaluated and documented.
Process Management
Written and approved policies, processes, and
procedures must exist for all critical functions
performed in the facility, and these functions
must be carried out under controlled condi-
tions. Each facility should have a systematic
approach for identifying, planning, and imple-
menting new (and making changes to existing)
policies, processes, and procedures that affect
the quality of the facility’s tests, products, or
services. Such activities should include a re-
view of at least the following:
䡲 11
䡲 Customer needs and expectations.
䡲 Accreditation and regulatory requirements.
䡲 Specifications to be met.
䡲 Risk assessment.
䡲 Performance measures.
䡲 Nonconformance analyses.
䡲 Current knowledge (eg, of other successful
practices).
䡲 Resource needs (eg, financial, facility, hu-
man, materials, and equipment).
䡲 Interrelationships of the new or changed
process(es) with other processes.
䡲 Documents needed for the new or changed
process(es).
The documents developed should be re-
viewed by management personnel with direct
authority over the process and by quality over-
sight personnel before implementation.
Changes in policies, processes, and proce-
dures should be documented, validated, re-
viewed, and approved. Additional information
on policies, processes, and procedures can be
found in the “Documents and Records” sec-
tion later in this chapter.
Once a process has been implemented,
the facility should have a mechanism to
ensure that procedures are performed as de-
fined and that critical equipment, reagents,
and supplies are used in conformance with
manufacturers’ written instructions and facili-
ty requirements. Table 1-1 lists elements that
constitute sound process control (among oth-
er elements of a quality management system).
A facility using critical equipment, reagents, or
supplies in a manner that is different from the
manufacturer’s directions should validate such
use. If the activity is covered under regulations
for blood and blood components or HCT/Ps,
the facility may be required to request FDA ap-
proval to operate at variance to requirements
(see 21 CFR 640.1202 or 21 CFR 1271.1554). If a
facility believes that changes to the manufac-
turer’s directions would be appropriate, it
should encourage the manufacturer to make
such changes in the labeling (ie, the package
insert or user manual).
12 䡲 AABB TECHNICAL MANUAL

TABLE 1-1. Components of a Quality Management System

Quality System Component Quality Functions and Responsibilities

Organization and leadership 䡲 Organization structure and function

䡲 Leadership roles and responsibilities, authority, and relationships
䡲 Establishment of a quality management system
䡲 Customer needs
䡲 Planned products and services
䡲 Documented, followed, and improved policies, processes, and
procedures
䡲 Quality representative
䡲 Management reviews
䡲 Provision of adequate resources
䡲 Adequate design and effective implementation
䡲 Conformance with requirements
䡲 Effective communication
䡲 Effective process improvement
Customer focus 䡲 Customer requirements
䡲 Agreements
䡲 Customer feedback
Facilities, work environment, and 䡲 Minimal health and safety risks
safety 䡲 Design and space allocations
䡲 Clean work environment
䡲 Controlled environment
䡲 Communication and information management systems
䡲 Storage facilities
䡲 Health and safety programs
䡲 Hazard discards
䡲 Emergency preparedness
Human resources 䡲 Adequate and qualified staff
䡲 Job descriptions and qualifications
䡲 Defined roles and responsibilities for all staff and their reporting
relationships
䡲 Staff selection
䡲 New hire orientation
䡲 Training on the quality system, job-related activities, computer use,
and safety
䡲 Staff competence
䡲 Continuing education
䡲 Staff identifying information
䡲 End-of-employment activities
Suppliers and materials 䡲 Supplier qualification
management 䡲 Qualifying materials
䡲 Agreement reviews
䡲 Inventory management
䡲 Adequate storage conditions
䡲 Receipt, inspection, and testing of incoming materials and products
䡲 Acceptance and rejection of materials and products
䡲 Tracing critical supplies and services
 CHAPTER 1 Quality Management Systems: Theory and Practice
TABLE 1-1. Components of a Quality Management System (Continued)
Quality System Component
Equipment management
Process management
Documents and records
Information management
Management of nonconforming
events
Monitoring and assessment
Process improvement
䡲 13
Quality Functions and Responsibilities
䡲 Selection and acquisition
䡲 Unique identification code
䡲 Verification of performance
䡲 Installation, operational, and performance qualification
䡲 Calibration
䡲 Preventive maintenance and repairs
䡲 Retirement
䡲 Process development
䡲 Change control
䡲 Process validation
䡲 Process implementation
䡲 Adherence to policies, processes, and procedures
䡲 Quality control program
䡲 Inspection of products and services
䡲 Concurrent creation of records
䡲 Requirements for critical activities
䡲 Traceability
䡲 Standardized formats
䡲 Document creation
䡲 Unique identification code
䡲 Review and approval process
䡲 Document use and maintenance
䡲 Change control
䡲 Record archiving and storage
䡲 Record retention and destruction
䡲 Confidentiality
䡲 Prevention of unauthorized access
䡲 Data integrity
䡲 Data backup
䡲 Alternative system
䡲 Detection of deviations and nonconformances
䡲 Complaint file
䡲 Adverse event reporting
䡲 Investigations
䡲 Immediate actions
䡲 Monitoring and assessment of specified requirements
䡲 Quality indicators
䡲 Internal and external assessments
䡲 Laboratory proficiency testing
䡲 Data analyses
䡲 Identifying opportunities for improvement
䡲 Systems approach to continual improvement
䡲 Root cause evaluation
䡲 Corrective action plans
䡲 Preventive action plans
䡲 Monitoring for effectiveness
14 䡲 AABB TECHNICAL MANUAL
Process Validation
Validation is used to demonstrate that a pro-
cess is capable of consistently and reliably
achieving planned results.13 It is critical to vali-
date processes in situations where it is not fea-
sible to measure or inspect each finished prod-
uct or service to fully verify conformance with
specifications. However, even when effective
end-product testing can be achieved, it is ad-
visable to validate important processes to gen-
erate information that can be used to optimize
performance. Prospective validation is used
for new or revised processes. Retrospective
validation may be used for processes that are
already in operation but were not adequately
validated before implementation. Concurrent
validation is used when required data cannot
be obtained without performance of a “live”
process. If concurrent validation is used, data
are reviewed at predefined periodic intervals
before full implementation receives final ap-
proval. Modifications to a validated process
may warrant revalidation, depending on the
nature and extent of the change. It is up to the
facility to determine the need for revalidation
on the basis of its understanding of how the
proposed changes may affect the process.
Test Method Validation
When the laboratory wishes to implement a
nonwaived test using an FDA-approved or
-cleared test system, CLIA requires that the
performance specifications established by the
manufacturer be verified by the laboratory be-
fore it reports patient results.1 At a minimum,
the laboratory must demonstrate that it can
obtain performance specifications compara-
ble to those of the manufacturer for accuracy,
precision, reportable range, and reference in-
tervals (normal values).
If the laboratory develops its own meth-
od, introduces a test system not subject to FDA
approval or clearance, or makes modifications
to an FDA-approved or -cleared test system, or
if the manufacturer does not provide perfor-
mance specifications, then the laboratory
must establish the test system performance
specifications before reporting patient results.1
At a minimum, the following must be estab-
lished for the test system:
䡲 Accuracy.
䡲 Precision.
䡲 Reportable range of test results for the test
system.
䡲 Reference intervals (normal values).
䡲 Analytical sensitivity.
䡲 Analytical specificity, including interfering
substances.
䡲 Any other performance characteristic re-
quired for test performance (eg, specimen
or reagent stability).
Based on performance specifications, the
laboratory must also establish calibration and
control procedures and document all activities
for test method validation. (See 42 CFR
493.1253.1)
Validation Plan
Validation should be planned if it is to be effec-
tive. Development of a validation plan is best
accomplished after one obtains an adequate
understanding of the system or framework
within which the process will occur. The plan
should include conducting the process as de-
signed. Additionally, a significant amount of
effort should be targeted at attempts to
“break” the process to identify weaknesses and
limitations. Many facilities develop a template
for the written validation plan to ensure that
all aspects are adequately addressed. Although
no single format for a validation plan is re-
quired, most plans include the following com-
mon elements:
䡲 System description.
䡲 Purpose or objectives.
䡲 Risk assessment.
䡲 Responsibilities.
䡲 Validation procedures.
䡲 Acceptance criteria.
䡲 Approval signatures.
䡲 Supporting documentation.
 CHAPTER 1 Quality Management Systems: Theory and Practice
The validation plan should be reviewed
and approved by quality oversight personnel
before the validation activities are carried out.
Staff responsible for carrying out the vali-
dation activities should be trained in the pro-
cess before the plan is executed. The results
and conclusions of these activities may be ap-
pended to the approved validation plan or
may be recorded in a separate document. This
documentation typically contains the follow-
ing elements:
䡲 Expected and observed results.
䡲 Interpretation of results as acceptable or
unacceptable.
䡲 Corrective action for and resolution of un-
expected results.
䡲 Explanation of and rationale for any devia-
tions from the validation plan.
䡲 Conclusions and limitations.
䡲 Approval signatures.
䡲 Supporting documentation.
䡲 Implementation timeline.
When a validation process does not pro-
duce the expected outcome, its data and cor-
rective actions must be documented as well.
The responsible quality oversight personnel
should provide final review and approval of
the validation plan, results, and corrective ac-
tions and determine whether new or modified
processes and equipment may be implement-
ed as planned or implemented with specified
limitations.
Equipment Validation
Validation of new equipment used in a process
should include installation, operational, and
performance qualifications, as follows20:
䡲 Installation qualification demonstrates that
the instrument is properly installed in envi-
ronmental conditions that meet the manu-
facturer’s specifications.
䡲 Operational qualification demonstrates
that the installed equipment operates as in-
tended. It focuses on the equipment’s capa-
bility to operate within the established lim-
䡲 15
its and specifications supplied by the
manufacturer.
䡲 Performance qualification demonstrates
that the equipment performs as expected
for its intended use in the processes estab-
lished by the facility and that the output
meets the facility’s specifications. It evalu-
ates the adequacy of equipment for use in a
specific process that uses the facility’s own
personnel, procedures, and supplies in a
normal working environment.
Computer System Validation
The FDA considers computerized systems to
include “hardware, software, peripheral devic-
es, networks, personnel, and documenta-
tion.”21 End-user validations of computer sys-
tems and the interfaces between systems
should be conducted in the environment in
which they will be used. Testing by the com-
puter software vendor or supplier is not a sub-
stitute for computer validation at the facility.
End-user acceptance testing may repeat some
of the validation performed by the developer,
such as load or stress testing and verification
of security, safety, and control features, to eval-
uate performance under actual operating con-
ditions. In addition, the end user should evalu-
ate the ability of personnel to use the
computer system as intended within the con-
text of actual work processes. The hardware
and software interface should be designed so
that staff can navigate successfully and re-
spond appropriately to messages, warnings,
and other functions. If changes to the comput-
er system result in changes to the way a pro-
cess is performed, process revalidation should
also be performed. As with process validation,
quality oversight personnel should review
and approve validation plans, results, and
corrective actions and should determine wheth-
er implementation may proceed with or with-
out limitations.
For additional information, facilities
should refer to FDA guidance on computer
system validation in the user’s facility.21 Those
who develop their own software should con-
sult Title 21 CFR Part 880 and FDA guidance
16 䡲 AABB TECHNICAL MANUAL
regarding general software validation princi-
ples.22
Quality Control
QC testing is performed to ensure the proper
functioning of materials, equipment, and
methods during operations. QC performance
expectations and acceptable ranges should be
defined and be made readily available to staff
so they will recognize, and respond appropri-
ately to, unacceptable results and trends. The
frequency for QC testing is determined by the
facility in accordance with the applicable
CMS, FDA, AABB, state, and manufacturer re-
quirements. QC results should be documented
concurrently with performance.2 Records of
QC testing should include the following:
䡲 Identification of personnel performing the
test.
䡲 Identification of reagents (including lot
numbers and expiration dates).
䡲 Identification of equipment.
䡲 Testing date and, when applicable, time.
䡲 Results.
䡲 Interpretation (eg, meets or fails to meet es-
tablished criteria).
䡲 Reviews.
Unacceptable QC results must be investi-
gated and corrective action must be imple-
mented, if indicated, before the QC procedure
is repeated or the operational process is con-
tinued. If products or services have been pro-
vided since the last acceptable QC results were
obtained, it may be necessary to evaluate the
conformance of these products or services.
Examples of QC performance intervals for
equipment and reagents are included in Ap-
pendix 1-3.
Documents and Records
Documentation provides a framework for
understanding and communicating about
processes throughout the organization. Doc-
uments provide a description of or instruc-
tions regarding what is supposed to happen.
Documents describe how processes are in-
tended to work, how they interact, where
they must be controlled, what their require-
ments are, and how to implement them. Rec-
ords provide evidence of what did happen
(ie, that a process was performed as intend-
ed), and provide information needed to as-
sess product and service quality. Together,
documents and records are used by quality
oversight personnel to evaluate the effective-
ness of a facility’s policies, processes, and
procedures. ISO 9001 provides an example of
quality system documentation that includes
the following items13:
䡲 The quality policy and objectives.
䡲 A description of the interactions between
processes.
䡲 Documented procedures for the control of
documents, records, and nonconforming
products and for corrective action, preven-
tive action, and internal quality audits.
䡲 Records related to the quality management
system, operational performance, and
product or service conformance.
䡲 All other “documents needed by the organi-
zation to ensure the effective planning, op-
eration, and control of its processes.”
Written policies, process descriptions,
procedures, work instructions, job aids, labels,
forms, and records are all part of the facility’s
document management system. They may be
paper based or electronic. A document man-
agement system provides assurance that doc-
uments are comprehensive, current, and
available and that records are accurate and
complete. A well-structured document man-
agement system links policies, process de-
scriptions, procedures, forms, and records to-
gether in an organized and workable system.
Documents
Documents should be developed in a format
that conveys information clearly and provides
staff with the necessary instructions and
templates for recording data. The CLSI offers
guidance regarding general levels of
documentation11 as well as detailed instruc-
tions on how to write procedures.23 General
types of documentation are described below.
 CHAPTER 1 Quality Management Systems: Theory and Practice
POLICIES. Policies communicate the organi-
zation’s highest-level goals, objectives, and in-
tent. The rest of the organization’s documenta-
tion interprets, and provides instructions
regarding implementation of, these policies.
PROCESSE S. Process documents describe a
sequence of actions and identify responsibili-
ties, decision points, requirements, and accep-
tance criteria. Process diagrams or flowcharts
are often used for this level of documentation.
It is helpful to show process control points on
a diagram as well as the flow of information
and handoffs between departments or work
groups.
PROCEDURES, WORK INSTRUCTIONS, AND
JOB AIDS. These documents provide step-by-
step directions on performing job tasks and
procedures. Procedures and work instructions
should include enough detail to perform a task
correctly but not so much as to be difficult to
read. The use of standardized formats helps
staff know where to find specific elements and
facilitates implementation and control. Job
aids are excerpted from an approved docu-
ment and condense information into a short-
er, more readily viewable format. External doc-
uments (eg, from a manufacturer’s manual or
package insert) may also be incorporated into
the facility’s procedures manual by reference.
Relevant procedures should be available to the
staff in each area in which the corresponding
job tasks are performed.2,5,8
FORMS. Forms provide templates for captur-
ing data on paper or electronically. These doc-
uments specify the data requirements called
for in SOPs and processes. Forms should be
carefully designed to be easy to use, minimize
the likelihood of errors, facilitate data and in-
formation retrieval, effectively capture out-
comes, and support process traceability. When
it is not immediately evident what data should
be recorded or how to record them, forms
should include instructions for their use.
Forms should indicate units of measure for
recording quantitative data.
LA BELS. Product labels, such as blood com-
ponent or HCT/P labels, are subject to many of
䡲 17
the requirements in a document management
system. Many facilities maintain a master set
of labels that can be used as a reference to veri-
fy that only currently approved stock is in use.
The accuracy of new stock labels should be
verified before this stock is put into inventory;
comparison against a master label provides a
mechanism for this verification. Change con-
trol procedures should be established for the
use of on-demand label printers to prevent
nonconforming modifications of label format
or content.
Each facility should have a defined pro-
cess for developing and maintaining docu-
ments. This process should identify basic ele-
ments required for documents; procedures for
review and approval of new or revised docu-
ments; a method for keeping documents cur-
rent; a process for control of document distri-
bution; and a process for archiving, protecting,
and retrieving obsolete documents. Training
should be provided to the staff responsible for
the content of new or revised documents. Doc-
ument management systems include these es-
tablished processes:
䡲 Verifying the adequacy of the document be-
fore its approval and issuance.
䡲 Periodically reviewing, modifying, and re-
approving documents as needed to keep
them current.
䡲 Identifying changes and revision status.
䡲 Ensuring that documents are legible, iden-
tifiable, and readily available in the loca-
tions in which they will be used.
䡲 Retaining and retrieving previous versions
for the required retention period.
䡲 Preventing unintended use of outdated or
obsolete documents.
䡲 Protecting documents from alteration,
damage, or unintended destruction.
External documents that are incorporated
by reference become part of the document
management system and should be identified
and controlled. The facility should have a
mechanism to detect changes to external doc-
uments in its system, such as manufacturers’
package inserts or user manuals, so that corre-
18 䡲 AABB TECHNICAL MANUAL
sponding changes to procedures and forms
can be made.
When new or revised policies, process de-
scriptions, procedures, or forms are added to
or replaced in the facility’s manual, these doc-
uments should be marked with the date on
which they were first put into use (ie, effective
date).
One copy of retired documents should be
retained as defined by existing and applicable
standards and regulations.
A master list of all current policies, pro-
cess descriptions, procedures, forms, and la-
bels is useful for maintaining document con-
trol. It should include document titles,
individuals or work groups responsible for
maintaining each document, revision dates,
unique document identifiers, and the areas in
which each document is used. It should also
identify the number and locations of con-
trolled copies in circulation. Copies of docu-
ments that are used in the workplace should
be identified and controlled to ensure that
none are overlooked when changes are imple-
mented.
Records
Records provide evidence that critical steps in
a procedure have been performed appropri-
ately and that products and services conform
to specified requirements. Records should be
created concurrently with the performance of
each significant step and should clearly indi-
cate the identity of the individuals who per-
formed each step and when each step was
completed.2,6,7 Data should be recorded in a
format that is clear and consistent. When
forms are used for capturing or recording data,
steps, or test results, the forms become rec-
ords.
The process for managing records should
address the following items:
䡲 Creation and identification of records.
䡲 Protection from accidental or unauthorized
modification or destruction.
䡲 Verification of completeness, accuracy, and
legibility.
䡲 Storage and retrieval.
䡲 Creation of copies or backups.
䡲 Retention periods.
䡲 Confidentiality.
Records review is an important tool to
help evaluate the effectiveness of the quality
management system. The facility should de-
fine a process and time frames for records re-
view to ensure accuracy, completeness, and
appropriate follow-up. It should determine
how reports and records are to be archived and
how to define their retention period. Specific
requirements for records to be maintained by
AABB-accredited facilities are included in the
relevant AABB standards.
Record-keeping systems should allow for
ready retrieval of records within time frames
established by the facility and permit trace-
ability of blood components, cellular therapy
products, tissues, and derivatives as required
by federal regulations.2,4 When copies of rec-
ords are retained, the facility should verify that
each copy contains the complete, legible, and
accessible content of the original record before
the original is destroyed.
If records are maintained electronically,
adequate backups should exist in case of sys-
tem failure. Electronic records should be read-
able for the entire duration of their retention
period. Obsolete computer software that is
necessary to reconstruct or trace records
should also be archived appropriately. If the
equipment or software used to access archived
data cannot be maintained, the records should
be converted to another format or copied to
another medium to permit continued access.
Converted data should be verified against the
original to ensure completeness and accuracy.
Electronic media such as magnetic tapes, opti-
cal disks, or online remote servers are widely
used for archiving documents. Records kept in
this manner must meet FDA requirements for
electronic record-keeping.24 Microfilm or mi-
crofiche may also be used to archive records.
The medium selected should be appropriate
to comply with the retention requirements.
Each facility must have a policy for alter-
ing or correcting records. 6 The date of the
changes and the identity of the individual
making each change must be documented. In
 CHAPTER 1 Quality Management Systems: Theory and Practice
some instances, it may also be important to in-
dicate the reason for the change. The original
wording must not be obliterated in written
records; the original may be crossed out with a
single line, but it should remain legible. Write-
overs and scratch-outs should not be used.
Electronic records must permit tracking of
both original and corrected data and must in-
clude the date and identity of the person who
made the change. There should be a process
for controlling changes. A method for refer-
encing changes to records that is linked to the
original record and a system for reviewing
changes for completeness and accuracy are es-
sential. Audit trails for changed data in com-
puterized systems are required by the FDA.24
The following issues might be considered
when planning record storage:
䡲 Storage of records in a manner that protects
them from damage and accidental or unau-
thorized destruction or modification.
䡲 Degree of accessibility of records in propor-
tion to frequency of their use.
䡲 Method and location of record storage re-
lated to the volume of records and the
amount of available storage space.
䡲 Availability of properly functioning equip-
ment, such as computer hardware, and
software to view archived records.
䡲 Documentation that all records copied,
transferred to microfiche, or converted to
digital files legitimately replace originals
that are stored elsewhere or destroyed.
䡲 Retention of original color-coded records
when only black-and-white reproductions
are available.
Considerations for electronic records in-
clude the following:
䡲 A method of verifying the accuracy of data
entry.
䡲 Prevention of unintended deletion of data
or access by unauthorized persons.
䡲 Adequate protection against inadvertent
data loss (eg, when a storage device is full).
䡲 Validated safeguards to ensure that a record
can be edited by only one person at a time.
䡲 Security of and access to confidential data.
䡲 19
The facility should maintain a record of
names; inclusive dates of employment; and
corresponding signatures, identifying initials,
or identification codes of personnel autho-
rized to create, sign, initial, or review reports
and records. Magnetically coded employee
badges and other computer-related identify-
ing methods are generally accepted in lieu of
written signatures, provided that the badges or
other methods meet electronic record-keeping
requirements.
Information Management
The quality management system should en-
sure the confidentiality and appropriate use of
data and information in both oral and written
communications. Privacy of patient and donor
records should be addressed to maintain the
security and confidentiality of such records.
The system should prevent unauthorized
access, modification, or destruction of the
data and information. Individuals who are au-
thorized to make changes to data should be
defined by name, code, or job responsibility.
Information systems should be designed with
security features to prevent unauthorized ac-
cess and use. Systems may include levels of se-
curity defined by job responsibility and re-
quire the use of security codes and passwords
or, for paper-based systems, locked cabinets
and keys.
The integrity of data should be main-
tained so that data are retrievable and usable.
Periodic integrity checks should be conducted
to ensure that critical data have not been inad-
vertently modified, been lost, or become
inaccessible. When data are sent manually or
electronically from one point to another, a
process should be in place to ensure that the
data accurately and reliably reach their final
destination in a timely manner.
Backup versions (eg, disks, tapes, or du-
plicate hard copies) of critical data should be
maintained in the event of an unexpected loss
from the storage medium. It is advisable to
store backup or archived computer records
and databases off-site at a sufficient distance
away to ensure that disasters will not affect
both the originals and the backups. The
20 䡲 AABB TECHNICAL MANUAL
backup storage facility should be secure. Envi-
ronmental conditions in the backup storage
facility should be maintained in a way that
protects and preserves the equipment and
media for the duration of their storage. Tem-
perature and humidity should be monitored
and controlled. Archival copies of computer
operating systems and applications software
required to view original records should be
stored in the same manner.
The facility should develop and maintain
alternative systems to ensure access to critical
data and information in the event that com-
puterized data or primary sources of informa-
tion are not available. The backup and recov-
er y procedures for computer downtime
should be defined, and validation documenta-
tion should show that the backup system
works properly. The associated processes
should be tested periodically to ensure that
the backup system remains effective. Special
consideration should be given to staff compe-
tence and readiness to use the backup system.
Management of Nonconforming
Events
The quality management system should in-
clude a process for detecting, investigating,
and responding to events that result in devia-
tions from accepted policies, processes, and
procedures or in failures to meet require-
ments, as defined by the facility, AABB stan-
dards, or applicable regulations.2-4 This pro-
cess should be implemented after, for
example, the discovery of nonconforming
products and services or of adverse reactions
to donation, blood components, cellular ther-
apy products, tissues, or derivatives. The facili-
ty should define how to perform the following
for nonconforming events:
䡲 Document and classify occurrences.
䡲 Determine the effect, if any, on the quality
of products or services.
䡲 Evaluate the effect on interrelated activities.
䡲 Analyze the event to understand root
causes.
䡲 Implement corrective action as appropri-
ate, including notification and recall, on the
basis of investigations and root cause anal-
yses.
䡲 Implement preventive actions as appropri-
ate on the basis of analyses of aggregate
data about events and their causes.
䡲 Report to external agencies when required.
䡲 Evaluate effectiveness of the corrective ac-
tions and preventive actions taken.
The CLSI has published a consensus stan-
dard on occurrence management that ex-
plores event management in more detail.25
Facility personnel should be trained to
recognize and report such occurrences. De-
pending on the severity of an event and risk to
patients, donors, and products as well as the
likelihood of recurrence, investigation of con-
tributing factors and underlying causes may
be warranted. The cGMP regulations require
investigation and documentation of results if a
specific event could adversely affect patient
safety or the safety, purity, or potency of blood
or components. 2 , 3 The cGTP regulations
require similar activities for deviations and
possible product contamination or communi-
cable disease transmission. 4 Tools and ap-
proaches for performing root cause analysis
and implementing corrective action are dis-
cussed in the section on “Process Improve-
ment.” A summary of each event, investiga-
tion, and any follow-up activities must be
prepared. Table 1-2 outlines suggested com-
ponents of an internal event report.
Fatalities related to blood collection or
transfusion or to HCT/Ps must be reported as
soon as possible to the FDA Center for Biolog-
ics Evaluation and Research (CBER). [See 21
CFR 606.170(b) and 1271.350(a)(i), respective-
ly.] Instructions for reporting to CBER are
available in published guidance27 and on the
FDA website.28 A written follow-up report must
be submitted within 7 days of the fatality and
should include a description of any new pro-
cedures implemented to avoid recurrence.
AABB Association Bulletin #04-06 provides ad-
ditional information on these reporting re-
quirements, including a form for reporting do-
nor fatalities.29
Regardless of their licensure and registra-
tion status with the FDA, all donor centers,
 CHAPTER 1 Quality Management Systems: Theory and Practice 䡲 21

TABLE 1-2. Components of an Internal Event Report26

Component Examples

Who 䡲 Reporting individual(s)

䡲 Individual(s) involved (by job title) in committing, compounding, discovering, investi-
gating, and initiating any immediate action
䡲 Patient or donor identification code
䡲 Reviewer(s) of report
What 䡲 Brief description of event
䡲 Effects on and outcomes for patient, donor, blood component, or tissue
䡲 Name of component and unit identification number
䡲 Manufacturer, lot number, and expiration dates of applicable reagents and supplies
䡲 Immediate actions taken
When 䡲 Date of report
䡲 Date and time event occurred
䡲 Date and time of discovery
䡲 Date (and time, if applicable) that immediate action was taken

And as applicable, date and time of:

䡲 Blood component collection, processing steps, and shipping
䡲 Order for blood component
䡲 Order for patient testing
䡲 Patient sample collection, transport, and receipt
䡲 Test performance and reporting
Where 䡲 Physical location of event
䡲 Where in process event was detected
䡲 Where in process event was initiated
Why and How 䡲 How event occurred
䡲 Contributing factors to event
䡲 Root cause(s)
Follow-up 䡲 External reports or notifications (eg, regulatory* or accreditation agencies, manufac-
turer, or patient’s physician)
䡲 Corrective actions
䡲 Implementation dates
䡲 Effectiveness of actions taken
䡲 Linkage to preventive action if appropriate
*All blood establishments (including licensed, registered but unlicensed, and unregistered transfusion services)2 (21 CFR
606.121) are required to notify the FDA of deviations from cGMP, applicable standards, or established specifications that
may affect the safety, purity, or potency of biological products or otherwise cause the biological products to be in violation
of the Food, Drug, and Cosmetic Act or the Public Health Service Act (21 CFR 600.14).2 The FDA has identified the following
examples as reportable events if components or products are released for distribution:
䡲 Arm preparation not performed or performed incorrectly.
䡲 Units released from donors who are (or should have been) either temporarily or permanently deferred because of their
medical history or a history of repeatedly reactive viral marker tests.
䡲 Shipment of a unit with repeatedly reactive viral markers.
䡲 ABO/Rh or infectious disease testing not performed according to the manufacturer’s package insert.
䡲 Units released from donors for whom test results were improperly interpreted because of testing errors related to
improper use of equipment.
䡲 Units released before completion of all tests (except as an emergency release).
䡲 Sample used for compatibility testing that contains incorrect identification.
䡲 Testing error that results in the release of an incorrect unit.
䡲 Incorrectly labeled blood components (eg, ABO group or expiration date).
(Continued)
22 䡲 AABB TECHNICAL MANUAL

TABLE 1-2. Components of an Internal Event Report26 (Continued)

䡲 Incorrect crossmatch label or tag.

䡲 Storage of biological products at an incorrect temperature.
䡲 Microbial contamination of blood components when the contamination is attributed to an error in manufacturing.
Deviations involving distributed HCT/Ps and relating to core cGTP requirements must also be reported to the FDA if they
occurred in the facility or in a facility that performed a manufacturing step for the facility under contract, agreement, or other
arrangement.4 Each report must contain a description of the HCT/P deviation, information relevant to the event and the
manufacture of the HCT/Ps involved, and information on all follow-up actions that have been or will be taken in response to
the deviation.
CFR = Code of Federal Regulations, FDA = Food and Drug Administration; cGMP = current good manufacturing practice;
HCT/Ps = human cells, tissues, and cellular and tissue-based products; cGTP = current good tissue practice.

blood banks, and transfusion services must
promptly report biological product devia-
tions—and information relevant to these
events—to the FDA2,30 using Form FDA-3486
when the event 1) is associated with manufac-
turing (ie, collecting, testing, processing, pack-
ing, labeling, storing, holding, or distributing);
2) represents a deviation from cGMP, estab-
lished specifications, or applicable regula-
tions or standards or that is unexpected or un-
foreseen; 3) may affect the product’s safety,
purity, or potency; 4) occurs while the facility
had control of, or was responsible for, the
product; and 5) involves a product that has left
the facility’s control (ie, has been distributed).
Using the same form, facilities must also
promptly report biological product deviations
associated with a distributed HCT/P if the
event represents a deviation from applicable
regulations, standards, or established specifi-
cations that relate to the prevention of com-
municable disease transmission or HCT/P
contamination. This requirement pertains to
events that are unexpected or unforeseeable
but may relate to the transmission or potential
transmission of a communicable disease or
may lead to HCT/P contamination.4 More in-
formation concerning biological product devi-
ation reporting can be found on the FDA web-
site.31
There must also be a mechanism to re-
port medical device adverse events to the FDA
and the device manufacturer. 8,32 The Joint
Commission encourages reporting of sentinel
events, including hemolytic transfusion reac-
tions involving the administration of blood or
components having major blood group in-
compatibilities.9,10
Hemovigilance processes also provide the
opportunity to detect, investigate, and re-
spond to adverse transfusion reactions and
events that result in deviations from safe blood
transfusion and collection practices. Adverse
transfusion reactions and events (or incidents)
can be reported voluntarily to the Centers for
Disease Control and Prevention (CDC) Nation-
al Healthcare Safety Network (NHSN) Hemo-
vigilance Module. This system was developed
through a public-private collaboration that in-
cluded the Department of Health and Human
Services and its agencies, and the private sec-
tor, including AABB, America’s Blood Centers,
and the American Red Cross. The AABB Center
for Patient Safety, a licensed Patient Safety Or-
ganization, works with hospitals to provide ad-
ditional analysis and benchmarking of hospi-
tal transfusion event reports, while protecting
data confidentiality. AABB also administers
the AABB United States Donor Hemovigilance
Program where blood collectors can report,
analyze, and benchmark adverse donor reac-
tions.
Each facility should track reported events
and look for trends. The use of classification
schemes may facilitate trend analysis and typi-
cally involves one or more of the following cat-
egories: the nature of the event, the process (or
procedure) in which the event occurred, the
outcome and severity of the event, and the
contributing factors and underlying causes. If
several events within a relatively short period
involve a particular process or procedure, that
process or procedure should be further inves-
 CHAPTER 1 Quality Management Systems: Theory and Practice 䡲 23

tigated. The most useful schemes involve the
use of multiple categories for each event,
which allows data to be sorted in a variety of
ways so that patterns that were previously not
obvious can emerge. (See example in Table 1-
3.) Such sorting or stratification can result in
identification of situations that require closer
monitoring or problems needing corrective or
preventive action. For smaller facilities that
may not have sufficient data to identify trends,
pooling data with a larger entity (eg, the labo-
ratory or all transfusion services in a health-
care system) or following national trends from
data provided by organizations such as the
AABB, CAP, or The Joint Commission, may also
prove helpful. The extent of monitoring and
the length of time to monitor processes de-
pends on the frequency and critical aspects of
the occurrences. Reporting and monitoring of
events are essential problem identification
methods for process improvement activities in
a quality management system.
Occasionally, there may be a need for a fa-
cility to deviate from approved procedures to
meet the unique medical needs of a particular
patient. When this situation arises, a medically
indicated exception should be planned and
approved in advance by the facility’s medical
director. The rationale and nature of the
planned exception should be documented.
Careful consideration should be given to
maintaining a controlled process and verifying
the safety and quality of the resulting product
or service. Any additional risk to the patient
must be disclosed.
Monitoring and Assessment
The quality management system should de-
scribe how the facility monitors and evaluates
its processes. Assessments are systematic ex-
aminations to determine whether actual activ-
ities comply with planned activities, are imple-
mented effectively, and achieve objectives.
Depending on the focus, assessments can

TABLE 1-3. Example of an Event Classification

Event: A unit of Red Blood Cells from a directed donor was issued to the wrong oncology patient. The unit
was not transfused.

Event Classification
䡲 Type of event: patient
䡲 Procedure involved: issuing products
䡲 Process involved: blood administration
䡲 Product involved: Red Blood Cells
䡲 Location: transfusion service
䡲 Other factors: directed donor
䡲 Other factors: oncology patient

Underlying Causes
䡲 Proximate cause: two patients with similar names had crossmatched blood available
䡲 Root cause: inadequate procedure for verification of patient identification during issue

Outcome
䡲 Severity: serious, FDA reportable
䡲 Patient: no harm, correct product was obtained and transfused
䡲 Product: no harm, product returned to inventory
䡲 Donor: not applicable

Successful Barriers
䡲 Problem detected during the patient identification verification step of blood administration

FDA = Food and Drug Administration.

24 䡲 AABB TECHNICAL MANUAL
include: 1) evaluation of process outputs (ie,
results); 2) the activities that make up a pro-
cess as well as its outputs; or 3) a group of re-
lated processes and outputs (ie, the system).
Assessments can be internal or external
and can include quality assessments, peer re-
views, self-assessments, and proficiency test-
ing. Evaluations typically include comparisons
of actual to expected results.
Internal Assessments
Internal assessments may include evaluations
of quality indicator data, targeted audits of a
single process, or system audits that are
broader in scope and may cover a set of inter-
related processes. These assessments should
be planned and scheduled. The details of who
performs the assessments and how they are
performed should be addressed. Assessments
should cover the quality system and the major
operating systems in the donor center and
transfusion or cellular therapy service.
In addition, there should be a process for
responding to the issues raised as a result of
the assessment, including review processes
and time frames. The results should be docu-
mented and submitted to management per-
sonnel who have authority over the process as-
sessed as well as to executive management.
Management should develop corrective ac-
tion plans with input from operational staff
and quality oversight personnel for any defi-
ciencies noted in the assessment. Quality
oversight personnel should track progress to-
ward implementation of corrective actions
and monitor them for effectiveness.
To make the best use of these assess-
ments, there should be a process to track,
monitor trends in, and analyze the problems
identified so that opportunities for improve-
ment can be recognized. Early detection of
trends makes it possible to develop preventive
actions before patient safety, blood, compo-
nents, tissues, or derivatives are adversely af-
fected. Evaluation summaries provide infor-
mation that is useful for addressing individual
or group performance problems and ensuring
the adequacy of test methods and equipment.
Any corrective or preventive action associated
with assessment results should be reviewed by
executive management.
Quality Indicators
Quality indicators are performance measures
designed to monitor one or more processes
during a defined time and are useful for evalu-
ating service demands, production, personnel,
inventory control, and process stability. These
indicators can be process based or outcome
based. Process-based indicators measure the
degree to which a process can be consistently
performed. An example of a process-based in-
dicator is turnaround time from blood product
ordering until transfusion. Outcome-based in-
dicators are often used to measure what does
or does not happen after a process is or is not
performed. The number of incorrect test result
reports is an example of such an indicator. For
each indicator, thresholds are set that repre-
sent warning limits, action limits, or both.
These thresholds can be determined from reg-
ulatory or accreditation requirements, bench-
marking, or internal facility data.
Tools frequently used for displaying qual-
ity indicator data are run charts and control
charts. In a run chart, time is plotted on the
x-axis and values on the y-axis. In control
charts, the mean of the data and the upper and
lower control limits, which have been calculat-
ed from the data, are added to the chart. Single
points outside the upper and lower control
limits result from special causes. Statistical
rules for interpreting consecutive points out-
side 1 standard deviation (SD), 2 SDs, and 3
SDs should be used to recognize a process that
is out of control. The root cause should be de-
termined, and corrective action should be ini-
tiated, if indicated.
Blood Utilization Assessment
The activities of blood usage review commit-
tees in the transfusion setting are an example
of internal assessment. Guidelines are avail-
able from the AABB for both adult and pediat-
ric utilization review.34-36 Peer review of trans-
fusion practices, required by the AABB, is also
required by The Joint Commission9 for hospi-
tal accreditation, by CMS1 for hospitals to
 CHAPTER 1 Quality Management Systems: Theory and Practice 䡲 25

qualify for Medicare reimbursement, and by evolving technologies and products, such as
some states for Medicaid reimbursement. growth factors and cytokines.
Transfusion audits provide reviews of pol-
icies and practices to ensure safe and appro- External Assessments
priate transfusions and are based on measur-
External assessments include inspections, sur-
able, predetermined performance criteria.
veys, audits, and assessments performed by
(See Chapter 28.) Transfusion services should
those not affiliated with the organization, such
investigate an adequate sample of cases (eg,
as the AABB, CAP, CMS, COLA, FACT, the FDA,
5% of cases within a defined time frame or 30
The Joint Commission, or state and regional
cases, whichever is larger). Audits assess a fa-
health departments. Participation in an exter-
cility’s performance and effectiveness in the
nal assessment program provides an indepen-
following6:
dent objective view of the facility’s performance.
External assessors often bring broad-based ex-
䡲 Ordering practices.
perience and knowledge of best practices that
䡲 Patient identification.
can be shared. Such assessments are increas-
䡲 Sample collection and labeling.
ingly being performed unannounced or with
䡲 Infectious and noninfectious adverse events.
minimal notification.
䡲 Near-miss events.
In the preparation phase, there is typically
䡲 Usage and discard.
some data gathering and information to sub-
䡲 Appropriateness of use.
mit to the organization performing the assess-
䡲 Blood administration policies.
ment. To prepare, facilities can perform inter-
䡲 Ability of services to meet patient needs.
nal audits and conduct drills to ensure that
䡲 Compliance with peer-review recommen-
staff can answer questions. For most external
dations.
assessments, there is an increased emphasis
䡲 Critical laboratory results before and after
on observations of the processes and dialogue
transfusion.
with nonmanagement staff, so preparation is
key. During the assessment phase, it is impor-
One method of assessing the blood ad-
tant to know who is responsible for the asses-
ministration process is to observe a predeter-
sors or inspectors while they are in the facility.
mined number of transfusions by following a
Clear descriptions of what information can be
unit of blood as it is issued for transfusion and
given to these individuals—and in what form—
is then transfused.34
help the facility through the assessment or
Assessments of transfusion safety policy
inspection process. After the assessment,
and practice may include reviews of transfu-
identified issues should be addressed. Usually,
sion reactions and transfusion-transmitted
a written response is submitted.
diseases. The review committee may monitor
policies and practices for notifying recipients
Proficiency Testing for Laboratories
or recipients’ physicians of recalled products
and notifying donors of abnormal test results. Proficiency testing (PT) is one means for deter-
Other assessments important in transfusion mining that test systems (including methods,
practice include reviews of policies for in- supplies, and equipment) are performing as
formed consent, indications for transfusion, expected. As a condition for certification, CMS
releases of directed donor units, and outpa- requires laboratories to participate successful-
tient or home transfusions. Additional assess- ly in an approved PT program for CLIA-
ments should include, where appropriate, 1) regulated testing. When no approved PT
therapeutic apheresis; 2) use of blood recov- program exists for a particular analyte, the lab-
ery devices; 3) procurement and storage of he- oratory must have another means to verify the
matopoietic progenitor cells; 4) perioperative accuracy of the test procedure at least twice
autologous blood collection; 5) procurement annually.1 Some accrediting agencies may re-
and storage of tissue; and 6) evaluation of quire more frequent verification of accuracy.
26 䡲 AABB TECHNICAL MANUAL
PT must be performed using routine work
processes and conditions if it is to provide
meaningful information. PT samples should
generally be handled and tested in the same
way as patient or donor specimens. However, a
CLIA-certified laboratory is prohibited from
discussing the PT or sending the samples to a
laboratory with a different CLIA number dur-
ing the active survey period, even if the two
laboratories are within the same organization
and that would be the routine manner for han-
dling patient or donor specimens. Supervisory
review of the summary evaluation report
should be documented along with investiga-
tion and corrective action for unacceptable re-
sults. Quality oversight personnel should
monitor the PT program and verify that test
systems are maintained in a state of control
and appropriate corrective action is taken
when indicated.
Process Improvement
Continual improvement is a fundamental goal
in any quality management system. In transfu-
sion and cellular therapies and clinical diag-
nostics, this goal is tied to patient safety goals
and expectations for the highest quality health
care. The importance of identifying, investi-
gating, correcting, and preventing problems
cannot be overstated. The process of develop-
ing corrective and preventive action plans in-
volves identification of problems and their
causes as well as identification and evaluation
of solutions to prevent future problems. This
process should also include evaluation of
near-miss events and a mechanism for data
collection and analysis as well as follow-up to
evaluate the effectiveness of the actions taken.
Statistical tools and their applications may be
found in publications from the AABB and the
TABLE 1-4. Applicable Joint Commission Performance Improvement Standards9,10
䡲 The organization collects data to monitor its performance, including the following:
– Blood and blood component use.
– All confirmed transfusion reactions.
䡲 The organization compiles and analyzes data.
䡲 The organization improves performance on an ongoing basis.
American Society for Quality.37-39 The Joint
Commission standards for performance im-
provement are outlined in Table 1-4.9,10
Corrective action is defined as action tak-
en to address the root causes of an existing
nonconformance or other undesirable situa-
tion to reduce or eliminate the risk of recur-
rence. Preventive action is defined as action
taken to reduce or eliminate the potential for a
nonconformance or other undesirable situa-
tion to prevent occurrence. Corrective action
can be thought of as a reactive approach to ad-
dress the root causes of actual nonconfor-
mances, deviations, complaints, and process
failures, whereas preventive action can be
thought of as a proactive approach to address
the underlying causes of anticipated prob-
lems identified through the analysis of data
and information.40 In contrast, remedial action
is defined as action taken to alleviate the
symptoms of existing nonconformances or any
other undesirable situation.41 Remedial action,
sometimes called correction, addresses only a
problem’s visible indicator and not the actual
cause. (See comparisons in Table 1-5.) Effec-
tive corrective and preventive action cannot
be implemented until the underlying cause is
determined and the process is evaluated in re-
lationship to other processes. Pending such
evaluation, it may be desirable to implement
interim remedial action.
Identification of Problems and Their
Causes
Sources of information for process improve-
ment activities include process deviations,
nonconforming products and services, cus-
tomer complaints, QC records, PT, internal au-
dits, quality indicators, and external assess-
ments. Active monitoring programs may be set
 CHAPTER 1 Quality Management Systems: Theory and Practice
TABLE 1-5. Comparison of Remedial, Corrective, and Preventive Actions40
Action Problem
Remedial Existent
Corrective Existent
Preventive Nonexistent
up to help identify problem areas. These pro-
grams should be representative of the facility
processes and consistent with organizational
goals, and they should reflect customer needs.
Preparation of a facility quality report at least
annually in which data from all these sources
are aggregated and analyzed can be valuable
to identify issues for performance improve-
ment.
Once identified, problems should be ana-
lyzed to determine their scope, potential ef-
fects on quality management and operational
systems, relative frequency, and extent of their
variation. Such an analysis is important to
avoid tampering with processes that are mere-
ly showing normal variations or problems with
little effect.
The underlying causes of an undesirable
condition or problem can be identified by an
individual or group. The more complex the
problem and the more involved the process,
the greater the need to enlist a team and to for-
malize the analysis. Three commonly used
tools for identifying underlying causes in an
objective manner are process flowcharting,
use of the “repetitive why,” and the cause-and-
effect diagram.
PROCESS FLOWCHART . A process flowchart
gives a detailed picture of the multiple activi-
ties and important decision points within that
process. By examining this picture, one may
identify problem-prone areas.
REPETITIVE WHY. The “repetitive why” is
used to work backward through the process.
One repeatedly asks “Why did this happen?”
until 1) no new information can be gleaned; 2)
the causal path cannot be followed because of
missing information; or 3) further investiga-
䡲 27
Approach Outcome
Reactive Alleviates symptoms
Reactive Prevents recurrence
Proactive Prevents occurrence
tion is impractical, impossible, or outside the
boundaries of the organization. Use of the “re-
petitive why” prevents the mistake of inter-
preting an effect as a cause.
CAUSE-A ND-E FFECT DIAGRA M. The cause-
and-effect diagram, also known as the Ishika-
wa or fish-bone diagram, uses a specialized
form of brainstorming that breaks down prob-
lems into “bite-sized” pieces. (An example of a
cause-and-effect diagram is shown in Fig 1-1.)
The method used in the diagram is designed to
focus ideas around the component parts of a
process as well as to give a pictorial represen-
tation of the ideas that are generated and their
interactions. When using the cause-and-effect
diagram, one looks at equipment, materials,
methods, environment, and human factors.
These three tools identify both active and
latent failures. Active failures are those that
have an immediate adverse effect. Latent fail-
ures are more global actions and decisions
with potential for damage that may lie dor-
mant and become evident only when triggered
by the presence of localized factors. The key to
successfully determining root causes is to
avoid stopping too soon or getting caught in
the trap of placing blame on an individual.
Most problems, particularly those that are
complex, have several root causes. A method
that can be of use when such problems occur
is the Pareto analysis. A chart of causes, laid
out in order of decreasing frequency, is pre-
pared. Those that occur most frequently are
considered the “vital few”; the rest are consid-
ered the “trivial many.” This method offers di-
rection on where to dedicate resources for
maximal effect. An example of a Pareto chart is
shown in Fig 1-2.
28 䡲 AABB TECHNICAL MANUAL

Root Cause Analysis of Failed Test Runs

FIGURE 1-1. Example of a cause-and-effect diagram.

SOP = standard operating procedure.

Identification and Evaluation of scale solutions can be tried on a limited basis

Solutions
Potential solutions to problems are identified
during the creative phase of process improve-
ment. Brainstorming and process flowcharting
can be particularly helpful in this phase.
Benchmarking with other organizations can
also be helpful. Possible solutions should be
evaluated relative to organizational con-
straints and should be narrowed down to
those that are most reasonable. Individuals
who implement the process are usually the
most knowledgeable about what will work.
They should be consulted when possible solu-
tions are being considered. Individuals with
knowledge of the interrelationships of pro-
cesses who have a more “global” view of the
organization should also be involved in this
step. Solutions may fail if representatives with
those perspectives are not involved.
Potential solutions should be tested be-
fore full implementation and a clear plan
should be created regarding methods, objec-
tives, timelines, decision points, and algo-
rithms for all possible results of a trial. Large-
and can be expanded if successful; small-scale
solutions can be implemented pending an ef-
fectiveness evaluation. Data should be collect-
ed to evaluate the effectiveness of the pro-
posed change. Data can be collected using the
methods employed initially to identify the
problems or methods specially designed for
the trial. Once solutions have been successful-
ly tested, full implementation can occur. After
implementation, data should be collected at
least periodically to ensure the continuing ef-
fectiveness and control of the changed pro-
cess.
Other Process Improvement Methods
Failure modes and effects analysis is a system-
atic stepwise approach for identifying all pos-
sible failures within a process, product, or
service; studying and prioritizing the conse-
quences, or effects, of those failures; and elim-
inating or reducing the failures, starting with
those of highest priority. Despite their relative
complexity, LEAN and Six Sigma process im-
provement methods from the manufacturing
 CHAPTER 1 Quality Management Systems: Theory and Practice 䡲 29

FIGURE 1-2. Example of a Pareto chart.

industry are finding increasing use in the
health-care setting. LEAN emphasizes speed
and efficiency. Six Sigma emphasizes precision
and accuracy. Six Sigma uses the data-driven
approach to problem solving of define, mea-
sure, analyze, improve, and control. Applica-
tion of these principles and techniques can
improve performance, reduce costs and waste,
cut time, and eliminate non-value-added ac-
tions. Additional information about both
methods can be found on the website of the
American Society for Quality.42

KEY POINTS

1. Organization and Leadership. A defined organizational structure in addition to top man-

agement’s support and commitment to the quality policy, goals, and objectives are key to
ensuring the success of the quality management system.
30 䡲 AABB TECHNICAL MANUAL

2. Customer Focus. Quality organizations should understand and meet or exceed customer
needs and expectations. These needs and expectations should be defined in a contract,
agreement, or other document developed with feedback from the customer.
3. Facilities, Work Environment, and Safety. Procedures related to general safety; biological,
chemical, and radiation safety; fire safety; and disaster preparedness are required. Space al-
location, building utilities, ventilation, sanitation, trash, and hazardous substance disposal
must support the organization’s operations.
4. Human Resources. Quality management of all personnel addresses adequate staffing levels
and staff selection, orientation, training, and competence assessment as well as specific
regulatory requirements.
5. Suppliers and Materials Management. Suppliers of critical materials and services (ie, those
affecting quality) should be qualified, and these requirements should be defined in con-
tracts or agreements. All critical materials should be qualified and then inspected and test-
ed upon receipt to ensure that specifications are met.
6. Equipment Management. Critical equipment may include instruments, measuring devices,
and computer hardware and software. This equipment must be uniquely identified and op-
erate within defined specifications, as ensured by qualification, calibration, maintenance,
and monitoring.
7. Process Management. A systematic approach to develop new, and control changes to, poli-
cies, processes, and procedures includes process validation, test method validation, com-
puter system validation, equipment validation, and QC. Validation must be planned and re-
sults reviewed and accepted.
8. Documents and Records. Documents include policies, process descriptions, procedures,
work instructions, job aids, forms, and labels. Records provide evidence that the process
was performed as intended and allow assessment of product and service quality.
9. Information Management. Unauthorized access, modification, or destruction of data and
information must be prevented and confidentiality of patient and donor records main-
tained. Data integrity should be assessed periodically and backup devices, alternative sys-
tems, and archived documents maintained.
10. Management of Nonconforming Events. Deviations from facility-defined requirements,
standards, and regulations must be addressed by documenting and classifying occurrences,
assessing effects on quality, implementing remedial actions, and reporting to external agen-
cies as required.
11. Monitoring and Assessment. Evaluation of facility processes includes internal and external
assessments, monitoring of quality indicators, blood utilization assessment, proficiency
testing, and analysis of data.
12. Process Improvement. Opportunities for improvement may be identified from deviation
reports, nonconforming products and services, customer complaints, QC records, profi-
ciency testing results, internal audits, quality indicator monitoring, and external assess-
ments. Process improvement includes determination of root causes, implementation of
corrective and preventive actions, and evaluation of the effectiveness of these actions.

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Printing Office, 2014 (revised annually).
 CHAPTER 1 Quality Management Systems: Theory and Practice
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䡲 31
18. Code of federal regulations. Title 21, CFR Part
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MJ. Biovigilance initiatives. ISBT Sci Ser 2008;
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34. Shulman IA, Lohr K, Derdiarian AK, Picukaric
JM. Monitoring transfusionist practices: A
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Transfusion 1994;34:11-15.
35. Roback J, Waxman D, for the Clinical Transfu-
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Guidelines for patient blood management and
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acceptability of American Association of Blood
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Transfusion 1993;33:168-71.
37. Vaichekauskas L. You need the tools to do the
job. In: Walters L, ed. Introducing the big Q: A
practical quality primer. Bethesda, MD: AABB
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38. Walters L. So many tools, so little understand-
ing. In: Walters L, Carpenter-Badley J, eds. S3:
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40. Motschman TL. Corrective versus preventive
action. AABB News 1999;21(8):5,33.
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 CHAPTER 1 Quality Management Systems: Theory and Practice 䡲 33

䡲 APPENDIX 1-1
Glossary of Commonly Used Quality Terms
Biovigilance Collection and analysis of adverse event data for the purpose of improving out-
comes in the use of blood products, organs, tissues, and cellular therapies.
Calibration Comparison of measurements performed with an instrument to those made
with a more accurate instrument or standard for the purpose of detecting,
reporting, and eliminating errors in measurement.
Change control Established procedures for planning, documenting, communicating, and exe-
cuting changes to infrastructure, processes, products, or services. Such proce-
dures include the submission, analysis, approval, implementation, and
postimplementation review of the change and decisions made about the change.
Formal change control provides a measure of stability and safety and avoids
arbitrary changes that might affect quality.
Control chart A graphic tool used to determine whether the distribution of data values gener-
ated by a process is stable over time. A control chart plots a statistic vs time and
helps to determine whether a process is in control or out of control according to
defined criteria (eg, a shift from a central line or a trend toward upper or lower
acceptance limits).
Design output Documents, records, and evidence in any other format used to verify that design
goals have been met. Design output should identify characteristics of a product
or service that are crucial to safety and function and to meeting regulatory
requirements. It should contain or make reference to acceptance criteria. Exam-
ples of design output include standard operating procedures; specifications for
supplies, reagents, and equipment; identification of quality control require-
ments; and results of verification and validation activities.
End-product test and Verification through observation, examination, or testing (or a combination) that
inspection the finished product or service conforms to specified requirements.
Near-miss event An unexpected occurrence that did not adversely affect the outcome but could
have resulted in a serious adverse event.
Process capability Ability of a controlled process to produce a service or product that fulfills
requirements or a statistical measure of the inherent process variability for a
given characteristic relative to design specifications. The most widely accepted
formula for process capability is Six Sigma.
Process control Activities intended to minimize variation within a process to produce a predict-
able output that meets specifications.
Qualification Demonstration that an entity is capable of fulfilling specified requirements and
verification of attributes that must be met or complied with for a person or thing
to be considered fit to perform a particular function. For example, equipment
may be qualified for an intended use by verifying performance characteristics,
such as linearity, sensitivity, or ease of use. An employee may be qualified on
the basis of technical, academic, and practical knowledge and skills developed
through training, education, and on-the-job performance.

(Continued)
34 䡲 AABB TECHNICAL MANUAL

䡲 APPENDIX 1-1
Glossary of Commonly Used Quality Terms (Continued)
Quality assurance Activities involving quality planning, control, assessment, reporting, and
improvement necessary to ensure that a product or service meets defined qual-
ity standards and requirements.
Quality control Operational techniques and activities used to monitor and eliminate causes of
unsatisfactory performance at any stage of a process.
Quality indicators Measurable aspects of processes or outcomes that provide an indication of the
condition or direction of performance over time. Quality indicators are used to
monitor progress toward stated quality goals and objectives.
Quality management The organizational structure, processes, and procedures necessary to ensure
that the overall intentions and direction of an organization’s quality program are
met and that the quality of the product or service is ensured. Quality manage-
ment includes strategic planning, allocation of resources, and other systematic
activities, such as quality planning, implementation, and evaluation.
Requirement A stated or obligatory need or expectation that can be measured or observed
and that is necessary to ensure quality, safety, effectiveness, or customer satis-
faction. Requirements can include things that the system or product must do,
characteristics that it must have, and levels of performance that it must attain.
Specification Description of a set of requirements to be satisfied by a product, material, or
process indicating, if appropriate, the procedures to be used to determine
whether the requirements are satisfied. Specifications are often in the form of
written descriptions, drawings, professional standards, and other descriptive
references.
Validation Demonstration through objective evidence that the requirements for a particular
application or intended use have been met. Validation provides assurance that
new or changed processes and procedures are capable of consistently meeting
specified requirements before implementation.
Verification Confirmation, by examination of objective evidence, that specified requirements
have been met.
 CHAPTER 1 Quality Management Systems: Theory and Practice 䡲 35

䡲 APPENDIX 1-2
Code of Federal Regulations Quality-Related References
Code of Federal Regulations, Title 21

Topic Biologics, Blood Drugs Tissues, HCT/Ps

Personnel 600.10, 606.20 211.25, 211.28 1271.170

Facilities 600.11, 606.40 211.42-58 1271.190
Environmental control 211.42 1271.195
and monitoring
Equipment 606.60 211.63-72, 211.105, 1271.200
211.182
Supplies and reagents 606.65 211.80 1271.210
Standard operating 606.100 211.100-101 1270.31, 1271.180
procedures
Process changes and 211.100-101 1271.225, 1271.230
validation
Quality assurance/quality 211.22
control unit
Label controls 610.60-64, 211.122-130 1271.250, 1271.370
606.120-122
Laboratory controls 606.140 211.160
Records and record 600.12, 606.160 211.192, 211.194, 1270.33, 1271.55
reviews 211.196 1271.270
Receipt, predistribution, 606.165 211.142, 211.150 1271.265
and distribution
Adverse reactions 606.170 211.198 1271.350
Tracking 211.188 1271.290
Complaints 606.170-171 211.198 1271.320
Reporting deviations 600.14, 606.171 1271.350
Storage 640.2, 640.11, 211.142 1271.260
640.25, 640.34,
640.54, 640.69
HCT/Ps = human cells, tissues, and cellular and tissue-based products.
36 䡲 AABB TECHNICAL MANUAL

䡲 APPENDIX 1-3
Suggested Quality Control Performance Intervals for Equipment and Reagents*

Equipment and Reagent Frequency of Quality Control

Refrigerators/freezers/platelet storage

Refrigerators

䡲 Recorder Daily
䡲 Manual temperature Daily
䡲 Alarm system board (if applicable) Daily
䡲 Temperature charts (review daily) Weekly
䡲 Alarm activation Quarterly
Freezers
䡲 Recorder Daily
䡲 Manual temperature Daily
䡲 Alarm system board (if applicable) Daily
䡲 Temperature charts (review daily) Weekly
䡲 Alarm activation Quarterly
Platelet incubators
䡲 Recorder Daily
䡲 Manual temperature Daily
䡲 Temperature charts (review daily) Weekly
䡲 Alarm activation Quarterly
䡲 Ambient platelet storage Every 4 hours

Laboratory equipment

Centrifuges/cell washers
䡲 Speed Quarterly
䡲 Timer Quarterly
䡲 Function Yearly
䡲 Tube fill level (serologic) Day of use
䡲 Saline fill volume (serologic) Weekly
䡲 Volume of antihuman globulin dispensed (if applicable) Monthly
䡲 Temperature check (refrigerated centrifuge) Day of use
䡲 Temperature verification (refrigerated centrifuge) Monthly
 CHAPTER 1 Quality Management Systems: Theory and Practice 䡲 37

䡲 APPENDIX 1-3
Suggested Quality Control Performance Intervals for Equipment and Reagents*
(Continued)
Equipment and Reagent Frequency of Quality Control

Heating blocks/waterbaths/view boxes

䡲 Temperature Day of use
䡲 Quadrant/area checks Periodically
Component thawing devices Day of use
pH meters Day of use
Blood irradiators
䡲 Calibration Yearly
䡲 Turntable (visual check each time of use) Yearly
䡲 Timer Monthly/quarterly
䡲 Source decay Dependent on source type
䡲 Leak test Twice yearly
䡲 Dose delivery check (with indicator) Each irradiator use
䡲 Dose delivery verification
– Cesium-137 Yearly
– Cobalt-60 Twice yearly
– Other source As specified by manufacturer
Thermometers (vs NIST-certified or traceable thermometer)
䡲 Liquid-in-glass Yearly
䡲 Electronic As specified by manufacturer
Timers/clocks Twice yearly
Pipette recalibration Quarterly
Sterile connecting device
䡲 Weld check Each use
䡲 Function Yearly
Blood warmers
䡲 Effluent temperature Quarterly
䡲 Heater temperature Quarterly
䡲 Alarm activation Quarterly

(Continued)
38 䡲 AABB TECHNICAL MANUAL

䡲 APPENDIX 1-3
Suggested Quality Control Performance Intervals for Equipment and Reagents*
(Continued)
Equipment and Reagent Frequency of Quality Control

Blood collection equipment

Whole blood equipment

䡲 Agitators Day of use
䡲 Balances/scales Day of use
䡲 Gram weight (vs NIST-certified) Yearly
Microhematocrit centrifuge
䡲 Timer check Quarterly
䡲 Calibration Quarterly
䡲 Packed cell volume Yearly
Cell counters/hemoglobinometers Day of use
Blood pressure cuffs Twice yearly
Apheresis equipment
䡲 Checklist requirements As specified by manufacturer

Reagents

Red cells Day of use

Antisera Day of use
Antiglobulin serum Day of use
Transfusion-transmissible disease marker testing Each test run

Miscellaneous

Copper sulfate Day of use

Shipping containers for blood and component transport Twice yearly
(usually at temperature extremes)
*The frequencies listed above are suggested intervals, not requirements. For any new piece of equipment, installation, oper-
ational, and performance qualifications must be performed. After the equipment has been suitably qualified for use, ongoing
QC testing should be performed. Depending on the operational and performance qualification methodology, the ongoing QC
may initially be performed more often than the ultimately desired frequency. Once a record of appropriate in-range QC
results has been established (during either equipment qualification or the ongoing QC), the frequency of testing can be
reduced. At a minimum, the frequency must comply with the manufacturer’s suggested intervals; if no such guidance is pro-
vided by the manufacturer, the intervals given in this table are appropriate to use.
NIST = National Institute of Standards and Technology, QC = quality control.
 C h a p t e r 2

Facilities, Work Environment,

and Safety

Betsy W. Jett, MT(ASCP), CQA(ASQ)CQM/OE;

Susan L. Wilkinson, EdD, MS, MT(ASCP)SBB; and
Tania L. Motschman, MS, MT(ASCP)SBB, CQA(ASQ)

T H E P H Y S I C A L W O R K environment sponsible for protecting their own safety and

can have a significant impact on the
safety, efficiency, and effectiveness of work
processes and on the quality of work process
outcomes. It should be designed and managed
in a way that meets operational needs and
provides for the safety of staff and visitors. The
layout of the physical space; management of
utilities, such as water and air ventilation; flow
of personnel, materials, and waste; and ergo-
nomic factors should all be considered in the
facility management plan.
In addition to providing adequate facili-
ties, the organization should develop and im-
plement a safety program that defines policies
and procedures for safe work practices and
emergency responses. Such a program also in-
cludes requirements for training, hazard com-
munication, use of engineering controls, and
protective equipment. All employees are re-
the safety of others by adhering to policies and
procedures set forth in the facility safety pro-
gram.
The AABB requires its accredited facilities
to plan, implement, and maintain a program
to minimize risks to the health and safety of
donors, patients, volunteers, and employees
from biological, chemical, and radiological
hazards.1,2 Other professional and accrediting
organizations, including the College of Ameri-
can Pathologists (CAP), the Clinical and Labo-
ratory Standards Institute, and The Joint Com-
mission, have similar or more detailed safety
program requirements.3-6
US federal regulations and recommenda-
tions intended to protect the safety of workers
and the public in health-care settings are listed
in Appendix 2-1. Appendix 2-1 also lists rele-
vant safety recommendations of trade and

Betsy W. Jett, MT(ASCP), CQA(ASQ)CQM/OE, Vice President for Quality and Regulatory Affairs, New York
Blood Center, New York, New York; Susan L. Wilkinson, EdD, MS, MT(ASCP)SBB, Interim Department Head,
Analytical and Diagnostic Sciences, College of Allied Health Sciences and Associate Professor Emerita, Uni-
versity of Cincinnati, Cincinnati, Ohio; and Tania L. Motschman, MS, MT(ASCP)SBB, CQA(ASQ), Quality
Director, Esoteric Business Unit, Laboratory Corporation of America, Burlington, North Carolina
The authors have disclosed no conflicts of interest.

39
40 䡲 AABB TECHNICAL MANUAL
professional associations. The contents of
these regulations and guidelines are discussed
in more detail in each section of this chapter.
US state and local government regulations
may have additional safety requirements, in-
cluding architectural and construction safety
considerations.
FAC IL I T IE S
Facility Design and Workflow
Effective design and maintenance of facilities
along with the physical organization of work
activities can help reduce or eliminate many
potential hazards. Facility design, workflow,
and maintenance also affect process efficien-
cy, productivity, error rates, employee and cus-
tomer satisfaction, and the quality of products
and services.
During the design phase for a new or ren-
ovated space, the location and flow of person-
nel, materials, and equipment should be con-
sidered in the context of the processes to be
performed. Adequate space must be allotted
for personnel movement, location of supplies
and large equipment, and private or distrac-
tion-free zones for certain manufacturing
tasks (eg, donor interviewing, record review,
and blood component labeling). The facility
must offer designated “clean” and “dirty” spac-
es and provide for controlled movement of
materials and waste in and out of these areas
to avoid contamination. Chemical fume hoods
and biological safety cabinets (BSCs) should
be located away from drafts and high-traffic
areas. The number and location of eyewash
stations and emergency showers must also be
considered in planning. Staff handling hazard-
ous materials must have ready access to hand-
washing sinks. In some cases, additional spe-
cial water sources for reagent preparation
must be provided. The location of very heavy
equipment, such as irradiators, should be tak-
en into account to ensure that the flooring has
sufficient load-bearing capacity.
Laboratories must be designed with ade-
quate illumination, electrical power, and con-
veniently located electrical outlets. Emergency
backup power sources, such as uninterrupt-
ible power supplies and backup generators,
should be considered to ensure that blood
components, cellular therapy products, and
critical reagents are not compromised during
power failures. The National Electrical Code is
routinely used as a national guideline for the
design of essential electrical distribution sys-
tems, with modifications approved by the local
building authority that has jurisdiction.7
Heating, ventilation, and air conditioning
must be adequate for the needs of the facility.
Environmental monitoring systems should be
considered for laboratories that require posi-
tive or negative air pressure differentials or
where air filtration systems are used to control
particle levels. The nationally accepted specifi-
cations for ventilation are published by the
American Society of Heating, Refrigerating,
and Air-Conditioning Engineers.8
Housekeeping
The workplace should be kept clean and free
of clutter. Work surfaces and equipment
should be regularly cleaned and disinfected.
Items that may accumulate dust and debris
should not be stored above clean supplies or
work surfaces. Exits and fire safety equipment
must not be blocked or obstructed in any way.
Receptacles and disposal guidelines for non-
hazardous solid waste and biohazardous,
chemical, and radiation waste should be clear-
ly delineated. Housekeeping responsibilities,
methods, and schedules should be defined for
every work area. Written procedures, initial
training, continuing education of personnel,
and ongoing monitoring of housekeeping ef-
fectiveness are essential to safe operations.
Clean Rooms
Clean-room facilities should be considered for
open processing activities that cannot be ac-
commodated in a BSC. Laboratories that pro-
cess cellular therapy products may choose to
adopt clean-room specifications and mainte-
nance practices to meet the requirements of
the Food and Drug Administration (FDA) cur-
rent good tissue practice regulations.9
International standards for clean rooms
are published by the International Organiza-
 CHAPTER 2 Facilities, Work Environment, and Safety
tion for Standardization and provide specifica-
tions for general manufacturing applications
to limit airborne particulates, contaminants,
and pollutants.10 These standards also provide
guidance for pharmaceutical and biotechnol-
ogy applications that include methods to as-
sess, monitor, and control biocontamination.11
Aspects of a biocontamination-control system
include 1) developing air-sampling plans us-
ing validated equipment; 2) assessing biocon-
tamination of surfaces, textiles, and liquids; 3)
evaluating laundering processes; and 4) main-
taining personnel training and work practices.
Restricted Areas
Hazardous areas should be clearly and uni-
formly identified with warning signs in accor-
dance with federal Occupational Safety and
Health Administration (OSHA) and Nuclear
Regulatory Commission (NRC) standards so
that personnel entering or working around
them are aware of existing biological, chemi-
cal, or radiation dangers.12-15 Staff members
not normally assigned to these areas should
receive adequate training to avoid endanger-
ing themselves. Risk areas can be stratified. For
example, high-risk areas might include those
that contain chemical fume hoods, BSCs, and
storage areas for volatile chemicals or radio-
isotopes. Technical work areas might be con-
sidered moderate risk and restricted to labora-
tory personnel. Administrative and clerical
areas are generally considered low risk and not
restricted. Guidelines for restricted access
based on biosafety levels are published by the
US Department of Health and Human Services
(DHHS).16 Where possible, functions not re-
quiring special precautions should be separat-
ed from those performed in restricted areas.
Organizations should consider establish-
ing specific safety guidelines for visitors with
business in restricted areas and verifying that
safety guidelines have been reviewed before
the visitors enter the area. Casual visitors
should not be allowed in restricted areas. Chil-
dren should not be allowed in areas where they
could be exposed to hazards and should be
closely supervised in those areas where their
presence is permitted.
䡲 41
Mobile Sites
Mobile blood collection operations can pre-
sent special challenges. An advance safety sur-
vey of the proposed collection site helps en-
sure that hazards are minimized.
Responsibility for site safety should be as-
signed to an individual with adequate knowl-
edge to recognize safety concerns and the au-
thority to address them in a timely manner. All
mobile personnel should be trained to recog-
nize unsafe conditions and understand how to
effectively implement infection-control poli-
cies and procedures in a variety of settings.
Hand-washing access is essential at col-
lection sites. Carpeted or difficult-to-clean
surfaces may be covered using an absorbent
overlay with waterproof backing to protect
them from possible blood spills. Portable
screens and crowd-control ropes are helpful in
directing traffic flow to maintain safe work ar-
eas. Food service areas should be physically
separated from areas for blood collection and
storage. Blood-contaminated waste must be
either returned to a central location for dispos-
al or packaged and decontaminated in accor-
dance with local regulations for medical waste.
Ergonomics
Consideration in physical design should be
given to ergonomics and to accommodations
for individuals covered under the Americans
with Disabilities Act [42 United States Code
(USC) Sections 2101-12213, 1990]. Several fac-
tors may contribute to employee fatigue, mus-
culoskeletal disorder syndromes, or injury, in-
cluding the following17:
䡲 Awkward postures—positions that place
stress on the body, such as reaching over-
head, twisting, bending, kneeling, or squat-
ting.
䡲 Repetition—performance of the same mo-
tions continuously or frequently.
䡲 Force—the amount of physical effort used
to perform work.
䡲 Pressure points—pressing of the body
against hard or sharp surfaces.
䡲 Vibration—continuous or high-intensity
hand/arm or whole-body vibration.
42 䡲 AABB TECHNICAL MANUAL
䡲 Other environmental factors—extreme high
or low temperatures or lighting that is too
dark or too bright.
Both the total time per work shift and the
length of uninterrupted work periods can
make significant contributions to physical
problems. Actions to correct problems associ-
ated with ergonomics may include the follow-
ing:
䡲 Engineering improvements to reduce or
eliminate the underlying cause, such as
making changes to equipment, worksta-
tions, or materials.
䡲 Administrative improvements, such as pro-
viding variety in tasks; adjusting work
schedules and work pace; providing recov-
ery or relaxation time; modifying work
practices; ensuring regular housekeeping
and maintenance of work spaces, tools, and
equipment; and encouraging exercise.
䡲 Provision of personal protective equipment
(PPE), such as gloves, knee and elbow pads,
protective footwear, and other items that
employees wear to protect themselves
against injury.
SA FET Y PROGRAM
An effective safety program starts with a well-
thought-out safety plan. This plan identifies
the applicable regulatory requirements and
describes how they will be met. A safety plan
includes procedures to:
䡲 Provide a workplace free of recognized haz-
ards.
䡲 Evaluate all procedures for potential expo-
sure risks.
䡲 Evaluate each employment position for po-
tential exposure risks.
䡲 Identify hazardous areas or materials with
appropriate labels and signs.
䡲 Educate staff, document training, and
monitor compliance.
䡲 Apply standard precautions (including uni-
versal and blood and body fluid precau-
tions) to the handling of blood, body fluids,
and tissues.
䡲 Dispose of hazardous waste appropriately.
䡲 Report incidents and accidents, and pro-
vide treatment and follow-up.
䡲 Provide ongoing review of safety policies,
procedures, operations, and equipment.
䡲 Develop facility-specific plans for disaster
preparedness and response, and test these
plans at defined intervals (Chapter 4).
Safety programs should consider the
needs of all persons affected by the work envi-
ronment. Most obvious is the safety of techni-
cal staff members, but potential risks for blood
donors, ancillary personnel, volunteers, visi-
tors, housekeeping staff, and maintenance
and repair workers must also be evaluated.
Appropriate provisions must be applied if
these individuals cannot be excluded from risk
areas.
Laboratories should consider appointing
a safety officer who can provide general guid-
ance and expertise.4 Typical duties of a safety
officer are to develop the safety program, over-
see orientation and training, perform safety
audits, survey work sites, recommend chang-
es, and serve on or direct the activities of safety
committees. Facilities using hazardous chemi-
cals and radioactive materials often assign
specially trained individuals to oversee chemi-
cal and radiation protection programs as
needed.12,15 Five basic elements must be ad-
dressed for each type of hazard covered in the
safety program:
䡲 Training.
䡲 Hazard identification and communication.
䡲 Engineering controls and PPE.
䡲 Safe work practices, including waste dis-
posal.
䡲 Emergency response plan.
Management controls should be estab-
lished to ensure that these elements are imple-
mented and maintained and that they are ef-
fective. Management is responsible for the
following:
䡲 Developing and communicating the writ-
ten plan.
 CHAPTER 2 Facilities, Work Environment, and Safety 䡲 43

䡲 Ensuring implementation and providing temporary work assignments if significant po-

adequate resources.
䡲 Providing access to employee health servic-
es related to prevention strategies and
treatment of exposures.
䡲 Monitoring compliance and effectiveness.
䡲 Evaluating and improving the safety plan.
Basic Elements of a Safety Program
Training
tential for exposure exists. Staff members who
do not demonstrate the requisite understand-
ing and skills must receive additional training.
Training should be provided not only to labo-
ratory staff, but also to housekeeping and oth-
er personnel who may come into contact with
hazardous substances or waste. Table 2-1 lists
topics to cover in work safety training pro-
grams.

Employees must be trained to recognize the Hazard Identification and

hazards in their workplace and take appropri-
ate precautions. Supervisors are responsible
for assessing and documenting each employ-
ee’s understanding of and ability to apply safe-
ty precautions before independent work is
permitted. Safety training must precede even
Communication
Employers are required to provide information
about workplace hazards to their staff to help
reduce the risk of occupational illnesses and
injuries. Staff need to know what hazardous

TABLE 2-1. Topics to Cover in a Work Safety Training Program

Work safety training programs should ensure that all personnel:

䡲 Have access to a copy of pertinent regulatory texts and an explanation of the contents.
䡲 Understand the employer’s exposure control plan and how to obtain a copy of the written plan.
䡲 Understand how hepatitis and human immunodeficiency virus (HIV) are transmitted and how often; be famil-
iar with the symptoms and consequences of hepatitis B virus (HBV), hepatitis C virus, and HIV infection.
䡲 Know that they are offered vaccination against HBV.
䡲 Recognize tasks that pose infectious risks, and distinguish them from other duties.
䡲 Know what protective clothing and equipment are appropriate for the procedures they will perform and how
to use them.
䡲 Know and understand the limitations of protective clothing and equipment (eg, different types of gloves are
recommended according to the permeability of the hazardous material to be used).
䡲 Know where protective clothing and equipment are kept.
䡲 Become familiar with and understand all requirements for work practices specified in standard operating
procedures for the tasks they perform, including the meaning of signs and labels.
䡲 Know how to remove, handle, decontaminate, and dispose of contaminated material.
䡲 Know the appropriate actions to take and the personnel to contact if exposed to blood or other biologic,
chemical, or radiologic hazards.
䡲 Know the corrective actions to take in the event of spills or personal exposure to fluids, tissues, and contam-
inated sharp objects; appropriate reporting procedures; and medical monitoring recommended when paren-
teral exposure may have occurred.
䡲 Know their right to access to medical treatment and medical records.
䡲 Know fire safety procedures and evacuation plans.
44 䡲 AABB TECHNICAL MANUAL
substances they are working with and where
those materials are located in the facility. This
communication is achieved by means of sign-
age, labels on containers, written information,
and training programs.
Engineering Controls and PPE
If the physical workspace cannot be designed
to eliminate the potential for exposure to haz-
ards, appropriate protective gear must be pro-
vided. Engineering controls are physical plant
controls or equipment, such as sprinkler sys-
tems, chemical fume hoods, and needleless
systems, that isolate or remove the hazard
from the workplace.
PPE is specialized clothing or equipment,
such as gloves, masks, and laboratory coats,
worn by employees for protection against a
hazard. Employees should remove their PPE,
such as gloves and laboratory coats, and
should wash their hands with soap and water
when leaving a laboratory area. General guid-
ance on the use of engineering controls and
PPE is included in Appendix 2-2.
Safe Work Practices
Employees must be trained in how to work
with hazardous materials in ways that protect
themselves, their coworkers, and the environ-
ment. Safe work practices are defined as tasks
performed in a manner that reduces the likeli-
hood of exposure to workplace hazards. Gen-
eral recommendations for safe work practices
are included in Appendix 2-2.
Emergency Response Plan
When engineering and work practice controls
fail, employees must know how to respond
promptly and appropriately. The purpose of
advance planning is to control a hazardous sit-
uation as quickly and safely as possible. Regu-
lar testing of the emergency response plan
identifies areas for improvement and builds
staff confidence in their ability to respond ef-
fectively in a real emergency. OSHA requires
facilities with more than 10 employees to have
a written emergency response plan. Verbal
communication of the plan is acceptable for
facilities with 10 or fewer employees.18
Management Controls
Supervisory personnel must monitor safety
practices in their areas of responsibility. Con-
tinuing attention to safety issues should be ad-
dressed in routine staff meetings and training
sessions. Periodic audits performed by a safety
professional increase safety awareness. Man-
agement should seek staff input on the design
and improvement of the facility’s safety plan.
The safety program policies, procedures,
guidelines, and supporting references should
be documented in writing and made available
to all personnel at risk. These documents
should be reviewed on a regular basis and up-
dated as technology evolves and new informa-
tion becomes available. Work sites and safety
equipment should be inspected regularly to
ensure compliance and response readiness.
Checklists may be helpful for documenting
safety inspections and assessing safety pre-
paredness.3,4,19
Employee Health Services
Hepatitis Prophylaxis
All employees routinely exposed to blood must
be offered hepatitis B virus (HBV) vaccine if
they do not already have HBV-protective anti-
bodies (ie, anti-HBs). OSHA requires that the
vaccine be offered at no cost to all employees
and, if any employee refuses the vaccine, that
the refusal be documented.14
Monitoring Programs
Employers must provide a system for monitor-
ing exposure to certain substances as defined
in the OSHA standard if there is reason to be-
lieve that exposure levels routinely exceed the
recommended action level.20
Medical First Aid and Follow-Up
When requested by a worker who has sus-
tained known or suspected blood exposure,
monitoring for HBV, hepatitis C virus (HCV),
and human immunodeficiency virus (HIV) in-
 CHAPTER 2 Facilities, Work Environment, and Safety
fection should be provided with appropriate
counseling. In some states, consent is required
for this voluntary testing; rejection of offered
testing must be documented. The usual
schedule includes immediate tests of the
worker and the source of the potentially infec-
tious material, with follow-up testing of the
worker at intervals after exposure.13,14 All as-
pects of accident follow-up should be appro-
priately documented.
The Centers for Disease Control and Pre-
vention (CDC) has published recommenda-
tions for both pre-exposure and post-exposure
prophylaxis if the contaminating material is
HBV-positive or if this information is un-
known.21 HBV immune globulin is usually giv-
en concurrently with HBV vaccine in cases of
penetrating injuries. When administered in
accordance with the manufacturer’s direc-
tions, both products are very safe and carry no
documented risk of infection with HBV, HCV,
or HIV.21 Post-exposure prophylaxis for HIV is
continually evolving; policies are generally
based on Public Health Service recommenda-
tions and current standards of practice.
Reporting Accidents and Injuries
When an injury occurs, relevant information
should be documented, including the date,
time, and place where the injury occurred; the
nature of the injury; descriptions of what hap-
pened from the injured person and any wit-
nesses; and first aid or medical attention pro-
vided. The supervisor should complete any
accident reports and investigation forms re-
quired by the institution’s insurer and worker’s
compensation agencies. Employers must re-
port fatalities and injuries resulting in the hos-
pitalization of three or more employees to
OSHA within 8 hours of the accident.22
OSHA requires health service employers
with 11 or more workers to maintain records of
occupational injuries and illnesses requiring a
level of care that exceeds the capabilities of a
person trained in first aid.23 Initial documenta-
tion must be completed within 6 days of the
incident. Records of first aid provided by a
nonphysician for minor injuries, such as cuts
or burns, do not need to be retained. All logs,
䡲 45
summaries, and supplemental records must
be preserved for at least 5 years beyond the
calendar year of occurrence. Medical records
of employees should be preserved for the du-
ration of employment plus 30 years, with few
exceptions.24
Latex Allergies
Adverse reactions associated with latex, pow-
dered gloves, or both include contact dermati-
tis, allergic dermatitis, urticaria, and anaphy-
laxis. Medical devices that contain latex must
bear a caution label. The National Institute for
Occupational Safety and Health (NIOSH) of-
fers the following recommendations to prevent
these allergic reactions25:
䡲 Make latex-free gloves available as an alter-
native to latex, and encourage the use of
latex-free gloves for activities and work en-
vironments where there is minimal risk of
exposure to infectious materials.
䡲 If latex gloves are used, consider providing
reduced-protein and powder-free gloves.
䡲 Use good housekeeping practices to remove
latex-containing dust from the workplace.
䡲 Use work practices that reduce the chance
of reaction, such as hand washing and
avoiding oil-based hand lotions.
䡲 Educate workers about latex allergy.
䡲 Evaluate current prevention strategies.
䡲 Periodically screen high-risk workers for la-
tex allergy symptoms.
䡲 If symptoms develop, have workers avoid
direct contact with latex and consult a phy-
sician about allergy precautions.
FIRE PREVENTION
Fire prevention relies on a combination of fa-
cility design that is based on the National Fire
Protection Association (NFPA) Life Safety
Code, which identifies processes to maintain
fire protection systems in good working order,
and fire safe-work practices.26 The Life Safety
Code includes both active and passive fire-
protection systems (eg, alarms, smoke detec-
tors, sprinklers, exit lights in corridors, and
fire-rated barriers).
46 䡲 AABB TECHNICAL MANUAL
Training
Fire safety training is recommended at the
start of employment and at least annually
thereafter. Training should emphasize preven-
tion and an employee’s awareness of the work
environment, including how to recognize and
report unsafe conditions, how to report fires,
where the nearest alarm and fire-containment
equipment are located and how to use it, and
what the evacuation policies and routes are.
All staff members in facilities accredited
by CAP or The Joint Commission are required
to participate in fire drills at least annually.3,5 In
areas where patients are housed or treated,
The Joint Commission requires quarterly drills
on each shift. Staff participation and under-
standing should be documented.
Hazard Identification and
Communication
Emergency exits must be clearly marked with
an exit sign. Additional signage must be posted
along the exit route to show the direction of
travel if it is not immediately apparent. All
flammable materials should be labeled with
appropriate hazard warnings, and flammable
storage cabinets should be clearly marked.
Engineering Controls and PPE
Laboratories storing large volumes of flamma-
ble chemicals are usually built with 2-hour fire
separation walls, or with 1-hour separation
walls if there is an automatic fire-extinguishing
system.4 Fire detection and alarm systems
should be provided in accordance with feder-
al, state, and local regulations. All fire equip-
ment should be inspected on a regular basis to
ensure that it is in good working order. Fire ex-
tinguishers should be readily available, and
the staff should be trained to use them proper-
ly. Housekeeping and inventory management
plans should be designed to control the accu-
mulations of flammable and combustible ma-
terials stored in the facility. In areas where
sprinkler systems are installed, all items
should be stored at least 18 inches below the
sprinkler heads. Facilities should consult local
fire codes, which may require greater clear-
ance.
Safe Work Practices
Emergency exit routes must be clear of any-
thing that would obstruct evacuation efforts.
Exit doors must not be locked in such a way as
to impede egress. Permanent exit routes must
be designed to allow free and unobstructed
exit from all parts of the facility to an area of
safety. Secondary exits may be required for ar-
eas larger than 1000 square feet; facilities
should consult local safety authorities with ju-
risdiction, such as the local fire marshal and
NFPA, for guidance on secondary exits.
Emergency Response Plan
The fire emergency response plan should en-
compass both facility-wide and area-specific
situations. It should describe reporting and
alarm systems; location and use of emergency
equipment; roles and responsibilities of the
staff during the response; “defend-in-place”
strategies; and conditions for evacuation,
evacuation procedures, and exit routes.5,18
When a fire occurs, the general sequence
for immediate response should be to 1) rescue
anyone in immediate danger; 2) activate the
fire alarm system and alert others in the area;
3) confine the fire by closing doors and shut-
ting off fans or other oxygen sources if possi-
ble; and 4) extinguish the fire with a portable
extinguisher if the fire is small, or evacuate if it
is too large to manage.
ELECTRICAL SAFETY
Electrical hazards, including fire and shock,
may arise from the use of faulty electrical
equipment; damaged receptacles, connectors,
or cords; or unsafe work practices. Proper use
of electrical equipment, periodic inspection
and maintenance, and hazard recognition
training are essential to help prevent accidents
that may result in electric shock or electrocu-
tion. The severity of shock depends on the
path that the electrical current takes through
the body, the amount of current flowing
 CHAPTER 2 Facilities, Work Environment, and Safety
through the body, and the length of time that
current is flowing through the body. Even low-
voltage exposures can lead to serious injury.27
Training
Safety training should be designed to make
employees aware of electrical hazards associ-
ated with receptacles and connectors. This
training should also help them recognize po-
tential problems, such as broken receptacles
and connectors, improper electrical connec-
tions, damaged cords, and inadequate
grounding.
Hazard Identification and
Communication
The safety plan should address the proper use
of receptacles and connectors. Equipment that
does not meet safety standards should be
marked to prevent accidental use.
Engineering Controls and PPE
OSHA requires that electrical systems and
equipment be constructed and installed in a
way that minimizes the potential for work-
place hazards. When purchasing equipment,
the facility should verify that it bears the mark
of an OSHA-approved independent testing
laboratory, such as Underwriters Laborato-
ries.28 Adequate working space should be pro-
vided around equipment to allow easy access
for safe operation and maintenance. Ground-
fault circuit interrupters should be installed in
damp or wet areas.
Safe Work Practices
Electrical safety practices focus on two factors:
1) proper use of electrical equipment and 2)
proper maintenance and repair of this equip-
ment. Staff should not plug or unplug equip-
ment from an electrical source with wet hands.
Overloading circuits with too many devices
may cause the current to heat the wires to a
very high temperature and generate a fire.
Damaged receptacles and faulty electrical
equipment must be tagged and removed from
service until they have been repaired and
䡲 47
checked for safety. Flexible cords should be se-
cured to prevent tripping and should be pro-
tected from damage from heavy or sharp ob-
jects. Flexible cords should be kept slackened
to prevent tension on electrical terminals, and
cords should be checked regularly for cut, bro-
ken, or cracked insulation. Extension cords
should not be used in lieu of permanent
wiring.
Emergency Response Plan
In case of an emergency in which it is not pos-
sible to decrease the power or disconnect
equipment, the power supply should be shut
off from the circuit breaker. If it is not possible
to interrupt the power supply, a nonconduc-
tive material, such as dry wood, should be
used to pry a victim from the source of cur-
rent.27 Victims must not be touched directly.
Emergency first aid for victims of electrical
shock must be sought. Water-based fire extin-
guishers should not be used on electrical fires.
BIOSAFET Y
The facility must define and enforce measures
to minimize the risk of exposure to biohazard-
ous materials in the workplace. Requirements
published by OSHA (Blood-borne Pathogens
Standard) and recommendations published by
the US DHHS provide the basis for an effective
biosafety plan.13,14,16
Blood-Borne Pathogens Standard
The OSHA Blood-Borne Pathogens Standard is
intended to protect employees in all occupa-
tions where there is a risk of exposure to blood
and other potentially infectious materials. It
requires the facility to develop an exposure
control plan and describes appropriate engi-
neering controls, PPE, and work practice con-
trols to minimize the risk of exposure. The
standard also requires employers to provide
HBV vaccinations to any staff members with
occupational exposure, provide medical fol-
low-up care in case of accidental exposure,
and keep records related to accidents and ex-
posures.
48 䡲 AABB TECHNICAL MANUAL
Standard Precautions
Standard precautions represent the most cur-
rent recommendations by the CDC to reduce
the risk of transmission of blood-borne patho-
gens and other pathogens in hospitals. Origi-
nally published in 1996 in the Guidelines for
Isolation Precautions in Hospitals, standard
precautions build on earlier recommenda-
tions, including body substance isolation
(1987), universal precautions (1986), and
blood and body fluid precautions (1983).13
Standard precautions apply to all patient care
activities, regardless of diagnosis, in which
there is a risk of exposure to 1) blood; 2) all
body fluids, secretions, and excretions, except
sweat; 3) nonintact skin; or 4) mucous mem-
branes.
The OSHA Blood-Borne Pathogens Stan-
dard refers to the use of universal precautions.
However, OSHA recognizes the more recent
guidelines from the CDC and, in Directive CPL
02-02-069, allows hospitals to use acceptable
alternatives, including standard precautions,
as long as all other requirements in the stan-
dard are met.29
Biosafety Levels
Recommendations for biosafety in laborato-
ries are based on the potential hazards per-
taining to specific infectious agents and the
activities performed.16 Biosafety recommen-
dations include guidance on both engineering
controls and safe work practices. The four bio-
safety levels are designated in ascending order,
with increasing protection for personnel, the
environment, and the community:
䡲 Biosafety Level 1 (BSL-1) involves work with
agents of no known or of minimal potential
hazard to laboratory personnel and the en-
vironment. Activities are usually conducted
on open surfaces, and no containment
equipment is needed.
䡲 BSL-2 work involves agents of moderate
potential hazard to personnel and the envi-
ronment, usually from contact-associated
exposure. Most blood bank laboratory ac-
tivities are considered BSL-2.
䡲 BSL-3 includes work with indigenous or ex-
otic agents that may cause serious or
potentially lethal disease as a result of ex-
posure to aerosols (eg, Mycobacterium tu-
berculosis) or by other routes (eg, HIV) that
would result in grave consequences to the
infected host. Recommendations for BSL-3
work are designed to contain biohazardous
aerosols and minimize the risk of surface
contamination.
䡲 BSL-4 applies to work with dangerous or
exotic agents that pose high individual risk
of life-threatening disease from aerosols
(eg, agents of hemorrhagic fevers or filovi-
ruses). BSL-4 is not applicable to routine
blood-bank-related activities.
The precautions described in this section
focus on BSL-2 requirements. Laboratories
should consult the CDC or National Institutes
of Health guidelines for precautions appropri-
ate for higher levels of containment.
Training
OSHA requires annual training for all employ-
ees whose tasks increase their risk of infectious
exposure.14,29 Training programs must be tai-
lored to the target group both in level and con-
tent. General background knowledge of bio-
hazards, understanding of control procedures,
or work experience cannot meet the require-
ment for specific training, although an assess-
ment of such knowledge is a first step in plan-
ning program content. Workplace volunteers
require at least as much safety training as paid
staff members who perform similar functions.
Hazard Identification and
Communication
The facility’s exposure control plan communi-
cates the risks present in the workplace and
describes controls to minimize exposure. BSL-
2 through BSL-4 facilities must have a biohaz-
ard sign posted at the entrance when infec-
tious agents are in use. The sign notifies
personnel and visitors of the presence of infec-
tious agents, provides a point of contact for
 CHAPTER 2 Facilities, Work Environment, and Safety
the area, and indicates any special protective
equipment or work practices required.
Biohazard warning labels must be placed
on containers of regulated waste; refrigerators
and freezers containing blood or other poten-
tially infectious material; and other containers
used to store, transport, or ship blood or other
potentially infectious materials. Blood compo-
nents that are labeled to identify their contents
and have been released for transfusion or oth-
er clinical use are exempted.
Engineering Controls and PPE
OSHA requires that hazards be controlled by
engineering or work practices whenever possi-
ble.14 Engineering controls for BSL-2 laborato-
ries include limited access to the laboratory
when work is in progress and BSCs or other
containment equipment for work that may in-
volve infectious aerosols or splashes. Hand-
washing sinks and eyewash stations must be
available. The work space should be designed
so that it can be easily cleaned, and bench tops
should be impervious to water and resistant to
chemicals and solvents.
To help prevent exposure or cross-con-
tamination, work area telephones can be
equipped with speakers to eliminate the need
to pick up the receiver. Computer keyboards
and telephones can be covered with plastic.
Such equipment should be cleaned on a regu-
lar basis and when visibly soiled.
BSCs are primary containment devices
for handling moderate-risk and high-risk or-
ganisms. There are three types—Classes I, II,
and III—with Class III providing the highest
protection to workers. In addition to protect-
ing personnel during the handling of biohaz-
ardous materials, a BSC may be used to pre-
vent contamination of blood and cellular
therapy products during open processing
steps. A comparison of the features and appli-
cations of the three classes of cabinets is pro-
vided in Table 2-2.
BSCs are not required by standard pre-
cautions, but centrifugation of open blood
samples or manipulation of units known to be
positive for HBV surface antigen or HIV are ex-
䡲 49
amples of blood bank procedures for which a
BSC could be useful. The effectiveness of the
BSC is a function of directional airflow inward
and downward through a high-efficiency fil-
ter. Efficacy is reduced by anything that dis-
rupts the airflow pattern (eg, arms moving rap-
idly in and out of the BSC, rapid movements
behind an employee using the BSC, down-
drafts from ventilation systems, or open labo-
ratory doors). Care should be taken not to
block the front intake and rear exhaust grills.
Performance of the BSC should be certified
annually.31
Injuries from contaminated needles and
other sharp objects (sometimes called
“sharps”) continued to be a major concern in
health-care settings even after the Blood-
Borne Pathogens Standard went into effect. In
2001, OSHA revised the standard to refer to
engineered sharps injury protections and
needleless systems.14 The revised standard re-
quires that employers implement appropriate
new control technologies and safer medical
devices in exposure control plans and that em-
ployers solicit input from their employees to
identify, evaluate, and select engineering and
work practice controls. Examples of safer de-
vices are needleless systems and self-sheath-
ing needles in which the sheath is an integral
part of the device.
Decontamination
Reusable equipment and work surfaces that
may be contaminated with blood require daily
cleaning and decontamination. Obvious spills
on equipment or work surfaces should be
cleaned up immediately; routine wipe-downs
with disinfectant should occur at the end of
each shift or a different frequency that
provides equivalent safety. Equipment that is
exposed to blood or other potentially infec-
tious material must be decontaminated before
it is serviced or shipped. When decontamina-
tion of all or a portion of the equipment is not
feasible, a biohazard label stating which por-
tions remain contaminated should be at-
tached before the equipment is serviced or
shipped.
 50
䡲

TABLE 2-2. Comparison of Classes I, II, and III Biological Safety Cabinets*

Category Main Features Intended Use Common Applications

Class I Unfiltered room air is drawn into the cabinet. Inward airflow Personal and environmental To enclose equipment (eg, centrifuges)
protects personnel from exposure to materials inside the protection or procedures that may generate aero-
cabinet. Exhaust is high-efficiency particulate air (HEPA) sols
filtered to protect the environment. It maintains airflow at a
minimum velocity of 75 linear feet per minute (lfpm) across
the front opening (face velocity).
Class II (general— Laminar flow (air moving at a constant velocity in one direc- Personal, environmental, and prod- Work with microorganisms assigned to
applies to all types of tion along parallel lines) is used. Room air is drawn into the uct protection Biosafety Levels 1, 2, or 3
Class II cabinets) front grille. HEPA-filtered air is forced downward in a laminar
flow to minimize cross-contamination of materials in the
cabinet. Exhaust is HEPA filtered.
AABB TECHNICAL MANUAL

Handling of products for which preven-

tion of contamination is critical, such
as cell culture propagation or manipu-
lation of blood components in an open
system
Class II, A Approximately 75% of air is recirculated after passing See Class II, general See Class II, general
through a HEPA filter. Face velocity = 75 lfpm.
Class II, B1 Approximately 70% of air exits through the rear grille, is See Class II, general Allows for safe manipulation of small
HEPA filtered, and is then discharged from the building. The quantities of hazardous chemicals and
other 30% is drawn into the front grille, is HEPA filtered, and biologics
is recirculated. Face velocity = 100 lfpm.
Class II, B2 All air is exhausted, and none is recirculated. A supply blower See Class II, general Provides both chemical and biological
draws air from the room or outside and passes it through a containment; is more expensive to
CHAPTER 2

HEPA filter to provide the downward laminar flow. Face operate because of the volume of con-
velocity = 100 lfpm. ditioned room air being exhausted
Class II, B3 Although similar in design to Type A, the system is ducted See Class II, general Allows for safe manipulation of small
and includes a negative pressure system to keep any possi- quantities of hazardous chemicals and
ble contamination within the cabinet. Face velocity = biologics
100 lfpm.
Class III Cabinet is airtight. Materials are handled with rubber gloves Maximum protection to personnel Work with Biosafety Level 4 micro-
attached to the front of the cabinet. Supply air is HEPA fil- and environment organisms
tered. Exhaust air is double HEPA filtered or may have one
filter and an air incinerator. Materials are brought in and out
of the cabinet either through a dunk tank or a double-door
pass-through box that can be decontaminated. Cabinet is
kept under negative pressure.
Facilities, Work Environment, and Safety

*Data from the US Department of Health and Human Services.30

䡲
51
52 䡲 AABB TECHNICAL MANUAL
Choice of Disinfectants
The Environmental Protection Agency (EPA)
maintains a list of chemical products that have
been shown to be effective hospital antimicro-
bial disinfectants.32 The Association for Profes-
sionals in Infection Control and Epidemiology
also publishes a guideline to assist health-care
professionals with decisions involving judi-
cious selection and proper use of specific dis-
infectants.33 For facilities covered under the
Blood-Borne Pathogens Standard, OSHA al-
lows the use of EPA-registered tuberculocidal
disinfectants, EPA-registered disinfectants
that are effective against both HIV and HBV, a
diluted bleach solution to decontaminate
work surfaces, or a combination of these.29
Before selecting a disinfectant product,
workers should consider several factors.
Among them are the type of material or sur-
face to be treated; the hazardous properties of
the chemical, such as corrosiveness; and the
level of disinfection required. After a product
has been selected, procedures need to be writ-
ten to ensure effective and consistent cleaning
and treatment of work surfaces. Some factors
to consider for effective decontamination in-
clude contact time, type of microorganisms,
presence of organic matter, and concentration
of the chemical agent. Workers should review
the basic information on decontamination
and follow the manufacturer’s instructions.
Storage
Hazardous materials must be segregated, and
areas for different types of storage must be
clearly demarcated. Blood must be protected
from unnecessary exposure to other materials
and vice versa. If transfusion products cannot
be stored in a separate refrigerator from re-
agents, specimens, and unrelated materials,
areas within the refrigerator must be clearly la-
beled, and extra care must be taken to reduce
the likelihood of spills and other accidents.
Storage areas must be kept clean and orderly;
food or drink is never allowed where biohaz-
ardous materials are stored.
PPE
When hazards cannot be eliminated, OSHA re-
quires employers to provide appropriate PPE
and clothing and to clean, launder, or dispose
of PPE at no cost to their employees.14 Stan-
dard PPE and clothing include uniforms, labo-
ratory coats, gloves, face shields, masks, and
safety goggles. Indications and guidelines for
their use are discussed in Appendix 2-2.
Safe Work Practices
Safe work practices appropriate for standard
precautions include the following:
䡲 Wash hands after touching blood, body flu-
ids, secretions, excretions, and contaminat-
ed items, whether or not gloves are worn.
䡲 Wear gloves when touching blood, body
fluids, secretions, excretions, and contami-
nated items, and change gloves between
tasks.
䡲 Wear a mask and eye protection or a face
shield during activities that are likely to
generate splashes or sprays of blood, body
fluids, secretions, and excretions.
䡲 Wear a gown during activities that are likely
to generate splashes or sprays of blood,
body fluids, secretions, or excretions.
䡲 Handle soiled patient-care equipment in a
manner that prevents exposure; ensure that
reusable equipment is not used for another
patient until it has been cleaned and repro-
cessed appropriately; and ensure that
single-use items are discarded properly.
䡲 Ensure that adequate procedures are de-
fined and followed for the routine care,
cleaning, and disinfection of environmen-
tal surfaces and equipment.
䡲 Handle soiled linen in a manner that pre-
vents exposure.
䡲 Handle needles, scalpels, and other sharp
instruments or devices in a manner that
minimizes the risk of exposure.
䡲 Use mouthpieces, resuscitation bags, or
other ventilation devices as an alternative
to mouth-to-mouth resuscitation meth-
ods.
䡲 Place in a private room patients who are at
risk of contaminating the environment or
 CHAPTER 2 Facilities, Work Environment, and Safety
are not able to maintain appropriate hy-
giene (eg, patients with tuberculosis).
Laboratory Biosafety Precautions
Several factors need to be considered when as-
sessing the risk of blood exposures among lab-
oratory personnel. Some of these factors in-
clude the number of specimens processed,
personnel behaviors, laboratory techniques,
and type of equipment.34 The laboratory direc-
tor may wish to institute BSL-3 practices for
procedures that are considered to be higher
risk than BSL-2. When there is doubt whether
an activity is BSL-2 or BSL-3, the safety precau-
tions for BSL-3 should be followed. BSL-2 pre-
cautions that are applicable to the laboratory
setting are summarized in Appendix 2-3.
Considerations for the Donor Room
The Blood-Borne Pathogens Standard ac-
knowledges a difference between hospital pa-
tients and healthy donors, in whom the preva-
lence of infectious disease markers is
significantly lower. The employer in a volun-
teer blood donation facility may determine
that routine use of gloves is not required for
phlebotomy as long as the following condi-
tions exist14:
䡲 The policy is periodically reevaluated.
䡲 Gloves are made available to those who
want to use them, and their use is not dis-
couraged.
䡲 Gloves are required when an employee has
cuts, scratches, or breaks in skin; when
there is a likelihood that contamination
will occur; while an employee is drawing
autologous units; while an employee is per-
forming therapeutic procedures; and dur-
ing training in phlebotomy.
Procedures should be assessed for risks of
biohazardous exposures and risks inherent in
working with a donor or patient during the
screening and donation processes. Some tech-
niques or procedures are more likely to cause
injury than others, such as using lancets for
finger puncture, handling capillary tubes,
crushing vials for arm cleaning, handling any
䡲 53
unsheathed needles, cleaning scissors, and
giving cardiopulmonary resuscitation.
In some instances, it may be necessary to
collect blood from donors known to pose a
high risk of infectivity (eg, collection of autolo-
gous blood or source plasma for the produc-
tion of other products, such as vaccines). The
FDA provides guidance on collecting blood
from such “high-risk” donors.35,36 The most re-
cent regulations and guidelines should be con-
sulted for changes or additions.
Emergency Response Plan
Table 2-3 lists steps to take when a spill occurs.
Facilities should be prepared to handle both
small and large blood spills. Good preparation
for spill cleanup includes several elements:
䡲 Work areas designed so that cleanup is rela-
tively simple.
䡲 A spill kit or cart containing all necessary
supplies and equipment with instructions
for their use. It should be placed near areas
where spills are anticipated.
䡲 Responsibility assigned for kit or cart main-
tenance, spill handling, record-keeping,
and review of significant incidents.
䡲 Personnel trained in cleanup procedures
and procedures for reporting significant in-
cidents.
Biohazardous Waste
Medical waste is defined as any waste (solid,
semisolid, or liquid) generated in the diagno-
sis, treatment, or immunization of human be-
ings or animals in related research, produc-
tion, or testing of biologics. Infectious waste
includes disposable equipment, articles, or
substances that may harbor or transmit patho-
genic organisms or their toxins. In general, in-
fectious waste should be either incinerated or
decontaminated before disposal in a sanitary
landfill.
If state law allows, blood and compo-
nents, suctioned fluids, excretions, and secre-
tions may be carefully poured down a drain
connected to a sanitary sewer. Sanitary sewers
may also be used to dispose of other potential-
ly infectious wastes that can be ground and
54 䡲 AABB TECHNICAL MANUAL

TABLE 2-3. Blood Spill Cleanup Steps

䡲 Evaluate the spill.

䡲 Wear appropriate protective clothing and gloves. If sharp objects are involved, gloves must be puncture
resistant, and a broom or other instrument should be used during cleanup to avoid injury.
䡲 Remove clothing if it is contaminated.
䡲 Post warnings to keep the area clear.
䡲 Evacuate the area for 30 minutes if an aerosol has been created.
䡲 Contain the spill if possible.
䡲 If the spill occurs in the centrifuge, turn the power off immediately and leave the cover on for 30 minutes.
The use of overwraps helps prevent aerosolization and contain the spill.
䡲 Use absorbent material to mop up most of the liquid contents.
䡲 Clean the spill area with detergent.
䡲 Flood the area with disinfectant and use it as described in the manufacturer’s instructions. Allow adequate
contact time with the disinfectant.
䡲 Wipe up residual disinfectant if necessary.
䡲 Dispose of all materials safely in accordance with biohazard guidelines. All blood-contaminated items must
be autoclaved or incinerated.

flushed into the sewer. State and local health
departments should be consulted about laws
and regulations pertaining to disposal of bio-
logic waste into the sewer.
Laboratories should clearly define what
will be considered hazardous waste. For exam-
ple, in the blood bank, all items contaminated
with liquid or semiliquid blood are biohazard-
ous. Items contaminated with dried blood are
considered hazardous if there is potential for
the dried material to flake off during handling.
Contaminated sharp objects are always con-
sidered hazardous because of the risk for per-
cutaneous injury. However, items such as used
gloves, swabs, plastic pipettes with excess liq-
uid removed, or gauze contaminated with
small droplets of blood may be considered
nonhazardous if the material is dried and will
not be released into the environment during
handling.
Guidelines for Biohazardous Waste
Disposal
Employees must be trained before handling or
disposing of biohazardous waste, even if it is
packaged. The following disposal guidelines
are recommended37:
䡲 Identify biohazardous waste consistently;
red seamless plastic bags (at least 2 mm
thick) or containers carrying the biohazard
symbol are recommended.
䡲 Place bags in a protective container with
closure upward to avoid breakage and leak-
age during storage or transport.
䡲 Prepare and ship waste transported over
public roads according to US Department
of Transportation (DOT) regulations.
䡲 Discard sharps (eg, needles, broken glass,
glass slides, and wafers from sterile con-
necting devices) in rigid, puncture-proof,
leakproof containers.
䡲 Put liquids in leakproof, unbreakable con-
tainers only.
䡲 Do not compact waste materials.
Storage areas for infectious material must
be secured to reduce accident risk. Infectious
waste must never be placed in the public trash
collection system. Most facilities hire private
 CHAPTER 2 Facilities, Work Environment, and Safety
carriers to decontaminate and dispose of in-
fectious or hazardous waste. The facility
should disclose all risks associated with the
waste in their contracts with private compa-
nies. The carrier is responsible for complying
with all federal, state, and local laws for bio-
hazardous (medical) waste transport, treat-
ment, and disposal.
Treating Infectious or Medical Waste
Facilities that incinerate hazardous waste
must comply with EPA standards of perfor-
mance for new stationary sources and emis-
sion guidelines for existing sources.38 In this
regulation, a hospital/medical/infectious waste
incinerator is any device that combusts any
amount of hospital waste or medical/infec-
tious waste.
Decontamination of biohazardous waste
by autoclaving is another common method for
decontamination or inactivation of blood
samples and blood components. The follow-
ing elements are considered in determining
processing time for autoclaving:
䡲 Size of load being autoclaved.
䡲 Type of packaging of item(s) being auto-
claved.
䡲 Density of items being autoclaved.
䡲 Number of items in a single autoclave load.
䡲 Placement of items in the autoclave to al-
low for steam penetration.
It is useful to place a biologic indicator in
the center of loads that vary in size and con-
tents to evaluate optimal steam penetration
times. The EPA provides detailed information
about choosing and operating such equip-
ment.37
For decontamination, material should be
autoclaved for a minimum of 1 hour. For steril-
ization, longer treatment times are needed. A
general rule for decontamination is to process
for 1 hour for every 10 pounds of waste. Usual-
ly, decontaminated laboratory wastes can be
disposed of as nonhazardous solid wastes. The
staff should check with the local solid waste
authority to ensure that the facility is in com-
pliance with regulations for the area. Waste-
䡲 55
containing broken glass or other sharp items
should be disposed of using a method consis-
tent with policies for the disposal of other
sharp or potentially dangerous materials.
CHEMICAL SAFET Y
One of the most effective preventive measures
that a facility can take to reduce hazardous
chemical exposure is to choose alternative
nonhazardous chemicals whenever possible.
When the use of hazardous chemicals is re-
quired, purchasing these supplies in small
quantities reduces the risks associated with
storing excess chemicals and then dealing
with their disposal.
OSHA requires that facilities using haz-
ardous chemicals develop a written chemical
hygiene plan (CHP) and that the plan be ac-
cessible to all employees. The CHP should out-
line procedures, equipment, PPE, and work
practices that are capable of protecting em-
ployees from hazardous chemicals used in the
facility.15,20 The CHP must also provide assur-
ance that equipment and protective devices
are functioning properly and that criteria to
determine implementation and maintenance
of all aspects of the plan are in place. Employ-
ees must be informed of all chemical hazards
in the workplace and be trained to recognize
chemical hazards, protect themselves when
working with these chemicals, and know
where to find information about particular
hazardous chemicals. Safety audits and annual
reviews of the CHP are important control steps
to help ensure that safety practices comply
with the policies set forth in the CHP and that
the CHP is up to date.
Establishing a clear definition of what
constitutes hazardous chemicals is some-
times difficult. Generally, hazardous chemicals
pose a significant health risk if an employee is
exposed to them or a significant physical risk,
such as fire or explosion, if they are handled or
stored improperly. Categories of health and
physical hazards are listed in Tables 2-4 and
2-5. The NIOSH Pocket Guide to Chemical Haz-
ards provides a quick reference for many com-
mon chemicals.39
56 䡲 AABB TECHNICAL MANUAL

TABLE 2-4. Categories of Health Hazards

Hazard Definition

Carcinogens Cancer-producing substances

Irritants Agents causing irritation (eg, edema or burning) to skin or mucous mem-
branes upon contact
Corrosives Agents causing destruction of human tissue at the site of contact
Toxic or highly toxic agents Substances causing serious biologic effects following inhalation, ingestion,
or skin contact with relatively small amounts
Reproductive toxins Chemicals that affect reproductive capabilities, including chromosomal
damages and effects on fetuses
Other toxins Hepatotoxins; nephrotoxins; neurotoxins; agents that act on the hemato-
poietic system; and agents that damage the lungs, skin, eyes, or mucous
membranes

The facility should identify a qualified
chemical hygiene officer to be responsible for
developing guidelines for hazardous materi-
als.20 The chemical hygiene officer is also ac-
countable for monitoring and documenting
accidents and for initiating process change as
needed.
Training
Employees who may be exposed to hazardous
chemicals must be trained before they begin
work in an area in which hazards exist. If a new
employee has received prior training, it may
not be necessary to retrain the individual, de-
pending on the employer’s evaluation of the
new employee’s level of knowledge. New em-
ployee training is likely to be necessary regard-
ing such specifics as the location of each rele-
vant material safety data sheet (MSDS), details
on chemical labeling, PPE to be used, and site-
specific emergency procedures.
Training must be provided for each new
physical or health hazard when it is intro-
duced into the workplace but not for each new
chemical that falls within a particular hazard
class.15 For example, if a new solvent is brought
into the workplace and the solvent has hazards
similar to existing chemicals for which training
has already been conducted, then the employ-
er need only make employees aware of the
new solvent’s hazard category (eg, corrosive or
irritant). However, if the newly introduced sol-
vent is a suspected carcinogen and carcino-
genic hazard training has not been provided,
then new training must be conducted for em-
ployees with potential exposure. Retraining is
advisable as often as necessary to ensure that
employees understand the hazards linked to
the materials with which they work, particu-
larly any chronic and specific target-organ
health hazards.
Hazard Identification and
Communication
Hazard Communication
Employers must prepare a comprehensive
hazard communication program for all areas
in which hazardous chemicals are used to
complement the CHP and “ensure that the
hazards of all chemicals produced or imported
are classified, and that information concern-
ing the classified hazard is transmitted to em-
ployers and employees.”15 The program should
include labeling of hazardous chemicals, in-
structions on when and how to post warning
labels for chemicals, directions for managing
MSDS reports for hazardous chemicals in the
facilities, and employee training. Safety mate-
rials made available to employees should in-
clude the following:
 CHAPTER 2 Facilities, Work Environment, and Safety 䡲 57

TABLE 2-5. Categories of Physical Hazards

Hazard Definition

Combustible or flammable
chemicals
Compressed gases
Explosives
Unstable (reactive) chemicals
Water-reactive chemicals
Chemicals that can burn (including combustible and flammable
liquids, solids, aerosols, and gases)
Gases or mixtures of gases in a container under pressure
Unstable or reactive chemicals that undergo violent chemical
changes at normal temperatures and pressure
Chemicals that could be self-reactive under certain conditions
(shocks, pressure, or temperature)
Chemicals that react with water to release a gas that either is
flammable or presents a health hazard

䡲 The facility’s written CHP.
䡲 The facility’s written program for hazard
communication.
䡲 Identification of work areas where hazard-
ous chemicals are located.
䡲 Required list of hazardous chemicals and
the relevant MSDS. (It is the responsibility
of the facility to determine which chemi-
cals may present a hazard to employees.
This determination should be based on the
quantity of chemical used; physical prop-
erties, potency, and toxicity of the chemi-
cal; manner in which the chemical is used;
and means available to control the release
of, or exposure to, the chemical.)
Hazardous Chemical Labeling and
Signs
The Hazard Communication Standard re-
quires manufacturers of chemicals and haz-
ardous materials to provide the user with basic
information about the hazards of these mate-
rials through product labeling and the MSDS.15
Employers are required to provide employees
who are expected to work with these hazard-
ous materials with information about what the
hazards of the materials are, how to read the
labeling, how to interpret symbols and signs
on the labels, and how to read and use the
MSDS.
MSDS forms typically include the follow-
ing:
䡲 Identification.
䡲 Hazard(s) identification.
䡲 Composition/information on ingredients.
䡲 First-aid measures.
䡲 Fire-fighting measures.
䡲 Accidental release measures.
䡲 Handling and storage considerations.
䡲 Exposure controls/personal protection in-
formation.
䡲 Physical and chemical properties.
䡲 Stability and reactivity.
䡲 Toxicologic information.
䡲 Ecologic information.
䡲 Disposal considerations.
䡲 Transport information.
䡲 Regulatory information.
䡲 Other information.
At a minimum, hazardous chemical con-
tainer labels must include the name of the
chemical, name and address of the manufac-
turer, hazard warnings, symbols, designs, and
other forms of warning to provide visual re-
minders of specific hazards. The label may
refer to any MSDS for additional information.
Labels applied by the manufacturer must re-
main on containers. The user may add storage
requirements and dates of receipt, opening,
and expiration. If chemicals are aliquotted into
58 䡲 AABB TECHNICAL MANUAL
secondary containers, the secondary contain-
er must be labeled with the name of the chem-
ical and appropriate hazard warnings. Addi-
tional information, such as precautionary
measures, concentration if applicable, and
date of preparation, are helpful but not man-
datory.
It is a safe practice to label all containers
with their content, even water. Transfer con-
tainers used for temporary storage need not be
labeled if the person performing the transfer
retains control and intends the containers to
be used immediately. Information regarding
acceptable standards for hazard communica-
tion labeling is provided by the NFPA40 and the
National Paint and Coatings Association.41
Signs meeting OSHA requirements must
be posted in areas where hazardous chemicals
are used. Decisions about where to post warn-
ing signs are based on the manufacturer’s rec-
ommendations regarding the chemical haz-
ards, the quantity of the chemical in the room
or laboratory, and the potency and toxicity of
the chemical.
MSDS
The MSDS identifies the physical and chemi-
cal properties of a hazardous chemical (eg,
flash point or vapor pressure), its physical and
health hazards (eg, potential for fire, explo-
sion, and signs and symptoms of exposure),
and precautions for the chemical’s safe han-
dling and use. Specific instructions in an indi-
vidual MSDS take precedence over generic
information in the hazardous materials pro-
gram.
Employers must maintain copies of each
required MSDS in the workplace for each haz-
ardous chemical and ensure that MSDS cop-
ies are readily accessible during each work
shift to employees when they are in their work
areas. When household consumer products
are used in the workplace in the same manner
that a consumer would use them (ie, when the
duration and frequency of use, and therefore
exposure, are not greater than those that the
typical consumer would experience), OSHA
does not require that an MSDS be provided to
purchasers. However, if exposure to such prod-
ucts exceeds that normally found in consumer
applications, employees have a right to know
about the properties of such hazardous chemi-
cals. OSHA does not require or encourage em-
ployers to maintain an MSDS for nonhazard-
ous chemicals.
Engineering Controls and PPE
Guidelines for laboratory areas in which haz-
ardous chemicals are used or stored must be
established. Physical facilities, and especially
ventilation, must be adequate for the nature
and volume of work conducted. Chemicals
must be stored according to chemical compat-
ibility (eg, corrosives, flammables, and oxidiz-
ers) and in minimal volumes. Bulk chemicals
should be kept outside work areas. NFPA stan-
dards and others provide guidelines for proper
storage.4,40,42
Chemical fume hoods are recommended
for use with organic solvents, volatile liquids,
and dry chemicals with a significant inhalation
hazard. 4 Although constructed with safety
glass, most fume hood sashes are not designed
to serve as safety shields. Hoods should be po-
sitioned in an area where there is minimal foot
traffic to avoid disrupting the airflow and com-
promising the containment field.
PPE that may be provided, depending on
the hazardous chemicals used, includes chem-
ical-resistant gloves and aprons, shatterproof
safety goggles, and respirators.
Emergency showers should be accessible
to areas where caustic, corrosive, toxic, flam-
mable, or combustible chemicals are used.4,43
There should be unobstructed access, within
10 seconds, to the showers from the areas
where hazardous chemicals are used. Safety
showers should be periodically flushed and
tested for function, and associated floor drains
should be checked to ensure that drain traps
remain filled with water.
Safe Work Practices
Hazardous material should not be stored or
transported in open containers. Containers
and their lids or seals should be designed to
prevent spills or leakage in all reasonably an-
ticipated conditions. Containers should be
 CHAPTER 2 Facilities, Work Environment, and Safety
able to safely store the maximum anticipated
volume and be easy to clean. Surfaces should
be kept clean and dry at all times.
When an employee is working with a
chemical fume hood, all materials should be
kept at least 6 inches behind the face opening.
The vertical sliding sash should be positioned
at the height specified on the certification
sticker. The airfoil, baffles, and rear ventilation
slot must not be blocked. Appendix 2-5 lists
suggestions for working safely with specific
chemicals
Emergency Response Plan
The time to prepare for a chemical spill is be-
fore it occurs. A comprehensive employee
training program should provide each employ-
ee with all tools necessary to act responsibly at
the time of a chemical spill. The employee
should know response procedures, be able to
assess the severity of a chemical spill, know or
be able to quickly look up the basic physical
characteristics of the chemicals, and know
where to find emergency response telephone
numbers. The employee should be able to as-
sess, stop, and confine the spill; either clean up
the spill or call for a spill cleanup team; and
follow procedures for reporting the spill. The
employee must know when to ask for assis-
tance, when to isolate the area, and where to
find cleanup materials.
Chemical spills in the workplace can be
categorized as follows44:
䡲 Incidental releases are limited in quantity
and toxicity and pose no significant safety
or health hazard to employees. They may
be safely cleaned up by employees familiar
with the hazards of the chemical involved
in the spill. Waste from the cleanup may be
classified as hazardous and must be dis-
posed of in the proper fashion. Appendix 2-
6 describes appropriate responses to inci-
dental spills.
䡲 Releases that may be incidental or may re-
quire an emergency response may pose an
exposure risk to employees depending on
the circumstances. Considerations such as
the hazardous substance properties, cir-
䡲 59
cumstances of release, and mitigating fac-
tors play a role in determining the appro-
priate response. The facility’s emergency
response plan should provide guidance on
how to determine whether a spill is inci-
dental or requires an emergency response.
䡲 Emergency response releases pose a threat to
health and safety regardless of the circum-
stances surrounding their release. These
spills may require evacuation of the imme-
diate area. The response typically comes
from outside the immediate release area by
personnel trained as emergency respond-
ers. These spills include those that involve
immediate danger to life or health, serious
threat of fire or explosion, and high levels
of toxic substances.
Appendix 2-7 addresses the management
of hazardous chemical spills. Spill cleanup kits
or carts tailored to the specific hazards present
should be available in each area. The kits or
carts may contain rubber gloves and aprons,
shoe covers, goggles, suitable aspirators, gen-
eral absorbents, neutralizing agents, a broom,
a dust pan, appropriate trash bags or cans for
waste disposal, and cleanup directions. Chem-
ical absorbents, such as clay absorbents or
spill blankets, can be used for cleaning up a
number of chemicals and thus may be easier
for employees to use in spill situations.
With any spill of a hazardous chemical,
but especially of a carcinogenic agent, it is es-
sential to refer to the MSDS and contact a des-
ignated supervisor or designee trained to han-
dle these spills and hazardous waste disposal.4
Facility environmental health and safety
personnel can also offer assistance. The em-
ployer must assess the extent of the employee’s
exposure. After an exposure, the employee
must be given an opportunity for medical con-
sultation to determine the need for a medical
examination.
Another source of workplace hazards is
the unexpected release of hazardous vapors
into the environment. OSHA has set limits for
exposure to hazardous vapors from toxic and
hazardous substances.45 The potential risk as-
sociated with a chemical is determined by the
manufacturer and listed on the MSDS.
60 䡲 AABB TECHNICAL MANUAL
Chemical Waste Disposal
Most laboratory chemical waste is considered
hazardous and is regulated by the EPA through
the Resource Conservation and Recovery Act
(42 USC §6901 et seq, 1976). This regulation
specifies that hazardous waste can be legally
disposed of only at an EPA-approved disposal
facility. Disposal of chemical waste into a sani-
tary sewer is regulated by the Clean Water Act
(33 USC §1251 et seq, 1977), and most US
states have strict regulations concerning dis-
posal of chemicals in the water system. Federal
and applicable state regulations should be
consulted when a facility is setting up and re-
viewing its waste disposal policies.
RADIATION SAF ET Y
Radiation can be defined as energy in the form
of waves or particles emitted and propagated
through space or a material medium. Gamma
rays are electromagnetic radiation, whereas al-
pha and beta emitters are examples of particu-
late radiation. The presence of radiation in the
blood bank, from either radioisotopes used in
laboratory testing or self-contained blood irra-
diators, requires additional precautions and
training.4,46
Radiation Measurement Units
The measurement unit quantifying the amount
of energy absorbed per unit mass of tissue is
the gray (Gy) or radiation absorbed dose (rad);
1 Gy = 100 rad.
Dose equivalency measurements are
more useful than simple energy measure-
ments because dose equivalency measure-
ments take into account the ability of different
types of radiation to cause biologic effects. The
ability of radiation to cause damage is as-
signed a number called a quality factor (QF).
For example, exposure to a given amount of al-
pha particles (QF = 20) is far more damaging
than exposure to an equivalent amount of
gamma rays (QF = 1). The common unit of
measurement for dose equivalency is the
roentgen or rad equivalent man (rem). Rem is
the dose from any type of radiation that pro-
duces biologic effects in humans equivalent to
1 rad of x-rays, gamma rays, or beta rays. To
obtain the dose from a particular type of radia-
tion in rem, the number of rad should be mul-
tiplied by the QF (rad × QF = rem). Because the
QF for gamma rays, x-rays, and most beta par-
ticles is 1, the dose in rad is equal to the dose in
rem for these types of radiation.
Biologic Effects of Radiation
Any harm to tissue begins with the absorption
of radiation energy and subsequent disruption
of chemical bonds. Molecules and atoms be-
come ionized or excited (or both) by absorbing
this energy. The direct action path leads to ra-
diolysis or formation of free radicals that, in
turn, alter the structure and function of mole-
cules in the cell.
Molecular alterations can cause cellular
or chromosomal changes, depending on the
amount and type of radiation energy ab-
sorbed. Cellular changes can be manifested as
a visible somatic effect (eg, erythema). Chang-
es at the chromosome level may be manifested
as leukemia or other cancers or possibly as
germ-cell defects that are transmitted to future
generations.
Several factors influence the level of bio-
logic damage from exposure, including the
type of radiation, part of the body exposed, to-
tal absorbed dose, and dose rate. The total ab-
sorbed dose is the cumulative amount of radi-
ation absorbed in the tissue. The greater the
dose, the greater the potential for biologic
damage. Exposure can be acute or chronic.
The low levels of ionizing radiation likely to oc-
cur in blood banks should not pose any detri-
mental risk.47-50
Regulations
The NRC controls the use of radioactive mate-
rials by establishing licensure requirements.
States and municipalities may also have re-
quirements for inspection, licensure, or both.
The type of license for using radioisotopes or
irradiators depends on the scope and magni-
tude of the use of radioactivity. US facilities
should contact the NRC and appropriate state
agencies for information on license require-
 CHAPTER 2 Facilities, Work Environment, and Safety
ments and applications as soon as such activi-
ties are proposed.
Each NRC-licensed establishment must
have a qualified radiation safety officer who is
responsible for establishing personnel protec-
tion requirements and for proper disposal and
handling of radioactive materials. Specific ra-
diation safety policies and procedures should
address dose limits, employee training, warn-
ing signs and labels, shipping and handling
guidelines, radiation monitoring, and expo-
sure management. Emergency procedures
must be clearly defined and readily available
to the staff.
In 2005, the NRC imposed additional se-
curity requirements for high-risk radioactive
sources, including those used in blood irradia-
tors. The purpose of the increased controls is
to reduce the risk of unauthorized use of ra-
dioactive materials that may pose a threat to
public health and safety. These 2005 measures
include controlled access, approval in writing
of individuals deemed trustworthy and reli-
able to have unescorted access, a system of
monitoring to immediately detect and re-
spond to unauthorized access, and documen-
tation of authorized personnel and monitor-
ing activities. 51 In 2007, a requirement for
fingerprinting was added.52
Exposure Limits
The NRC sets standards for protection against
radiation hazards arising from licensed activi-
ties, including dose limits.12 Such limits, or
maximal permissible dose equivalents, are a
measure of the radiation risk over time and
serve as standards for exposure. The occupa-
tional total effective-dose-equivalent limit is
5 rem/year, the shallow dose equivalent limit
(skin) is 50 rem/year, the extremity dose equiv-
alent limit is 50 rem/year, and the eye dose
equivalent limit is 15 rem/year.12,47 Dose limits
for an embryo or fetus must not exceed 0.5
rem during pregnancy.12,47,53 Employers are ex-
pected not only to maintain radiation expo-
sure below allowable limits, but also to keep
exposure levels as far below these limits as can
reasonably be achieved.
䡲 61
Radiation Monitoring
Monitoring is essential for early detection and
prevention of problems resulting from radia-
tion exposure. Monitoring is used to evaluate
the facility’s environment, work practices, and
procedures and to comply with regulations
and NRC licensing requirements. Monitoring
is accomplished with the use of dosimeters,
bioassays, survey meters, and wipe tests.4
Dosimeters, such as film or thermolumi-
nescent badges, rings, or both, measure per-
sonnel radiation doses. The need for dosime-
ters depends on the amount and type of
radioactive materials in use; the facility radia-
tion safety officer determines individual do-
simeter needs. Film badges must be changed
at least quarterly and in some instances
monthly, be protected from high temperature
and humidity, and be stored at work away
from sources of radiation.
Bioassays, such as thyroid and whole
body counting or urinalysis, may be used to
determine whether there is radioactivity inside
the body and if so, how much. If necessary,
bioassays are usually performed quarterly and
after an incident where accidental intake may
have occurred.
Survey meters are sensitive to low levels
of gamma or particulate radiation and provide
a quantitative assessment of radiation hazard.
Survey meters can be used to monitor storage
areas for radioactive materials or wastes, test-
ing areas during or after completion of a pro-
cedure, and packages or containers of radio-
active materials. Survey meters must be
calibrated annually by an authorized NRC li-
censee. Selection of appropriate meters should
be discussed with the radiation safety officer.
In areas where radioactive materials are
handled, all work surfaces, equipment, and
floors that may be contaminated should be
checked regularly with a wipe test. In the wipe
test, a moistened absorbent material (the
wipe) is passed over the surface and then mea-
sured for radiation.
Training
Personnel who handle radioactive materials or
work with blood irradiators must receive radi-
62 䡲 AABB TECHNICAL MANUAL
ation safety training before beginning work.
This training should address the presence and
potential hazards of radioactive materials in
the employee’s work area, general health pro-
tection issues, emergency procedures, and ra-
diation warning signs and labels in use.
Instruction in the following areas is also sug-
gested:
䡲 NRC regulations and license conditions.
䡲 The importance of observing license condi-
tions and regulations and of reporting vio-
lations or conditions of unnecessary expo-
sure.
䡲 Precautions to minimize exposure.
䡲 Interpretation of results of monitoring de-
vices.
䡲 Requirements for pregnant workers.
䡲 Employees’ rights.
䡲 Documentation and record-keeping re-
quirements.
The need for refresher training is deter-
mined by the license agreement between the
NRC and the facility.
Engineering Controls and PPE
Although self-contained blood irradiators
present little risk to laboratory staff and film
badges are not required for routine operation,
blood establishments with irradiation pro-
grams must be licensed by the NRC.48
The manufacturer of the blood irradiator
usually accepts responsibility for radiation
safety requirements during transportation, in-
stallation, and validation of the unit as part of
the purchase contract. The radiation safety of-
ficer can help oversee the installation and vali-
dation processes and should confirm that
appropriate training, monitoring systems,
procedures, and maintenance protocols are in
place before use and that they reflect the man-
ufacturer’s recommendations. Suspected mal-
functions must be reported immediately so
that appropriate actions can be initiated.
Blood irradiators should be located in se-
cure areas so that only trained individuals
have access. Fire protection for the unit must
also be considered. Automatic fire detection
and control systems should be readily avail-
able in the immediate area. Blood compo-
nents that have been irradiated are not radio-
active and pose no threat to the staff or the
general public.
Safe Work Practices
Each laboratory should establish policies and
procedures for the safe use of radioactive mate-
rials. These policies and procedures should in-
clude requirements for following general labo-
ratory safety principles, appropriate storage of
radioactive solutions, and proper disposal of
radioactive wastes. Radiation safety can be im-
proved with the following procedures:
䡲 Minimizing the time of exposure by work-
ing as efficiently as possible.
䡲 Maximizing the distance from the source of
the radiation by staying as far from the
source as possible.
䡲 Maximizing shielding (eg, by using a self-
shielded irradiator or wearing a lead apron)
when working with certain radioactive ma-
terials. These requirements are usually stip-
ulated in the license conditions.
䡲 Using good housekeeping practices to min-
imize the spread of radioactivity to uncon-
trolled areas.
Emergency Response Plan
Radioactive contamination is the dispersal of
radioactive material into or onto areas in
which it is not intended—for example, the
floor, work areas, equipment, personnel cloth-
ing, or personnel skin. The NRC regulations
state that gamma or beta radioactive contami-
nation cannot exceed 2200 disintegrations per
minute (dpm) per 100 cm2 in the posted (re-
stricted) area or 220 dpm/100 cm2 in an unre-
stricted area, such as a corridor. For alpha
emitters, these values are 220 dpm/100 cm2
and 22 dpm/100 cm2, respectively.54
If a spill occurs, employees’ contaminated
skin surfaces must be washed several times,
and the radiation safety officer must be noti-
fied immediately to provide further guidance.
Others must not be allowed to enter the area
until emergency response personnel arrive.
 CHAPTER 2 Facilities, Work Environment, and Safety
Radioactive Waste Management
Policies for the disposal of radioactive waste,
whether liquid or solid, should be established
with input from the radiation safety officer.
Liquid radioactive waste may be collected
into large sturdy bottles labeled with an appro-
priate radiation waste tag. The rules for sepa-
ration by chemical compatibility apply. Bottles
must be carefully stored to protect against
spillage or breakage. Dry or solid waste may be
sealed in a plastic bag and tagged as radiation
waste. The isotope, its activity, and the date on
which the activity was measured should be
recorded on the bag. Radioactive waste must
never be discharged into the facility’s drain
system without prior approval by the radiation
safety officer.
SH I PPING H AZ AR DOUS
M AT E R I A L S
Hazardous materials commonly shipped by
transfusion medicine, cellular therapy, and
clinical diagnostic services include infectious
substances, biologic substances, liquid nitro-
gen, and dry ice.
The US DOT regulations for transporta-
tion of hazardous materials are harmonized
with the international standards published an-
nually by the International Air Transport Asso-
ciation (IATA). 55,56 These regulations provide
instructions for identifying, classifying, pack-
aging, marking, labeling, and documenting
hazardous materials to be offered for ship-
ment on public roadways or by air.
Specimens are classified as Category A if
they are known or likely to contain infectious
substances in a form that is capable of causing
permanent disability or life-threatening or fa-
tal disease in otherwise healthy humans or an-
imals when an exposure occurs. The proper
shipping name for Category A specimens is
“infectious substances, affecting humans”
(UN2814) or “infectious substances, affecting
animals only” (UN2900).
Specimens that may contain infectious
substances but do not have the level of risk
described above are classified as Category B,
and the proper shipping name is “biological
䡲 63
substance, Category B” (UN3373). HIV or
HBV in culture are classified as Category A in-
fectious substances, but these viruses present
in a patient blood specimen are classified as
Category B.
Patient specimens with minimal likeli-
hood of containing pathogens are exempt
from hazardous materials regulations if the
specimens are properly packaged and marked.
Blood components, cellular therapy products,
and tissue for transfusion or transplantation
are not subject to hazardous material regula-
tions. Method 1-1 provides additional ship-
ping instructions for safe transport of these
materials. However, the most recent revision of
the IATA or US DOT regulations should be con-
sulted for the most current classification,
packaging, and labeling requirements as well
as for limitations on the volumes of hazardous
materials that can be packaged together in one
container.
G E N E R A L WA S T E M A N AG E M E N T
Those responsible for safety at a facility must
be concerned with protecting the environ-
ment as well as all staff members. Every effort
should be made to establish facility-wide pro-
grams to reduce solid wastes, including non-
hazardous and, especially, hazardous wastes
(ie, biohazardous, chemical, and radioactive
wastes).
A hazardous waste-reduction program in-
stituted at the point of use of the material
achieves several goals. It reduces the institu-
tional risk for occupational exposures to haz-
ardous agents, reduces “cradle-to-grave” lia-
bility for disposal, and enhances compliance
with environmental requirements to reduce
pollution generated from daily operations of
the laboratory.37,57,58
Facilities can minimize pollution of the
environment by practicing the “three R’s”: re-
duce, reuse, and recycle. Seeking suitable al-
ternatives to materials that create hazardous
waste and separating hazardous waste from
nonhazardous waste can reduce the volume of
hazardous waste and decrease costs for its dis-
posal.
64 䡲 AABB TECHNICAL MANUAL

Changes in techniques or materials to re- In some states, copper sulfate contaminated

duce the volume of infectious waste or render
it less hazardous should be carefully consid-
ered, and employees should be encouraged to
identify safer alternatives wherever possible.
Facilities should check with state and
local health and environmental authorities
about current requirements for storage and
disposal of a particular multihazardous waste
before creating that waste. If creating the mul-
tihazardous waste cannot be avoided, the vol-
ume of waste generated should be minimized.
with blood is considered a multihazardous
waste. The disposal of this waste poses several
problems with transportation from draw sites
to a central facility for disposal of the final con-
tainers. State and local health departments
must be involved in reviewing transportation
and disposal practices where this is an issue,
and procedures must be developed in accor-
dance with state and local regulations as well
as those of the US DOT.

KEY POINTS

1. Facilities should be designed and maintained in a way that supports the work being done in
the physical space. Designing the space to accommodate planned work flow, the need to re-
strict certain areas, the movement of materials and waste, equipment location, special air-
handling requirements, and other critical aspects of the operation help ensure safety for
staff and visitors as well as the quality of products and services.
2. The facilities safety program should 1) strive to reduce hazards in the workplace, 2) ensure
that staff are trained to handle known hazards and potential risks, 3) ensure that known
hazards are clearly identified and marked, and 4) describe policies and procedures for
workplace safety and emergency response.
3. Safety programs should address fire, electrical, biological, chemical, and radioactive haz-
ards that may be found in the facility.
4. For each type of hazard, five basic elements that must be covered are 1) training; 2) hazard
identification and communication; 3) engineering controls and PPE; 4) safe work practices,
including waste disposal; and 5) an emergency response plan.
5. Management controls ensure that the safety program is implemented, maintained, and ef-
fective. Management is responsible for 1) developing and communicating the written plan,
2) ensuring implementation of the plan and providing adequate resources for this imple-
mentation, 3) providing access to employee health services for prevention strategies and
treatment of exposures, 4) monitoring compliance and effectiveness, and 5) evaluating and
improving the safety plan.

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䡲 65
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30. US Department of Health and Human Servic-
es. Primary containment for biohazards: Se-
lection, installation, and use of biological safe-
ty cabinets. Washington, DC: US Government
Printing Office, 2009. [Available at http://
www.cdc.gov/biosafety/publications (ac-
cessed January 6, 2013).]
31. Richmond JY. Safe practices and procedures
for working with human specimens in bio-
medical research laboratories. J Clin Immuno-
assay 1988;11:115-19.
32. ATP— tested actively registered hospital disin-
fectant products. (March 2010) Washington,
DC: US Environmental Protection Agency,
2012. [Available at http://www.epa.gov/
oppad001/chemregindex.htm (accessed Janu-
ary 6, 2013).]
66 䡲 AABB TECHNICAL MANUAL
33. Rutala WA. APIC guideline for selection and
use of disinfectants. Am J Infect Control 1996;
24:313-42.
34. Evans MR, Henderson DK, Bennett JE. Poten-
tial for laboratory exposures to biohazardous
agents found in blood. Am J Public Health
1990;80:423-7.
35. Memorandum: Guideline for collection of
blood products from donors with positive tests
for infectious disease markers (“high risk” do-
nors). (October 26, 1989) Silver Spring, MD:
CBER Office of Communication, Outreach,
and Development, 1989.
36. Memorandum: Revision to 26 October 1989
guidelines for collection of blood or blood
products from donors with positive tests for
infectious disease markers (“high-risk” do-
nors). Silver Spring, MD: CBER Office of Com-
munication, Outreach, and Development,
1991. [Available at http://www.fda.gov/biolog
icsbloodvaccines/guidancecomplianceregula
toryinformation/other recommendations for
manufacturers/memorandumtobloodestab
lishments.default.htm.]
37. US Environmental Protection Agency. EPA
guide for infectious waste management. EPA/
530-SW-86-014. NTIS #PB86-199130. Wash-
ington, DC: National Technical Information
Service, 1986.
38. Code of federal regulations. Title 40, CFR Part
264. Washington, DC: US Government Print-
ing Office, 2013 (revised annually).
39. NIOSH pocket guide to chemical hazards.
Washington, DC: National Institute for Occu-
pational Safety and Health, 2010. [Available at
http://www.cdc.gov/niosh/npg (accessed No-
vember 8, 2010).]
40. NFPA 704—Standard for the identification of
the hazards of materials for emergency re-
sponse. Quincy, MA: National Fire Protection
Association, 2012.
41. Hazardous Materials Identification System.
HMIS implementation manual. 3rd ed. Neen-
ah, WI: JJ Keller and Associates, Inc., 2001.
42. Lisella FS, Thomasston SW. Chemical safety in
the microbiology laboratory. In: Fleming DO,
Richardson JH, Tulis JJ, Vesley D, eds. Labora-
tory safety, principles, and practices. 2nd ed.
Washington, DC: American Society for Micro-
biology Press, 1995:247-54.
43. American national standards for emergency
eyewash and shower equipment. ANSI Z358.1-
2009. New York: American National Standards
Institute, 2009.
44. Inspection procedures for 29 CFR 1910.120
and 1926.65, paragraph (q): Emergency re-
sponse to hazardous substance releases.
OSHA Directive CPL 02-02-073. Washington,
DC: Occupational Safety and Health Adminis-
tration, 2007.
45. Code of federal regulations. Title 29, CFR Part
1910.1000. Washington, DC: US Government
Printing Office, 2013 (revised annually).
46. Cook SS. Selection and installation of self-
contained irradiators. In: Butch S, Tiehen A,
eds. Blood irradiation: A user’s guide. Bethes-
da, MD: AABB Press, 1996:19-40.
47. Beir V. Health effects of exposure to low levels
of ionizing radiation. Washington, DC: Nation-
al Academy Press, 1990:1-8.
48. Regulatory Guide 8.29: Instruction concerning
risks from occupational radiation exposure.
Washington, DC: Nuclear Regulatory Commis-
sion, 1996.
49. NCRP Report No. 115: Risk estimates for radia-
tion protection: Recommendations of the Na-
tional Council on Radiation Protection and
Measurements. Bethesda, MD: National
Council on Radiation Protection and Measure-
ments, 1993.
50. NCRP Report No. 105: Radiation protection for
medical and allied health personnel: Recom-
mendations of the National Council on Radia-
tion Protection and Measurements. Bethesda,
MD: National Council on Radiation Protection
and Measurements, 1989.
51. EA-05 090. Enforcement action: Order impos-
ing increased controls (licensees authorized to
possess radioactive material quantities of con-
cern). (November 14, 2005) Rockville, MD: US
Nuclear Regulatory Commission, 2005.
52. RIS 2007-14. Fingerprinting requirements for
licensees implementing the increased control
order. (June 5, 2007) Rockville, MD: US Nucle-
ar Regulatory Commission, 2007.
53. US Nuclear Regulatory Commission Regulato-
ry Guide 8.13: Instruction concerning prenatal
radiation exposure. Washington, DC: NRC,
1999.
54. Nuclear Regulatory Commission regulatory
guide 8.23: Radiation surveys at medical insti-
tutions. Washington, DC: NRC, 1981.
55. Code of federal regulations. Title 49, CFR Parts
171.22. Washington, DC: US Government
Printing Office, 2014 (revised annually).
56. Dangerous goods regulations manual. 54th ed.
Montreal, PQ, Canada: International Air Trans-
port Association, 2014 (revised annually).
 CHAPTER 2 Facilities, Work Environment, and Safety 䡲 67

57. United States Code. Pollution prevention act. Clinical and Laboratory Standards Institute,
42 U.S.C. §§13101 and 13102 et seq. 2011.
58. Clinical laboratory waste management. Ap-
proved guideline. 3rd ed. GP05-A3. Wayne, PA:
68 䡲 AABB TECHNICAL MANUAL
䡲 APPENDIX 2-1
Safety Regulations and Recommendations Applicable to Health-Care Settings
Agency/Organization
Federal Regulations and
Recommendations
Nuclear Regulatory Commission
Occupational Safety and Health
Administration
Department of Transportation
Environmental Protection Agency
(EPA)
Centers for Disease Control and
Prevention
Food and Drug Administration
Reference Title
10 CFR 20 Standards for Protection Against Radia-
tion
10 CFR 36 Licenses and Radiation Safety Require-
ments for Irradiators
Guide 8.29 Instructions Concerning Risks from
Occupational Radiation Exposure
29 CFR 1910.1030 Occupational Exposure to Bloodborne
Pathogens
29 CFR 1910.1020 Access to Employee Exposure and Medi-
cal Records
29 CFR 1910.1096 Ionizing Radiation
29 CFR 1910.1200 Hazard Communication Standard
29 CFR 1910.1450 Occupational Exposure to Hazardous
Chemicals in Laboratories
49 CFR 171-180 Hazardous Materials Regulations
EPA Guide for Infectious Waste Manage-
ment
Guideline for Isolation Precautions in
Hospitals
21 CFR 606.3-606.171 Current Good Manufacturing Practice for
Blood and Blood Components
21 CFR 630.6 General Requirements for Blood, Blood
Components, and Blood Derivatives
21 CFR 640.1-640.120 Additional Standards for Human Blood
and Blood Products
21 CFR 211.1-211.208 Current Good Manufacturing Practice for
Finished Pharmaceuticals
21 CFR 1270 Human Tissue Intended for Transplanta-
tion
21 CFR 1271 Human Cells, Tissues, and Cellular and
Tissue-Based Products
 CHAPTER 2 Facilities, Work Environment, and Safety 䡲 69

䡲 APPENDIX 2-1
Safety Regulations and Recommendations Applicable to Health-Care Settings
(Continued)
Agency/Organization Reference Title

Trade and Professional

Organizations
National Fire Protection
Association (NFPA)
National Paint and Coatings
Association
International Air Transport
Association
CFR = Code of federal regulations.
NFPA 70 National Electrical Code
NFPA 70E Electrical Safety Requirements for
Employee Workplaces
NFPA 101 Life Safety Code
NFPA 99 Standards for Health Care Facilities
NFPA 704 Standard for Identification of the Hazards
of Materials for Emergency Response
Hazardous Materials Identification
System Implementation Manual
Dangerous Goods Regulations
70 䡲 AABB TECHNICAL MANUAL

䡲 APPENDIX 2-2
General Guidelines for Safe Work Practices, Personal Protective Equipment, and
Engineering Controls
UNIFORMS AND LABORATORY COATS

Personnel should wear closed laboratory coats or full aprons over long-sleeved uniforms or gowns when they
are exposed to blood, corrosive chemicals, or carcinogens. The material of required coverings should be
appropriate for the type and amount of hazard exposure. Plastic disposable aprons may be worn over cotton
coats when there is a high probability of large spills or splashing of blood and body fluids; nitrile rubber
aprons may be preferred when caustic chemicals are poured.
Protective coverings should be removed before the employee leaves the work area and should be discarded or
stored away from heat sources and clean clothing. Contaminated clothing should be removed promptly,
placed in a suitable container, and laundered or discarded as potentially infectious. Home laundering of gar-
ments worn in Biosafety Level 2 areas is not permitted because unpredictable methods of transportation and
handling can spread contamination and home laundering techniques may not be effective.1

GLOVES

Gloves or equivalent barriers should be used whenever tasks are likely to involve exposure to hazardous materials.

TYPES OF GLOVES

Glove type varies with the task:

䡲 Sterile gloves: for procedures involving contact with normally sterile areas of the body.
䡲 Examination gloves: for procedures involving contact with mucous membranes, unless otherwise indicated,
and for other patient care or diagnostic procedures that do not require the use of sterile gloves.
䡲 Rubber utility gloves: for housekeeping chores involving potential blood contact, instrument cleaning and
decontamination procedures, and handling concentrated acids and organic solvents. Utility gloves may be
decontaminated and reused but should be discarded if they show signs of deterioration (eg, peeling, cracks,
or discoloration) or if they develop punctures or tears.
䡲 Insulated gloves: for handling hot or frozen material.

GUIDELINES FOR USE

The following guidelines should be used to determine when gloves are necessary1:
䡲 For donor phlebotomy when the health-care worker has cuts, scratches, or other breaks in his or her skin.
䡲 For phlebotomy of autologous donors or patients (eg, therapeutic apheresis procedures or intraoperative red
cell collection).
䡲 For persons who are receiving training in phlebotomy.
䡲 When handling open blood containers or specimens.
䡲 When collecting or handling blood or specimens from patients or donors known to be infected with a blood-
borne pathogen.
䡲 When examining mucous membranes or open skin lesions.
䡲 When handling corrosive chemicals and radioactive materials.
䡲 When cleaning up spills or handling waste materials.
䡲 When the likelihood of exposure cannot be assessed because of lack of experience with a procedure or situation.
 CHAPTER 2 Facilities, Work Environment, and Safety 䡲 71

䡲 APPENDIX 2-2
General Guidelines for Safe Work Practices, Personal Protective Equipment, and
Engineering Controls (Continued)
The Occupational Safety and Health Administration (OSHA) does not require the routine use of gloves by phle-
botomists working with healthy prescreened donors or the changing of unsoiled gloves between donors if
gloves are worn.1,2 Experience has shown that the phlebotomy process is low risk because donors typically
have low rates of infectious disease markers. Also, exposure to blood is rare during routine phlebotomy, and
other alternatives can be used to provide barrier protection, such as using a folded gauze pad to control any
blood flow when the needle is removed from the donor’s arm.
Employers whose policies and procedures do not require routine gloving should periodically reevaluate the
potential need for gloves. Employees should never be discouraged from using gloves, and gloves should
always be available.

GUIDELINES FOR USE

Guidelines for the safe use of gloves by employees include the following3,4:
䡲 Securely bandage or cover open skin lesions on hands and arms before putting on gloves.
䡲 Change gloves immediately if they are torn, punctured, or contaminated; after handling high-risk samples; or
after performing a physical examination (eg, on an apheresis donor).
䡲 Remove gloves by keeping their outside surfaces in contact only with outside and by turning the glove inside
out while taking it off.
䡲 Use gloves only when needed, and avoid touching clean surfaces such as telephones, doorknobs, or com-
puter terminals with gloves.
䡲 Change gloves between patient contacts. Unsoiled gloves need not be changed between donors.
䡲 Wash hands with soap or other suitable disinfectant after removing gloves.
䡲 Do not wash or disinfect surgical or examination gloves for reuse. Washing with surfactants may cause
“wicking” (ie, enhanced penetration of liquids through undetected holes in the glove). Disinfecting agents
may cause deterioration of gloves.
䡲 Use only water-based hand lotions with gloves, if needed; oil-based products cause minute cracks in latex.

FACE SHIELDS, MASKS, AND SAFETY GOGGLES

Where there is a risk of blood or chemical splashes, the eyes and mucous membranes of the mouth and nose
should be protected.5 Permanent shields fixed as a part of equipment or bench design are preferred (eg,
splash barriers attached to tubing sealers or centrifuge cabinets). All barriers should be cleaned and disin-
fected on a regular basis.
Safety glasses alone provide impact protection from projectiles but do not adequately protect eyes from bio-
hazardous or chemical splashes. Full-face shields or masks and safety goggles are recommended when per-
manent shields cannot be used. Many designs are commercially available; eliciting staff input on comfort and
selection can increase use.
Masks should be worn whenever there is danger from inhalation. Simple, disposable dust masks are adequate
for handling dry chemicals, but respirators with organic vapor filters are preferred for areas where noxious
fumes are produced (eg, for cleaning up spills of noxious materials). Respirators should be fitted to their
wearers and checked annually.
(Continued)
72 䡲 AABB TECHNICAL MANUAL

䡲 APPENDIX 2-2
General Guidelines for Safe Work Practices, Personal Protective Equipment, and
Engineering Controls (Continued)
HAND WASHING

Frequent, thorough hand washing is the first line of defense in infection control. Blood-borne pathogens gen-
erally do not penetrate intact skin, so immediate removal reduces the likelihood of transfer to a mucous mem-
brane or broken skin area or of transmission to others. Thorough washing of hands (and arms) also reduces
the risks from exposure to hazardous chemicals and radioactive materials.
Employees should always wash their hands before leaving a restricted work area or using a biosafety cabinet,
between medical examinations, immediately after becoming soiled with blood or hazardous materials, after
removing gloves, or after using the toilet. Washing hands thoroughly before touching contact lenses or apply-
ing cosmetics is essential.
OSHA allows the use of waterless antiseptic solutions for hand washing as an interim method.2 These solu-
tions are useful for mobile donor collections or in areas where water is not readily available for cleanup pur-
poses. If such methods are used, however, hands must be washed with soap and running water as soon as
possible thereafter. Because there is no listing or registration of acceptable hand-wipe products similar to the
one that the Environmental Protection Agency maintains for surface disinfectants, consumers should request
data from the manufacturer to support advertising claims.

EYEWASHES

Laboratory areas that contain hazardous chemicals must be equipped with eyewash stations.3,6 Unobstructed
access within a 1- second walk from the location of chemical use must be provided for these stations. Eye-
washes must operate so that both of the user’s hands are free to hold open the eyes. Procedures and indica-
tions for use must be posted, and routine function checks must be performed. Testing eyewash fountains
weekly helps ensure proper function and flushes out stagnant water. Portable eyewash systems are allowed
only if they can deliver flushing fluid to the eyes at a rate of at least 1.5 liters per minute for 15 minutes. They
should be monitored routinely to ensure the purity of their contents.
Employees should be trained in the proper use of eyewash devices, although prevention—through consistent
and appropriate use of safety glasses or shields—is preferred. If a splash occurs, the employee should be
directed to keep his or her eyelids open and to use the eyewash according to procedures, or the employee
should go to the nearest sink and direct a steady, tepid stream of water into his or her eyes. Solutions other
than water should be used only in accordance with a physician’s direction.
After eyes are adequately flushed (many facilities recommend 15 minutes), follow-up medical care should be
sought, especially if pain or redness develops. Whether washing the eyes is effective in preventing infection
has not been demonstrated, but it is considered desirable when accidents occur.
1. Code of federal regulations. Title 29, CFR Part 1910.1030. Fed Regist 1991;56:64175-82.
2. Occupational Safety and Health Administration. Enforcement procedures for the occupational exposure to bloodborne pathogens.
OSHA Instruction CPL 02-02-069. Washington, DC: US Government Printing Office, 2001.
3. Clinical laboratory safety: Approved guideline. 3rd ed (GP17-A3). Wayne, PA: Clinical and Laboratory Standards Institute, 2012.
4. Medical glove powder report. (September 1997) Rockville, MD: Food and Drug Administration, 2009. [Available at http://www.fda.
gov/MedicalDevices/DeviceRegulationand Guidance/GuidanceDocuments/ucm113316.htm (accessed January 6, 2013).]
5. Inspection checklist: General laboratory. Chicago: College of American Pathologists, 2012.
6. American national standards for emergency eyewash and shower equipment. ANSI Z358.1-2009. New York: American National Stan-
dards Institute, 2009.
 CHAPTER 2 Facilities, Work Environment, and Safety 䡲 73

䡲 APPENDIX 2-3
Biosafety Level 2 Precautions
Biosafety Level 2 precautions as applied in the blood establishment setting include at least the following1,2:
䡲 High-risk activities are appropriately segregated from lower-risk activities, and the boundaries are clearly
defined.
䡲 Bench tops are easily cleaned and are decontaminated daily with a hospital disinfectant approved by the Envi-
ronmental Protection Agency.
䡲 Laboratory rooms have closable doors and sinks. An air system with no recirculation is preferred but not
required.
䡲 Workers are required to perform procedures that create aerosols (eg, opening evacuated tubes, centrifuging,
mixing, or sonicating) in a biological safety cabinet or equivalent or to wear masks and goggles in addition to
gloves and gowns during such procedures. (Note: Open tubes of blood should not be centrifuged. If whole
units of blood or plasma are centrifuged, overwrapping is recommended to contain leaks.)
䡲 Gowns and gloves are used routinely and in accordance with general safety guidelines. Face shields or their
equivalents are used where there is a risk from splashing.
䡲 Mouth pipetting is prohibited.
䡲 No eating, drinking, smoking, applying cosmetics, or manipulating contact lenses occurs in the work area. All
food and drink are stored outside the restricted area, and laboratory glassware is never used for food or
drink. Personnel are instructed to avoid touching their face, ears, mouth, eyes, or nose with their hands or
other objects, such as pencils and telephones.
䡲 Needles and syringes are used and disposed of in a safe manner. Needles must never be bent, broken,
sheared, replaced in a sheath, or detached from a syringe before being placed in puncture-proof, leakproof
containers for controlled disposal. Procedures are designed to minimize exposure to sharp objects.
䡲 All blood specimens are placed in well-constructed containers with secure lids to prevent leaking during
transport. Blood is packaged for shipment in accordance with regulatory agency requirements for etiologic
agents or clinical specimens, as appropriate.
䡲 Infectious waste is not compacted and is decontaminated before its disposal in leakproof containers. Proper
packaging includes double, seamless, tear-resistant, orange or red bags that are enclosed in protective car-
tons. Both the cartons and the bags inside display the biohazard symbol. Throughout delivery to an incinera-
tor or autoclave, waste is handled only by suitably trained persons. If a waste management contractor is
used, the agreement should clearly define the respective responsibilities of the staff and the contractor.
䡲 Equipment to be repaired or submitted for preventive maintenance, if potentially contaminated with blood,
must be decontaminated before its release to a repair technician.
䡲 Accidental exposure to suspected or actual hazardous material is reported to the laboratory director or
responsible person immediately.
1. Clinical laboratory safety: Approved guideline. 3rd ed (GP17-A3). Wayne, PA: Clinical and Laboratory Standards Institute, 2012.
2. Fleming DO. Laboratory biosafety practices. In: Fleming DO, Richardson JH, Tulis JJ, Vesley DD, eds. Laboratory safety, principles,
and practices. 2nd ed. Washington, DC: American Society for Microbiology Press, 1995:203-18.
74 䡲 AABB TECHNICAL MANUAL

䡲 APPENDIX 2-4
Sample List of Hazardous Chemicals that May Be Encountered in a Blood Bank
Chemical Hazard

Ammonium chloride Irritant

Bromelin Irritant, sensitizer
Calcium chloride Irritant
Carbon dioxide, frozen (dry ice) Corrosive
Carbonyl iron powder Oxidizer
Chloroform Toxic, suspected carcinogen
Chloroquine Irritant, corrosive
Chromium-111 chloride hexahydrate Toxic, irritant, sensitizer
Citric acid Irritant
Copper sulfate (cupric sulfate) Toxic, irritant
Dichloromethane Toxic, irritant
Digitonin Toxic
Dimethyl sulfoxide Irritant
Dry ice (carbon dioxide, frozen) Corrosive
Ethidium bromide Carcinogen, irritant
Ethylenediaminetetraacetic acid Irritant
Ethyl ether Highly flammable and explosive, toxic, irritant
Ficin (powder) Irritant, sensitizer
Formaldehyde solution (34.9%) Suspected carcinogen, combustible, toxic
Glycerol Irritant
Hydrochloric acid Highly toxic, corrosive
Imidazole Irritant
Isopropyl (rubbing) alcohol Flammable, irritant
Liquid nitrogen Corrosive
Lyphogel Corrosive
2-Mercaptoethanol Toxic, stench
Mercury Toxic
Mineral oil Irritant, carcinogen, combustible
Papain Irritant, sensitizer
Polybrene Toxic
Sodium azide Toxic, irritant, explosive when heated
Sodium ethylmercurithiosalicylate (thimerosal) Highly toxic, irritant
 CHAPTER 2 Facilities, Work Environment, and Safety 䡲 75

䡲 APPENDIX 2-4
Sample List of Hazardous Chemicals that May Be Encountered in a Blood Bank
(Continued)
Chemical Hazard

Sodium hydrosulfite Toxic, irritant

Sodium hydroxide Corrosive, toxic
Sodium hypochlorite (bleach) Corrosive
Sodium phosphate Irritant, hygroscopic
Sulfosalicylic acid Toxic, corrosive
Trichloroacetic acid Corrosive, toxic
Trypsin Irritant, sensitizer
Xylene Highly flammable, toxic, irritant
76 䡲 AABB TECHNICAL MANUAL

䡲 APPENDIX 2-5
Chemical Categories and How to Work Safely with Them
Chemical Category Hazard Precautions Special Treatment

Acids, alkalis, and corro- Irritation, severe burns, During transport, protect Store concentrated acids
sive compounds tissue damage large containers with in acid safety cabinets.
plastic or rubber bucket Limit volumes of con-
carriers. centrated acids to 1
During pouring, wear eye liter per container.
protection and chemi- Post cautions for materi-
cal-resistant-rated als in the area.
gloves and gowns as Report changes in
recommended. appearance (perchloric
Always add acid to water, acid may be explosive if
never add water to acid. it becomes yellowish or
When working with large brown) to chemical
jugs, have one hand on safety officer.
the neck and the other
hand at the base, and
position them away
from the face.
Acrylamide Neurotoxic, carcino- Wear chemically rated Store in a chemical cabi-
genic, absorbed gloves. net.
through the skin Wash hands immediately
after exposure.
Compressed gases Explosive Label contents. Transport using hand
Leave valve safety covers trucks or dollies.
on until use. Place cylinders in a stand
Open valves slowly for or secure them to pre-
use. vent tipping over.
Label empty tanks. Store in well-ventilated
separate rooms.
Do not store oxygen
close to combustible
gas or solvents.
Check connections for
leaks using soapy
water.
 CHAPTER 2 Facilities, Work Environment, and Safety 䡲 77

䡲 APPENDIX 2-5
Chemical Categories and How to Work Safely with Them (Continued)
Chemical Category Hazard Precautions Special Treatment

Flammable solvents Classified according to Use extreme caution Make every attempt to
flash point—see mate- when handling. replace hazardous
rial safety data sheet, Post “No Smoking” signs materials with less haz-
classified according to in working area. ardous materials
volatility Keep a fire extinguisher Store containers larger
and solvent cleanup kit than 1 gallon in a flam-
in the room. mable solvent storage
Pour volatile solvents room or a fire safety
under a suitable hood. cabinet.
Use eye protection and Ground metal containers
chemical-resistant neo- by connecting the can
prene gloves when to a water pipe or
pouring. ground connection. If
No flame or other source the recipient container
of possible ignition is also metal, it should
should be in or near be electrically con-
areas where flammable nected to the delivery
solvents are being container during pour-
poured. ing.
Label as “flammable.”
Liquid nitrogen Freeze injury, severe Use heavy insulated The tanks should be
burns to skin or eyes gloves and goggles securely supported to
when working with liq- avoid being tipped
uid nitrogen. over.
The final container of liq-
uid nitrogen (freezing
unit) must be securely
supported to avoid
being tipped over.
78 䡲 AABB TECHNICAL MANUAL

䡲 APPENDIX 2-6
Incidental Spill Response*
Chemicals Hazards PPE Control Materials

Acids If inhaled, causes severe Acid-resistant gloves Acid neutralizers or

Acetic irritation. Apron and coveralls absorbent material
Hydrochloric Contact causes burns to Goggles and face shield Absorbent boom
Nitric skin and eyes. Acid-resistant foot covers Leakproof containers
Perchloric Spills are corrosive. Absorbent pillow
Sulfuric Fire or contact with metal Mat (cover drain)
Photographic chemi- may produce irritating Shovel or paddle
cals (acidic) or poisonous gas.
Nitric, perchloric, and
sulfuric acids are
water-reactive oxidiz-
ers.
Bases and caustics Spills are corrosive. Gloves, impervious apron Base control/neutralizer
Potassium hydroxide Fire may produce irritat- or coveralls Absorbent pillow
Sodium hydroxide ing or poisonous gas. Goggles or face shield, Absorbent boom
Photographic chemicals impervious foot covers Drain mat
(basic) Leakproof container
Shovel or paddle
Chlorine Inhalation can cause Gloves (double set of 4H Chlorine control powder
Bleach respiratory irritation. undergloves and butyl Absorbent pillow
Sodium hypochlorite Liquid contact can pro- or nitrile overgloves), Absorbent material
duce irritation of the impervious apron or Absorbent boom
eyes or skin. coveralls Drain mat
Toxicity is caused by Goggles or face shield Vapor barrier
alkalinity, possible Impervious foot covers Leakproof container
chlorine gas genera- (neoprene boots for Shovel or paddle
tion, and oxidant prop- emergency response
erties. releases)
Self-contained breathing
apparatus (emergency
response releases)
Cryogenic gases Contact with liquid nitro- Full face shield or gog- Hand truck (to transport
Carbon dioxide gen can produce frost- gles, neoprene boots, cylinder outdoors if
Nitrous oxide bite. gloves (insulated to necessary)
Liquid nitrogen Release can create an provide protection from Soap solution (to check
oxygen-deficient atmo- the cold) for leaks)
sphere. Putty (to stop minor pipe
Nitrous oxide has anes- and line leaks)
thetic effects.
 CHAPTER 2 Facilities, Work Environment, and Safety 䡲 79

䡲 APPENDIX 2-6
Incidental Spill Response* (Continued)
Chemicals Hazards PPE Control Materials

Flammable gases Simple asphyxiant Face shield and goggles, Hand truck (to transport
Acetylene (displaces air). neoprene boots, double cylinder outdoors if
Oxygen gases Inhaled vapors have an set of gloves, coveralls needed)
Butane anesthetic potential. with hood and feet Soap solution (to check
Propane Flammable gases pose an for leaks)
extreme fire and explo-
sion hazard.
Release can create an
oxygen-deficient
atmosphere.
Flammable liquids Vapors are harmful if Gloves (double set of 4H Absorbent material
Acetone inhaled (central ner- undergloves and butyl Absorbent boom
Xylene vous system depres- or nitrile overgloves), Absorbent pillow
Methyl alcohol toluene sants). impervious apron or Shovel or paddle (non-
Ethyl alcohol Liquid is harmful if coveralls, goggles or metal, nonsparking)
Other alcohols absorbed through the face shield, impervious Drain mat
skin. foot covers Leakproof container
Substances are extremely
flammable.
Liquid evaporates to form
flammable vapors.
Formaldehyde and Vapors are harmful if Gloves (double set of Aldehyde neutralizer or
glutaraldehyde inhaled; liquids are 4H undergloves and absorbent
4% formaldehyde harmful if absorbed butyl or nitrile over- Absorbent boom
37% formaldehyde through skin. gloves), impervious Absorbent pillow
10% formalin Substances are irritants apron or coveralls, Shovel or paddle
2% glutaraldehyde to skin, eyes, and goggles, impervious (nonsparking)
respiratory tract. foot covers Drain mat
Formaldehyde is a sus- Leakproof container
pected human carcino-
gen.
37% formaldehyde
should be kept away
from heat, sparks, and
flames.

(Continued)
80 䡲 AABB TECHNICAL MANUAL

䡲 APPENDIX 2-6
Incidental Spill Response* (Continued)
Chemicals Hazards PPE Control Materials

Mercury Mercury and mercury Gloves (double set of 4H Mercury vacuum or spill
Cantor tubes vapors are rapidly undergloves and butyl kit
Thermometers absorbed in respira- or nitrile overgloves), Scoop
Barometers tory tract, gastrointesti- impervious apron or Aspirator
Sphygmomanometers nal (GI) tract, or skin. coveralls, goggles, Hazardous waste contain-
Mercuric chloride Short-term exposure may impervious foot covers ers
cause erosion of respi- Mercury indicator powder
ratory or GI tracts, nau- Absorbent material
sea, vomiting, bloody Spatula
diarrhea, shock, head- Disposable towels
ache, or metallic taste. Sponge with amalgam
Inhalation of high con- Vapor suppressor
centrations can cause
pneumonitis, chest
pain, dyspnea, cough-
ing, stomatitis, gingivi-
tis, and salivation.
Avoid evaporation of
mercury from tiny
globules by quick and
thorough cleaning.
*This list of physical and health hazards in not intended as a substitute for the material safety data sheet (MSDS) informa-
tion. In case of a spill or if any questions arise, always refer to the chemical-specific MSDS for more complete information.
 CHAPTER 2
䡲 APPENDIX 2-7
Managing Hazardous Chemical Spills
Actions
Deenergize.
Isolate, evacuate, and secure
the area.
Have the appropriate per-
sonal protective equipment
(PPE).
Contain the spill.
Confine the spill.
Neutralize the spill
Clean up the spill.
Facilities, Work Environment, and Safety 䡲 81
Instructions for Hazardous Liquids, Gases, and Mercury
Liquids: For 37% formaldehyde, deenergize and remove all sources of igni-
tion within 10 feet of spilled hazardous material. For flammable liquids,
remove all sources of ignition.
Gases: Remove all sources of heat and ignition within 50 feet for flammable
gases.
Remove all sources of heat and ignition for nitrous oxide release.
Isolate the spill area and evacuate everyone from the area surrounding the
spill except those responsible for cleaning up the spill. (For mercury, evacuate
within 10 feet for small spills or 20 feet for large spills.) Secure the area.
See Appendix 2-2 for recommended PPE.
Liquids or mercury: Stop the source of spill if possible.
Gases: Assess the scene; consider the circumstances of the release (quantity,
location, and ventilation). If circumstances indicate that it is an emergency
response release, make appropriate notifications; if the release is determined
to be incidental, contact the supplier for assistance.
Liquids: Confine the spill to the initial spill area using appropriate control
equipment and material. For flammable liquids, dike off all drains.
Gases: Follow the supplier’s suggestions or request outside assistance.
Mercury: Use appropriate materials to confine the spill (see Appendix 2-6).
Expel mercury from the aspirator bulb into a leakproof container, if applicable.
Liquids: Apply appropriate control materials to neutralize the chemical (see
Appendix 2-6).
Mercury: Use a mercury spill kit if needed.
Liquids: Scoop up solidified materials, booms, pillows, and any other materi-
als. Put used materials into a leakproof container. Label the container with the
name of the hazardous materials. Wipe up residual material. Wipe the spill
area surface three times with a detergent solution. Rinse the areas with clean
water. Collect the supplies used (eg, goggles or shovels) and remove gross
contamination; place equipment to be washed and decontaminated into a
separate container.
Gases: Follow the supplier’s suggestions or request outside assistance.
Mercury: Vacuum up the spill using a mercury vacuum, or scoop up mercury
paste after neutralization and collect the paste in a designated container. Use
a sponge and detergent to wipe and clean the spill surface three times to
remove absorbent. Collect all contaminated disposal equipment and put it
into a hazardous waste container. Collect supplies and remove gross contam-
ination; place equipment that will be thoroughly washed and decontaminated
into a separate container.
(Continued)
82 䡲 AABB TECHNICAL MANUAL

䡲 APPENDIX 2-7
Managing Hazardous Chemical Spills (Continued)
Actions Instructions for Hazardous Liquids, Gases, and Mercury

Dispose. Liquids: Dispose of material that was neutralized as solid waste. Follow the
facility’s procedures for disposal. For flammable liquids, check with the facil-
ity safety officer for appropriate waste determination.
Gases: The manufacturer or supplier will instruct the facility about disposal if
applicable.
Mercury: Label with appropriate hazardous waste label and Department of
Transportation diamond label.
Report. Follow appropriate spill documentation and reporting procedures. Investigate
the spill; perform a root cause analysis if needed. Act on opportunities for
improving safety.
 C h a p t e r
Regulatory Issues in Blood Banking
Glenn Ramsey, MD
I N T H E U N I T E D S T A T E S , when fed-
I eral laws are enacted, they are published
chronologically as statutes and placed into the
appropriate 1 of 50 subject areas (titles) of the
United States Code (USC).1 The government
agency responsible for the law—for blood-
related laws, the Food and Drug Administra-
tion (FDA)—then writes regulations (rules) to
enforce the law. Proposed rules for public
comment and final rules, along with back-
ground and interpretive information, are pub-
lished chronologically in the daily Federal Reg-
ister.2 The current edition of all rules is collated
annually in the appropriate title of the Code of
Federal Regulations (CFR); Title 21 addresses
food and drugs, and Title 42 focuses on health
care.3
Some rules have associated guidance
documents to provide current thinking about
regulatory topics. Guidance documents con-
tain recommendations, not requirements, but
they are usually followed by industry. Memo-
randa to blood establishments were issued be-
fore 1998, but some are still active references.
These publications can be accessed from the
FDA blood and blood products webpage.4
Glenn Ramsey, MD, Medical Director, Blood Banks, Northwestern Memorial Hospital and Ann & Robert H.
Lurie Children’s Hospital of Chicago, and Department of Pathology, Feinberg School of Medicine, Northwest-
ern University, Chicago, Illinois
The author has disclosed no conflicts of interest.
3
The FDA regulates drugs and medical de-
vices under the Food, Drug, and Cosmetic Act
(USC Title 21, Sections 301-399d). 5 Blood
products are defined as drugs because they are
intended to cure, mitigate, treat, or prevent
disease (21 USC 321).6,7 The law requires blood
product manufacturers to register with the
FDA, obtain biologics licenses, and follow cur-
rent good manufacturing practice (cGMP) reg-
ulations. It also prohibits adulteration and
misbranding, authorizes inspections, and de-
fines civil and criminal penalties for violations.
The act controls the use of unapproved drugs
and devices during their investigational phas-
es and public health emergencies.
Medical devices include instruments, in-
vitro reagents, and their parts and accessories
for the diagnosis and treatment of disease. The
FDA classifies devices as Class I, II, or III, in as-
cending order of associated risk [21 USC
360(c)].8,9 Class I devices have no potential un-
reasonable risk. Class II devices must meet
performance standards beyond basic FDA reg-
ulations. Some Class I devices and most Class
II devices must be cleared by the FDA based on
substantial equivalence with another device
83
84 䡲 AABB TECHNICAL MANUAL
already on the market. This process is called
510(k) clearance, referring to the applicable
section of the act describing the submission of
applications to the FDA for such devices [21
USC 360(k)].8 Class III devices pose potential
unreasonable risk and require specific pre-
market approval from the FDA, as do unprece-
dented new devices before their class is deter-
mined.
B IOLO G IC A L P RO DU C TS
Biological products come from living sources
and include blood, blood components, deriva-
tives, therapeutic serum, and vaccines appli-
cable to the prevention or treatment of dis-
ease. They are regulated by Section 351 (42
USC 262) of the Public Health Service Act,
which addresses licensure, labeling, inspec-
tions, recalls, and penalties for violations in
producing biological drugs. This set of regula-
tions provides the core of federal law that is
most specific to blood products.10 Section 361,
Regulations to Control Communicable Diseas-
es (42 USC 264), grants the Surgeon General
inspection and quarantine powers to prevent
the spread of infectious diseases and is in-
voked in the regulation of tissues (see below).11
The FDA’s key regulations enforcing the
Food and Drug Act and the Public Health Ser-
vice Act are in Title 21 of the CFR, Food and
Drugs, and in particular, Parts 200 through 299
for drugs, 600 through 680 for biologicals, 800
through 898 for devices, and 1270 through
1271 for tissues (Table 3-1).12 The FDA website
has links to pertinent laws in the USC and reg-
ulations in the CFR.13
Within the FDA, the Center for Biologics
Evaluation and Research (CBER) regulates
blood products and most other biological
therapies.14 The Center for Devices and Radio-
logical Health (CDRH) regulates most medical
devices, but CBER retains primary jurisdiction
over medical devices used with blood and cel-
lular products.15 The FDA’s Office of Regulatory
Affairs (ORA) has responsibility for all field op-
erations, which include inspections and inves-
tigations.
As part of the development process for
FDA regulations and guidance documents,
several forums are offered for input from the
public and regulated groups.16 Proposed rules
and draft guidance documents are published
with an invitation for written comments,
which are filed in public dockets.17 When final
rules are published in the Federal Register, the
accompanying commentaries offer response
to key questions. The FDA also receives peti-
tions to write or change regulations. Expert
opinions on current issues are sought from
several advisory committees, including the
FDA’s committees on blood products and on
cellular, tissue, and gene therapies, and the
Department of Health and Human Services
(DHHS) Committee on Blood Safety and Avail-
ability. Public meetings hosted by the FDA on
selected topics provide an additional opportu-
nity for input.
Facilities may apply to CBER for approval
of exceptions to the regulations for blood
products or alternative procedures. These ap-
provals are periodically published, although
the circumstances for these approvals may not
apply to other facilities.18
LICENSURE AND
REGISTRATION
Blood establishments are classified into three
categories by the FDA: 1) licensure and regis-
tration for interstate commerce, 2) registration
for manufacturers not involved in interstate
commerce, and 3) exemption from registration
for transfusion services as described in the
regulations.19 Licensed and/or registered facil-
ities are inspected by the FDA. For transfusion
services that perform minimal manufacturing
and are certified for reimbursement by the
Centers for Medicare and Medicaid Services
(CMS), the FDA accepts CMS-sanctioned labo-
ratory inspections and approval.20 However,
the FDA still has jurisdiction and may conduct
its own inspections if warranted. All military
blood facilities, including transfusion services,
register with and are inspected by the FDA.21
Facilities use the Biologics License Appli-
cation (BLA, Form 356h) to apply for their
establishment and product licenses for
interstate commerce. 22 Subsequent license
amendments are based on the potential for
 CHAPTER 3 Regulatory Issues In Blood Banking 䡲 85

TABLE 3-1. Regulations of Interest in Title 21 of the CFR (Food and Drugs)

Topic Section Topic Section

FDA general Donor notification 630.6

Enforcement 1-19 Blood product 640
standards
Research and 50-58 Donor eligibility 640.3
development
Labeling 201 Blood collection 640.4
cGMP for drugs 210-211 Blood testing 640.5
Biological products 600-680 Red Blood Cells 640.10-.17
General 600 Platelets 640.20-.27
Licensing 601 Plasma 640.30-.34
cGMP for blood 606 Cryoprecipitated AHF 640.50-.56
components
Personnel, 606.20-.65 Exceptions, alternatives 640.120
resources
Standard operating Medical devices 800-898
procedures 606.100

Labeling 606.120-.122 Adverse events 803

Compatibility testing 606.151 Hematology and 864
pathology
Records 606.160-.165 Tissues
Adverse reactions 606.170 Tissues for transplan- 1270
tation
Product deviations 606.171 Cellular and tissue-based 1271*
products
Establishment 607 General provisions 1271.1-.20
registration
General standards 610 Registration and 1271.21-.37
products
Donor testing 610.40 Donor eligibility 1271.45-.90
Donor deferral 610.41 cGTP 1271.145-.320
Look-back 610.46-.48 Section 361 provi- 1271.330-.440
sions†
Dating periods 610.53
*The following citations represent Subparts A, B, C, D, and E-F, respectively.
†
Autologous and related donors (see text).
CFR = Code of Federal Regulations, FDA = Food and Drug Administration, cGMP = current good manufacturing practice;
cGTP = current good tissue practice.
86 䡲 AABB TECHNICAL MANUAL
adverse effects of the changes.23 Unlicensed
blood products, whether manufactured by a li-
censed or an unlicensed facility, may be
shipped across state lines for a medical emer-
gency if necessary, but this shipping should be
unscheduled and infrequent. 24 The shipping
facility must retain documentation of the
emergency for FDA review.
Facilities must register annually with the
FDA if they collect, manufacture, prepare, or
process blood products (Form FDA 2830).25,26
Transfusion services are exempt from this re-
quirement if they are approved for reimburse-
ment by CMS and their preparation activities
are basic, such as preparing Red Blood Cells
from whole blood, converting unused plasma
to recovered plasma, pooling components, re-
ducing leukocytes with bedside filters, or col-
lecting blood only in emergencies. However,
facilities that routinely collect blood (includ-
ing autologous units) or perform such proce-
dures as irradiation, washing, laboratory leu-
kocyte reduction, freezing, deglycerolization,
and rejuvenation must register as community
or hospital blood banks. Transfusion services
acting as depots for forwarding products to
other hospitals must register as distribution
centers. Clinical laboratories performing in-
fectious disease testing required for blood do-
nors must register unless they are CMS ap-
proved. If blood irradiation is performed
outside the blood bank or transfusion service,
such as in a nuclear medicine department,
that facility must register as well.
F D A I NS PE C T I O NS
New facilities are generally inspected within 1
year of establishment by a team from CBER
and ORA. Subsequent routine inspections are
generally performed by ORA every 2 years or
earlier depending on the facility’s compliance
history.
ORA publishes online manuals and in-
structions for FDA investigators for inspec-
tions in general and for inspections of licensed
and unlicensed blood banks in particular.25,26
The blueprints for blood bank inspections are
the general regulations for cGMP and drugs
(21 CFR 210-211) and the specific require-
ments for blood components (21 CFR 606).27
Routine inspections address the FDA’s five re-
quired layers of blood safety—donor screen-
ing, donor testing, product testing, quarantin-
ing, and monitor ing and investigating
problems. Investigators review the following
operational systems that create these safety
layers: quality assurance, donor eligibility,
product testing, quarantine/inventory man-
agement, and production and processing.
Full inspections of all systems are desig-
nated Level I. After a favorable inspection pro-
file, facilities with four or five systems some-
times have streamlined Level II inspections of
three systems. (Focused inspections for prob-
lems or fatalities need not follow these for-
mats.) Within each system, the investigators
review standard operating procedures, per-
sonnel and training, facilities, equipment cali-
bration and maintenance, and records. Specif-
ic requirements for individual systems and
processes are discussed in detail in their re-
spective chapters of this book.
If the investigator judges that significant
objectionable practices, violations, or condi-
tions are present under which a drug or device
is or could be adulterated or injurious to
health, these observations are written and pre-
sented to the facility (Form 483). Investigators
are instructed to seek and record the manage-
ment’s intentions to make corrections.25 How-
ever, most facilities also respond in writing im-
mediately with questions or corrective actions.
Final determination that a violation has oc-
curred is made by ORA.
The FDA can take a number of steps in re-
sponse to a violation, including no action at
all. For significant violations, the FDA may is-
sue a warning letter, which is an advisory ac-
tion to provide the facility with the opportuni-
ty for voluntary compliance. Enforcement
actions are categorized as administrative, judi-
cial, or recall. Administrative actions include
formal citations of violation and—for licensed
facilities—suspension or revocation of a li-
cense. Judicial actions range from seizures of
products to court injunctions, civil monetary
penalties, and criminal prosecution. The FDA
may also conduct recalls if necessary. ORA’s
 CHAPTER 3 Regulatory Issues In Blood Banking
Regulatory Procedures Manual provides details
on available sanctions.28
BLOOD -RE L ATE D DEVICES
CBER has lead responsibility in collaboration
with CDRH for equipment marketed for trans-
fusion and the collection and processing of
blood products, hematopoietic stem cells, and
autotransfusions.29,30 These devices include di-
agnostic tests for infectious diseases in blood
donors and pretransfusion testing in patients.
Blood bank computer software programs are
CBER-approved devices.
Most blood-related devices are in Class II
and cleared by 510(k) equivalence. Examples
of Class I devices are copper sulfate solutions
for hemoglobin screening, blood grouping
view boxes, and heat sealers. Human immuno-
deficiency virus (HIV) and hepatitis B and C
virus donor tests are regulated as Class II or III
devices. Screening tests for blood donations
are regulated as BLAs. BLAs and Class III de-
vices require specific premarket approval.
Each category of device is assigned a code, and
all cleared or approved vendors and products
for that code are searchable in the Establish-
ment Registration and Device Listing data-
base on the CDRH website.31
Serious adverse events related to medical
devices must be reported (21 CFR 803).32 User
facilities must report deaths or serious inju-
ries in which a device was or may have been a
factor. Serious injury is defined as being life
threatening, causing permanent impairment
or damage, or needing medical or surgical in-
tervention. Reports of serious injuries are sent
to the device maker using FDA Medwatch
Form 3500A within 10 working days of the
event, and deaths must also be reported to the
FDA. In years when a Form 3500A report is
submitted, an annual summary must be sent
to the FDA by January 1 of the following year
(Form 3419). 22 Users may voluntarily report
other device-related adverse events to the FDA
(Form 3500). 22 All possible adverse events,
whether reported or not, must be investigated
and their records must be kept on file for
2 years.
䡲 87
HEMATO POIETIC PROGENITOR
C E LL S A S T I SS U E S
Unmanipulated marrow cell transplants are
regulated by the Health Resources and Servic-
es Administration of DHHS, which also over-
sees marrow and cord blood donations and
transplant procedures coordinated by the Na-
tional Marrow Donor Program. Other hemato-
poietic progenitor cells (HPCs) collected be-
fore May 25, 2005 are regulated by FDA rules
for tissue transplants in 21 CFR 1270.12. The
FDA’s regulations in 21 CFR 1271 for human
cells, tissues, and cellular and tissue-based
products (HCT/Ps) apply to HPCs collected on
or after May 25, 2005.12,33,34
HCT/P manufacturers must comply with
rules for 1) facility registration and product
listing; 2) donor eligibility, including testing for
communicable diseases; and 3) production ac-
cording to current good tissue practice (cGTP)
regulations (Table 3-1). Registrants using FDA
Form 3356 report each type of HPC they man-
ufacture in three categories: 1) HCT/Ps as de-
scribed in 21 CFR 1271.10(a), 2) biologics, and
3) drugs. Table 3-2 outlines how the FDA de-
fines, regulates, and (when applicable) ap-
proves each type of HPC. Basic peripheral
blood stem cells (PBSCs) from autologous or
first- or second-degree related donors are in
the first category; these products are regulated
mainly with regard to preventing transmission
of communicable diseases or contamination
(Public Health Service Act, Section 361, 42 USC
264).11 Typical PBSCs from unrelated donors
are biologics, and the FDA is considering li-
censure requirements for them. Since October
20, 2011, unrelated umbilical cord cells must
be licensed or used under an investigational
new drug application (see Chapter 30). Any
HPCs that are more than minimally manipu-
lated (eg, by expansion, property alteration, or
gene therapy) or used for a different function
(eg, tissue engineering) are categorized as a
drug and must have premarket approval.
The FDA specifies required infectious dis-
ease tests for tissue donors and posts lists of
tests that have been licensed or cleared for this
indication.45 The elements of cGTP are analo-
gous to those of cGMP for blood products. The
TABLE 3-2. US Regulations for Manufacturers of Hematopoietic Progenitor Cells35,36
88

Key Regulations (21 CFR FDA Premarket Licensure, FDA Compliance Program
Type of HPC Product Jurisdiction Category except as noted) Approval, or Clearance? Manual37,38
䡲

Unmanipulated marrow Health Resources and Ser- 42 US Code 274(k) No Not applicable
vices Administration
HPCs* collected before May 25, 2005 Tissues, 21 CFR 1270 (before 1270, 1271 Subparts A-B No 7341.002A39
May 25, 2005) (see Table 3-1)
Autologous or allogeneic related- PHS Act Section 361: HCT/Ps‡ 1271.10(a)§ (must meet all No 7341.00237
donor† HPCs criteria); 1271 A-F 7342.007 Addendum40
(imported products)
Unrelated-donor peripheral blood PHS Act Section 351: 1271 A-D Planned 7341.00237
HPCs Biologics
Unrelated-donor umbilical cord cells Section 351: Biologics 1271 A-D Yes (after October 20, 2011): 7341.00237
Licensure Guidance41 licensure or IND application for
IND Guidance42 nonqualifying products
HPCs with altered characteristics or Cellular and Gene Therapy, 1271 A-D Yes: IND and BLA 7345.84843
AABB TECHNICAL MANUAL

different function (including somatic CBER

cell therapy), or HPCs combined with
drug
HPCs combined with medical device CBER and CDRH, as deter- 1271 A-D Yes: investigational device 7382.84544
mined exemption and BLA
*From manipulated marrow, peripheral blood, and umbilical cord blood (includes donor lymphocyte infusions).
†
First- or second-degree relative.
‡
As defined by 2005 tissue regulations [21 CFR 1271.3(d)]
§
21 CFR 1271.10(a) as applied to Section 361 (see full rule for details) requires that HPCs be 1) minimally manipulated (biological characteristics unaltered), 2) for homologous use only
(similar function as original tissue), 3) not combined with another article (except water; crystalloids; or sterilizing, preserving, or storage agents with no new safety concerns), and 4) used
in autologous transfusion or in allogeneic transfusion of donations from a first- or second-degree relative.
HPC = hematopoietic progenitor cell, CFR = Code of Federal Regulations, FDA = Food and Drug Administration, PHS = Public Health Service, HCT/Ps = human cells, tissues, and cellular and
tissue-based products, IND = investigational new drug, CBER = Center for Biologics Evaluation and Research, CDRH = Center for Devices and Radiological Health, BLA = biologics license
application.
 CHAPTER 3 Regulatory Issues In Blood Banking
Circular of Information for the Use of Cellular
Therapy Products is jointly written by the
AABB and other organizations for users of
these products.46 The AABB and the Founda-
tion for Accreditation of Cellular Therapy set
voluntary standards and accredit facilities for
collection and processing of HPCs (see Chap-
ters 29 and 30 for information on these stan-
dards and accreditation procedures).
The FDA tissue regulations do not apply
to facilities that receive, store, and administer
tissues but do not perform any manufacturing
steps. However, The Joint Commission (TJC)
has hospital standards for handling and trac-
ing tissues and investigating adverse events
(TS.03.01.01 to 03.03.01) (see Chapter 32 for
information on these standards).47
M A N AG I N G R E C A L LS A ND
W IT H D R AWA L S
The FDA’s requirements for monitoring and
investigating problems with drugs extend to
the time after a product’s release. When blood
banks discover after distribution that a blood
product was in violation of rules, standards,
specifications, or cGMP regulations, they must
report the problem to the FDA and their con-
signee. These problems comprise a subset of
biological product deviations (BPDs)—events
in which the safety, purity, or potency of a dis-
tributed blood product may be affected—all of
which must be reported to the FDA.48
A recall is defined as the removal or cor-
rection of a marketed product that is in viola-
tion of the law (21 CFR 7.3).49 The FDA classi-
fies recalls by severity. Most blood component
recalls are in Class III, not likely to cause ad-
verse health consequences. Class II recalls are
for products that may cause temporary ad-
verse effects or remotely possible serious
problems. Class I recalls involve a reasonable
probability of serious or fatal adverse effects.
All recalls are published by the FDA and in-
volve about 1 in 5800 blood components is-
sued in the United States.50
Market withdrawals are required for
products found to be in minor violation of the
law. Problems beyond the control of the man-
ufacturer, such as postdonation donor infor-
䡲 89
mation, are often in this category. Withdrawals
are much more common than recalls but are
not published.
Recommendations for managing com-
mon notices have been published in the Unit-
ed States and Canada.50,51 Transfusion services
need to have procedures and training for rapid
responses as advised when recalls and with-
drawals are received along with records of ac-
tions, reviews, and follow-up actions, as indi-
cated. Accreditation requirements of the AABB
(Standard 7.1) and the College of American Pa-
thologists (CAP) (Checklists TRM.42120 and
TRM.42170) address aspects of this topic.52,53
Most of these blood components are
transfused before a notice is received of their
nonconformance. In some recent blood guid-
ance documents on infectious diseases, the
FDA has included recommendations on
whether or not to notify the recipient’s physi-
cian about transfused units. In cases of possi-
ble recent infectious disease exposure in
donors or transfusion recipients, the serocon-
version window periods for tests should be
consulted for scheduling prospective testing
or reviewing retrospective results, such as for a
donor who has been retested since an expo-
sure.50
The most common reasons for BPDs and
blood component recalls are given in Table 3-
3. Look-backs investigations on units from do-
nors found after donation to have HIV or hep-
atitis C virus are discussed in Chapter 5.
ME DI CAL L ABO RATO RY L AW S
AND REGUL ATIONS
CMS regulates all US medical laboratories un-
der the Clinical Laboratory Improvement
Amendments [CLIA, 42 USC 263(a) and 42 CFR
493] to Section 353 of the Public Health Service
Act.54,55
The law and regulations establish the re-
quirements and procedures for laboratories to
be certified under CLIA as both a general re-
quirement and a prerequisite for receiving
Medicare and Medicaid payments.
To be certified, laboratories must have
adequate facilities and equipment, superviso-
ry and technical personnel with training and
90 䡲 AABB TECHNICAL MANUAL
TABLE 3-3. Most Common Reasons for Biological Product Deviations and Blood Component
Recalls
Biological Product Deviations
(Ranked by number of events)
Malaria travel/residence
Variant Creutzfeldt-Jakob disease travel/residence
Postdonation illness
Tattoo
Donor deferral missed via incorrect donor
identification
Biological product deviations shown are from licensed establishments.48 Blood component recalls are compiled from Food
and Drug Administration Enforcement Reports.50 Market withdrawals are not included in public recalls data.50
experience appropriate to the complexity of
testing, a quality management system (see
Chapter 1), and successful ongoing perfor-
mance in CMS-approved proficiency testing
(PT).56 All laboratories must register with CMS,
submit to inspection by CMS or its designees,
and obtain recertification every 2 years.
All laboratory tests are rated for complexi-
ty by the FDA for CMS as waived, moderate
complexity, and high complexity. Waived tests
are simple and easily performed with limited
technical training. Examples include over-the-
counter tests, urinalysis dipsticks, copper sul-
fate specific-gravity hemoglobin screens, spun
microhematocrits, and some simple devices
for measuring hemoglobin. Laboratories that
perform only waived tests register with CMS
for a certificate of waiver. The Centers for Dis-
ease Control and Prevention provides techni-
cal and advisory support to CMS for laboratory
regulation and has published practice recom-
mendations for waived testing sites.57
Nonwaived tests are classified as being of
moderate or high complexity based on a scor-
ing system of needs for training, preparation,
interpretive judgment, and other factors (42
CFR 493.17).54 The CDRH website provides the
searchable CLIA database on the complexity
levels of specific tests.31
Blood banks and transfusion services
have three pathways to obtain a CLIA certifi-
Blood Component Recalls
(Ranked by number of units)
Collection sterility and arm preparation
Storage temperature
Production according to current good manufacturing
practice
Donor suitability
Product quality control
cate to perform testing: 1) certificate of com-
pliance: approval via state health department
inspections using CMS requirements; 2) certif-
icate of accreditation: approval via a CMS-
approved accrediting organization; and 3)
CMS-exempt status: licensure programs for
nonwaived laboratories in New York and
Washington States that are accepted by CMS.58
The CLIA regulations delineate general
requirements for facilities; quality systems, in-
cluding quality assurance and quality control
systems; and management and technical per-
sonnel qualifications. High-complexity tests
require more stringent personnel qualifica-
tions. Immunohematology laboratories have
standards for blood supply agreements, com-
patibility testing, blood storage and alarms,
sample retention, positive identification of
blood product recipients, investigation of
transfusion reactions, and documentation (42
CFR 493.1103 and 493.1271).54 Viral and syphi-
lis serologic tests are part of the immunology
requirements. CMS has published guidelines
for conducting surveys (inspections).59
CMS has approved six laboratory accredi-
tation organizations with requirements that
meet CMS regulations: the AABB, American
Osteopathic Association, American Society for
Histocompatibility and Immunogenetics
(ASHI), CAP, COLA (formerly the Commission
on Office Laboratory Accreditation), and The
 CHAPTER 3 Regulatory Issues In Blood Banking 䡲 91

Joint Commission. 60 The Joint Commission
has cooperative agreements with ASHI, CAP,
and COLA to accept their laboratory accredita-
tions in facility surveys.61
AABB and CAP can coordinate joint sur-
veys by facilities seeking both types of accredi-
tation. CMS may perform its own follow-up
surveys to validate those of the accreditation
organizations.
CMS requires successful PT for ongoing
laboratory certification of nonwaived testing.
Within each laboratory section, CMS regula-
tions specify tests and procedures (regulated
analytes) that must pass approved PT if the
laboratory performs them. The CMS website
has a list of approved PT providers.62 PT is dis-
cussed in Chapter 1.
HOS PITAL R EG UL ATI ONS AND
ACC R E D I TAT I O N
CMS approves hospitals for Medicare reim-
bursement through state surveys or accredita-
tion programs from The Joint Commission, the
American Osteopathic Association, and DNV
Healthcare. These inspections cover CMS
regulations for blood administration and the
evaluation of transfusion reactions [42 CFR
482.23(c)].63 The Joint Commission has stan-
dards for preventing misidentification of
laboratory specimens and transfusion recipi-
ents (NPSG.01.01.01, .01.03.01), checking
blood products in the Universal Protocol pre-
procedure verification process (“time-out”)
(UP.01.01.01), and assessing transfusion appro-
priateness (MS.05.01.01).47 The Joint Commis-
sion includes hemolytic transfusion reactions
in its Sentinel Events reporting program.64
Facilities also should be familiar with all
relevant state and local laws and regulations,
including professional licensure requirements
for medical and laboratory personnel. In some
situations, facilities providing products or ser-
vices in other states must comply with local
regulations in the customer’s location.
CO N CLUS IO N
Blood components and other blood products
are regulated as pharmaceutical agents, and
blood banks and transfusion services are also
regulated as medical laboratories. These many
requirements make compliance complex.
However, close regulation underscores the rec-
ognized importance to public health of safe,
effective blood products provided via good
laboratory practices using the quality manage-
ment systems described in Chapter 1 and the
procedures discussed throughout this manual.

KEY POINTS

1. The FDA regulates the manufacture and use of drugs and medical devices, including blood
components, blood-derived biological products, and their ancillary testing and transfusion
equipment. The FDA website provides links to blood-related regulations in Title 21 CFR and
to explanatory guidance documents.
2. The FDA requires licensure and registration for blood manufacturers engaged in interstate
commerce and registration alone for manufacturing activities, such as irradiation or wash-
ing. Transfusion services performing only basic component preparation need not register
but must have laboratory approval from CMS.
3. Licensed and registered blood facilities are inspected by the FDA every 2 years, with a focus
on current cGMP (21 CFR 210-211) and blood component requirements (21 CFR 606). Ob-
servations of significant noncompliance or hazards are reported to the facility for its re-
sponse and correction and may result in a spectrum of sanctions, possibly including license
revocation and criminal prosecution.
4. HPCs for transplantation are regulated as tissues by the FDA, with an emphasis on prevent-
ing transmissions of infections and contaminations in autologous or closely related alloge-
neic transplants. The FDA has a licensure process for unrelated umbilical cord blood HPCs.
92 䡲 AABB TECHNICAL MANUAL

5. The FDA requires drug (and blood) manufacturers to conduct recalls or market withdrawals
when noncompliance is found after products are issued, such as for donor malaria risk or
potentially compromised component sterility. Transfusion services should have procedures
for evaluating and managing the potential impact of these recalls and withdrawals on pa-
tients transfused with such products.
6. All US medical laboratories must be approved by CMS every 2 years. Laboratory regulations
emphasize the need for adequate facilities and qualified personnel commensurate with the
complexity of testing, and they require ongoing successful performance in proficiency test-
ing by CMS-approved vendors. Laboratory approval by CMS is granted via inspections per-
formed by CMS-approved accrediting agencies or state health departments.
7. Health-care facilities also have CMS regulations for their activities, and The Joint Commis-
sion and other organizations accredit many hospitals for CMS compliance. CMS and The
Joint Commission have requirements for monitoring transfusion practices, evaluating ad-
verse transfusion reactions, and preventing mistransfusions.

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 CHAPTER 3 Regulatory Issues In Blood Banking
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 C h a p t e r 4

Disaster Management

Ruth D. Sylvester, MS, MT(ASCP)SBB;

William FitzGerald, LTC USA (Ret);
Wendy Trivisonno; and Theresa Wiegmann, JD

M U C H H A S B E E N learned over the often overlooked in traditional disaster plans.

past decade about preparing for and re-
sponding to disasters. A disaster is defined by
the United Nations International Strategy for
Disaster Reduction as a serious event that dis-
rupts the normal functioning of a community
or society and that exceeds the affected area’s
ability to cope.1 Disasters can be small, only
affecting a single business, or large, encom-
passing an entire region. Whether a disaster
involves natural forces (eg, hurricanes, earth-
quakes, floods, or pandemic influenza) or is
the result of human activity (eg, terrorism or
industrial accidents), organizations around
the world have put forth tremendous resourc-
es to counteract and mitigate the threats relat-
ed to these events.
The goal of this chapter is to provide an
overview of disaster management, which en-
compasses much more than just preparing for a
disaster. This chapter addresses the entire cycle
of disaster management, including elements
This chapter is more strategic than procedural.
Many of the specific and tactical information
and procedures mentioned in this chapter are
described in detail in the AABB Disaster Opera-
tions Handbook, which should be considered a
complementary text to this chapter.2
Even though this chapter focuses primar-
ily on emergency management strategies used
in the United States, many of the methods can
also be adapted for use in other countries.
Hospital-based blood banks and transfusion
services should note that many of the activities
discussed in this chapter are intended for
stand-alone organizations (eg, blood collec-
tion centers) and may already be addressed in
a hospital’s overall disaster plan. However,
hospital staff members should review both
their hospital and departmental disaster plans
to ensure that blood-related issues (including
those pertaining to cellular and related biolog-
ic therapies) are sufficiently covered.

Ruth D Sylvester, MS, MT(ASCP)SBB, Director, Regulatory Services, America’s Blood Centers, Washington,
District of Columbia; William FitzGerald, LTC USA (Ret), Senior Advisor, Biomedical Preparedness, American
Red Cross, Washington, District of Columbia; Wendy Trivisonno, Director, Strategic Biologic Sourcing and
Logistics, Blood Centers of America, West Warwick, Rhode Island; and Theresa Wiegmann, JD, Director of
Public Policy, AABB, Bethesda, Maryland
The authors have disclosed no conflicts of interest.

97
98 䡲 AABB TECHNICAL MANUAL
BACKGROUND
Disaster Management Cycle
Disaster management includes plans for, re-
sponses to, and recovery from disasters. Plan-
ning by its nature is cyclic and, as with many
activities, future responses to disasters can be
improved by learning from past events. The di-
saster management cycle includes four func-
tional areas: mitigation, preparedness, re-
sponse, and recovery.3 Organizations should
consider each of these areas when creating an
effective disaster management program.
䡲 Mitigation efforts focus on making perma-
nent changes to plants and property to re-
duce the overall exposure to various known
hazards. Mitigation strategies typically in-
volve the “where” and “how” of building
physical structures and protecting employ-
ees and assets (eg, building code require-
ments, flood plain analysis, and storm shel-
ters). Mitigation techniques also involve
taking relatively minor steps to reduce loss
or injury (eg, fastening bookshelves or file
cabinets to a wall).
䡲 Preparedness focuses on areas of risk that
cannot be addressed through mitigation ef-
forts alone. Emergencies are dynamic and
complex events, and careful preparation ef-
forts are needed to reduce the loss of life and
property while sustaining business opera-
tions. Preparation efforts begin with a thor-
ough risk analysis of all known hazards (both
natural and human) that could affect an or-
ganization (eg, fires, severe storms, earth-
quakes, pandemics, and terrorism). Based
on the risk assessment, planning efforts
should focus on the people and organiza-
tional systems that are most likely to be af-
fected. Effective preparation involves a con-
tinual improvement process whereby the
disaster plan is routinely tested by a series of
real or simulated events (drills) through
which gaps are identified and remedied.
䡲 Response takes place during an emergency
and typically involves time-sensitive action
steps taken by the staff to protect life and
property and to stabilize the organization
(eg, communication with staff, evacuation
procedures, and responses to customers
and the media). Effective response strate-
gies center on clearly outlined processes or
procedures that can be practiced during
drills and be implemented at any time of
the day or night (eg, during night and week-
end shifts). These strategies are also based
on well-defined lines of authority.
䡲 Recovery efforts begin once the initial re-
sponse actions have taken place. These ef-
forts focus on restoring critical systems (eg,
communications, water, power, and sewage)
to resume and maintain business opera-
tions. Recovery efforts complete the cycle of
disaster management by providing insights
into additional mitigation strategies that
can be deployed for future emergencies.
Risk Assessment
The cornerstone of an effective disaster man-
agement program is a thorough assessment of
the known hazards that can affect the organi-
zation. Risk can be defined as the potential
losses (physical, economic, and social) associ-
ated with a hazard and can be described in
terms of expected probability and frequency,
causative factors, and locations or areas affect-
ed.4 The goal of risk assessment is to identify
and reduce the number of potential risks asso-
ciated with a hazard given its probability of oc-
currence and scope. Potential hazards can be
grouped in different ways, such as natural vs
man-made or internal vs external to an organi-
zation. Using the latter categorization, hazards
that are generated externally include natural
events, such as earthquakes, floods, wildfires,
pandemics, and hurricanes, as well as human
events, such as terrorism or industrial acci-
dents, including chemical, hazardous materi-
al, and nuclear power plant emergencies.
Another class of potential hazards to con-
sider involves events that may occur internal-
ly at the organization. Examples of internal
hazards include internal flooding caused by
burst water pipes or fire sprinkler malfunc-
tions, fires, natural gas leaks, workplace vio-
lence, and hazardous spills. Each of these haz-
ards may involve service disruptions and
evacuations of staff, donors, and patients. An
 CHAPTER 4
example of a question to answer in a risk as-
sessment of internal disasters is, what is the
potential risk to stored blood components and
patients if the hospital’s blood bank becomes
flooded or must be relocated because of
smoke damage from a fire?
There are many approaches to conduct-
ing an effective risk assessment. This text does
not provide guidance on a specific risk assess-
ment process. However, a sample risk assess-
ment chart is provided in Table 4-1, which
highlights the major functional areas and haz-
ards to consider. For instance, a facility located
in an area of seismic activity would list earth-
quakes as a potential external hazard and
would need to determine the immediate ef-
fect of an earthquake on its operational status
(to humans, property, and business) and the
resources needed to recover and restore oper-
ations. Conversely, an organization located far
from hurricane and flood zones would reflect
that situation in its risk assessment chart. The
AABB Disaster Operations Handbook contains
a list of common hazards, including impact
factors as well as preparedness and response
strategies.2
Emergency Operations in the United
States
It is important for staff members to under-
stand the relationships between emergency
TABLE 4-1. Risk Assessment Chart with Sample Data4
Probability of Human
Occurrence Effect
High Low High Low
5 1 5 1
External Hazards
Earthquake 5 3
Hurricane 1 1
Internal Hazards
Flooding 4 1
Workplace violence 2 5
Disaster Management 䡲 99
operations at different levels of government
and an organization’s disaster management
program. An understanding of the entire spec-
trum of emergency operations allows an orga-
nization to communicate effectively when
working with emergency management profes-
sionals during a disaster event or a drill.
Disasters are inherently local events re-
quiring citizens, local organizations, and first
responders to work together. Key to effective
emergency response is the integrated plan-
ning for disasters that starts at the national
level and extends to the local emergency man-
agement agency (EMA). State and national
support systems are available to assist if a local
municipality is overwhelmed by a disaster and
with the development of local response plans
and training. These national and local systems
include the following:
䡲 National Incident Management System
(NIMS). The NIMS is a nationally accepted
system for helping responders from all lev-
els of government, various jurisdictions,
and the private sector to communicate and
coordinate their response efforts.5 The
NIMS uses a unified approach for incident
management that offers standard com-
mand and management structures and em-
phasizes preparedness, mutual aid, and re-
source management. Organizations should
Recovery
Property Business Resources
Effect Effect Needed Total
High Low High Low High Low
5 1 5 1 5 1
4 5 4 21
1 2 2 7
4 3 2 14
2 4 1 14
100 䡲 AABB TECHNICAL MANUAL
be familiar with NIMS protocols so that
they are prepared to communicate effec-
tively with responding organizations [eg,
police and fire departments and the Federal
Emergency Management Agency (FEMA)].
Online training courses are available on the
FEMA website.6
䡲 National Response Framework (NRF). The
NRF is a federally operated all-hazards re-
sponse plan that is activated in response to
a presidentially declared disaster under the
Robert T. Stafford Disaster Relief and Emer-
gency Assistance Act (amended June
2006).7,8 Under this act, the federal govern-
ment, coordinated by the Department of
Homeland Security (DHS), provides per-
sonnel, assets, and financial support to
state and local governments that are over-
whelmed during a major disaster. The need
for blood is covered under the medical and
public health section of the NRF (Emergen-
cy Support Function No. 8).8(p33),9 Blood col-
lection and hospital facilities should be fa-
miliar with the NRF to request and receive
assistance during major emergencies.
䡲 National Terrorism Advisory System (NTAS).
The DHS assesses threats posed by terror-
ists and communicates those threats to the
US public, government agencies, and the
private sector through the NTAS alerts.10
This system replaces the color-coded
Homeland Security Advisory System
(HSAS) used in the past. The NTAS uses
only two tiers of alerts:
– Imminent Threat Alerts warn of “credi-
ble, specific, and impending terrorist
threat[s] against the United States.”
– Elevated Threat Alerts warn of credible,
but not specific, threats against the Unit-
ed States.
NTAS alerts are brief, clearly articulating
what can be done to prevent and even, if
possible, mitigate a threat’s impact and re-
spond if necessary. Unlike the previously
used HSAS that issued ongoing warnings,
NTAS alerts are issued for a specific period
and expire once that period has ended.
䡲 National Disaster Medical System (NDMS).
The NDMS is a cooperative asset-sharing
program directed by the Department of
Health and Human Services (DHHS). The
NDMS augments local medical care when
an emergency exceeds the scope of a com-
munity’s health-care system. The NDMS
consists of more than 9000 medical and
support personnel from federal, state, and
local governments and the private sector as
well as civilian volunteers.11
䡲 AABB Interorganizational Task Force on
Domestic Disasters and Acts of Terrorism
(AABB Disaster Task Force). The AABB
Disaster Task Force provides support to
blood collection and transfusion facilities
during major emergencies, including dur-
ing NRF activation under the Stafford Act.
Facilities should review the AABB Disaster
Task Force response plan and integrate its
response processes into their organization-
al disaster plans.2
䡲 EMAs. EMAs are local, tribal, state, and fed-
eral agencies tasked with helping individu-
als and organizations deal with all cycles of
disaster management. These agencies are
staffed by both full-time and volunteer per-
sonnel (emergency managers) and use
standard methods when responding to a
disaster.11
Major Disaster Management Factors
for Blood and Transfusion
In conjunction with comprehensive disaster
planning strategies, blood collection and hos-
pital facilities should consider the disaster
management factors, described in this section,
that are key for blood and blood components
during all phases from collection to transfu-
sion.12-16
Most of the blood and blood components
used for transfusion in the United States are
collected, processed, tested, and stored at re-
gional blood centers and must be delivered to
hospitals before transfusion. During an emer-
gency, this “just-in-time” delivery system re-
sults in the need for close coordination be-
tween blood centers and the hospitals they
serve. This involves robust communication
and information systems, transportation and
logistics support, and critical utilities, such as
fuel and electrical power, to ensure that blood
 CHAPTER 4
can be transported and stored at required tem-
peratures.
Emergency management personnel are
often unaware of issues related to the collec-
tion, processing, storage, and distribution of
blood and blood components and may as-
sume that blood is collected and stored direct-
ly in hospitals. As a result, blood-related issues
(eg, the need for transportation and logistics
support) are often overlooked during real and
simulated disasters. A continuing process of
education for emergency managers at both the
local and national levels is imperative to in-
crease their awareness of issues related to
blood. Blood centers should pursue active par-
ticipation in EMA planning activities and
participate in activities of the emergency oper-
ations center to ensure continuous represen-
tation of blood-related issues.
Historically, an average of about 200 units
of blood are needed by victims of most disas-
ters. However, the public response to disasters
has traditionally resulted in increases in blood
donations regardless of the true medical need
for transfusions. A sudden increase in unneed-
ed blood donations can disrupt both local and
national blood supplies. Efforts should be
made to establish the medical need for blood
during a disaster and communicate this need
to national blood organizations and the AABB
Disaster Task Force, blood donors, and the
public through a clear and consistent messag-
ing strategy.12-16
In disasters resulting in injuries that do
require blood transfusions to treat victims, the
available local blood inventory is the primary
source for the initial treatments. Because
blood collected in response to a disaster takes
24 to 48 hours to process, it is critical for the lo-
cal blood supply to remain at ample levels to
treat victims of potential hazards as defined in
the organizational risk assessment. The AABB
Disaster Task Force recommends maintaining
a 5- to 7-day supply [combined inventory at
the blood collector and hospital(s) it serves] to
address potential disasters.2 Efforts are made
to resupply the affected facilities if needed
from neighboring blood collection centers or
through national support by the AABB Disas-
ter Task Force. However, delivering blood from
Disaster Management 䡲 101
outside the immediate vicinity to the affected
hospital might take 12 to 24 hours.
Disasters resulting from the use of biolog-
ic, chemical, radiologic, or nuclear agents may
result in widespread donor deferrals and the
potential quarantine of blood components al-
ready in the manufacturing process or storage
until the nature and effect of the agent can be
determined. Radiologic-nuclear attacks also
would dramatically increase the demand for
platelets, other blood components, and stem
cells for several weeks following an event. In
addition, in the event of an influenza pandem-
ic, blood supplies may be severely strained for
weeks due to high staff absenteeism and donor
shortages as these individuals become ill, need
to stay home to take care of loved ones, or fear
exposure to influenza in public places. An in-
fluenza pandemic could also severely affect
the availability of needed supplies as manu-
facturers face similar absenteeism and trans-
portation difficulties.17,18
Hospitals and transfusion services should
develop blood shortage management plans to
maximize the probability of optimal blood dis-
tribution and use during a severe shortage. It is
critical that detailed blood triage plans devel-
oped by hospital transfusion services, man-
agement staff, and physicians who make
transfusion decisions be available and be reg-
ularly reviewed in advance of a severe disaster.
Blood centers should work with their hospital
customers to ensure that they have triage
plans in place. The Minnesota Department of
Health’s Minnesota Healthcare System Pre-
paredness Program has developed an exten-
sive set of materials to help hospitals prepare
to respond to disasters including “When
Healthcare Resources are Scarce,” a set of
guidelines to assist health-care facilities in
planning for events that would overwhelm the
system’s resources, including blood and blood
components.19, 20
Finally, certain disasters may affect a
blood collector’s ability to collect and process
blood during the entire event, thus straining
the local blood supply for days or weeks. For
instance, as a hurricane approaches, a blood
collector may suspend collections for a few
days before and after the storm has passed,
102 䡲 AABB TECHNICAL MANUAL
resulting in a significant loss of expected col-
lections. Elective surgeries typically are post-
poned during this period, helping alleviate the
strain on supply. However, lessons learned
from previous disasters indicate that blood
collection and transfusion facilities should
prepare for the potential acute shortage of
blood components in the days following a hur-
ricane or disaster of similar magnitude, paying
special attention to the supply of platelets. Fa-
cilities should augment supplies before a pre-
dictable hazard (eg, a hurricane or severe win-
ter storm) and contact the AABB Disaster Task
Force for support if routine supply channels
are inadequate to bolster supplies before or af-
ter the event.
BUS I N ES S O PE R AT IO N S
PL AN N ING
Business operations planning is generally a
three-step process: assembling a planning
team; analyzing the current situation, includ-
ing hazards and capabilities; and developing a
plan to continue operations in an emergency.
Once planning has been completed, imple-
mentation of the plan (which includes train-
ing, testing, evaluating, and revising the plan)
can proceed.21,22
Disaster Planning Team
The first step in disaster planning is to identify
the person or team of people in an organiza-
tion who are responsible for emergency opera-
tions planning. The number of people in-
volved will depend on the size and complexity
of the operation. Assistance will be needed
from key people, such as the organization’s ac-
countant, attorney, human resources staff, and
insurance agent. In addition, establishing a re-
lationship with local EMAs can be vital to the
success of a disaster plan in an actual emer-
gency.
Once the responsible person has been
identified or the team has been formed, a proj-
ect plan should be developed to identify the
key steps and a timeline for completing the di-
saster plan. Business continuity planning has
proliferated over the past 5 years as organiza-
tions have seen human and natural disasters
wreak havoc in the United States and abroad.
References, training materials, and toolkits
with step-by-step instructions and templates
are widely available on the Internet and should
be acquired and used when developing a di-
saster plan.21-25
Analysis of Current Situation
The success of disaster planning depends on a
risk analysis of the current situation (see sec-
tion on “Risk Assessment”). This assessment
includes determining the hazards to which an
organization is most vulnerable, the probabili-
ty of each hazard, and the anticipated effect of
each event on human and physical resources
and on business operations.4,21 Effective pre-
vention strategies can be used for risks such as
fires, power interruptions, facility security
breaches, and workplace violence. What can-
not be prevented should be mitigated. For ex-
ample, conducting essential operations away
from floodplains, having multiple sources (but
not necessarily multiple suppliers) of critical
supplies, and relocating vital records (includ-
ing critical information technology resources)
to upper floors in flood-prone areas are all mit-
igation strategies. Plans should be developed
for events that have a high likelihood of occur-
ring as well as low-probability events with po-
tentially catastrophic consequences (eg, Cate-
gory 5 hurricanes or pandemic influenza).
Continuity of Operations Plan
A Continuity of Operations Plan (COOP) is
designed to ensure continued operation of
essential functions in the event of an emergen-
cy or disaster.21,25 A COOP should cover the
emergencies that are most likely to occur or
that could have a significant effect. A COOP
should address at least the areas identified in
Table 4-2. These elements are discussed in this
section.
Hospital-based blood banks and transfu-
sion services may be covered by the overall
hospital or laboratory COOP; however, the var-
ious COOPs should be reviewed to ensure that
issues specific to blood components and
transfusion are sufficiently covered. Such
 CHAPTER 4 Disaster Management 䡲 103

TABLE 4-2. Essential Elements of a Continuity of Operations Plan19,20,23

1. Identify essential processes and functions required for continued operations.
2. Develop decision trees for implementing the Continuity of Operations Plan.
3. Consider alternate facility options.
4. Establish safety and security plans for staff facilities and fleet vehicles.
5. Identify and ensure protection and survivability of vital records and databases.
6. Review insurance coverage and ensure that it is adequate for potential risks.
7. Establish minimum cash reserves necessary to continue operations for 90 days.
8. Develop an emergency communications plan.
9. Establish a chain of command and an order of succession for decision-making authority.
10. Develop a plan for dealing with the news media.
11. Identify designated spokespersons, and train them to handle risk and emergency communications.
12. Plan to maintain supplies and logistical support for operations.
13. Evaluate utility needs and develop contracts or memoranda of agreement to ensure replenishment and
restoration of essential utilities.
14. Review information technology systems and develop redundancies to ensure that vital systems and their
supporting subsystems remain operational during an emergency.
15. Identify staffing issues, including essential personnel and key contacts necessary to carry out essential
functions as well as employee compensation and benefits during and after the disaster.
16. Develop procedures for transitioning back to normal operations after a disaster.

plans are required by the AABB in its Standards
for Blood Banks and Transfusion Services as
well as by The Joint Commission.26,27
Essential Functions
Essential functions are those that must be car-
ried out to ensure business continuity, such as
the storage of blood and blood components
and their transportation and delivery to hospi-
tals. Even though blood collections, compo-
nent preparation, and testing may be inter-
rupted, the continued viability of a blood
center depends on its ability to provide hospi-
tal customers with blood. Blood can be im-
ported from outside the affected area to en-
sure an adequate inventory until collections
can resume. Likewise, hospitals must be cer-
tain that they can receive, store, and transfuse
blood to patients in need.
COOP Decision Trees
COOP operations and decisions are best
thought out in advance during normal opera-
tions. Flowcharts and decision trees that list
options can be developed to guide leaders
during emergency operations. Such flow
charts and decision trees can be developed by
groups and refined using lessons learned from
exercises and real-world events. An example of
a decision tree developed to support blood
bank operations by the California Blood Bank
Society is available on the Internet.28
Alternate Facilities
Alternate facilities that can be used to contin-
ue operations should be identified during the
planning phase of a COOP. Thought should be
given to the need for equipment, supplies, in-
formation technology support, and other
104 䡲 AABB TECHNICAL MANUAL
items at the alternate location. Consideration
should also be given to practical locations for
alternate facilities. For example, an alternate
facility across the street will most likely be sim-
ilarly affected by an external disaster. If fund-
ing is not available to pay for an alternate
facility, the organization should consider es-
tablishing formal agreements with other orga-
nizations in the area to provide alternate space
if one facility is incapacitated.
Security and Safety
In a disaster, security of critical resources—
ranging from the facility and fleet to staff and
information systems—can become a major
concern.22 For example, crowd control may be-
come an issue if large numbers of donors gath-
er at collection locations or in the event of
special screening requirements during a pan-
demic influenza outbreak. In natural disasters
in which fuel shortages occur, the security of
fuel supplies can become a major challenge. A
COOP should include the measures that will
be necessary to ensure the security of all criti-
cal resources.
Planners should review their normal op-
erations security plan and determine whether
the plan adequately addresses potential
threats and hazards to the local area. Coordi-
nation with EMA officials can provide infor-
mation on threat or risk assessments. Planners
should consider the implementation of mea-
sures to increase the security of their facilities,
staff, volunteers, and donors during changes
in the NTAS.10
Lastly, planners should ensure that the fa-
cility complies with all local building codes
and applicable Occupational Safety and
Health Administration (OSHA) regulations.
The actions implemented should be dis-
cussed with the local EMA and the insurance
company, and any suggestions that they pro-
vide should be addressed.22
Vital Records
The protection of vital records is especially
critical in an industry that depends on records
to document the safety and availability of
blood components. In addition to donor rec-
ords, the organizational human resource files,
legal records, payroll records, and financial
records (such as accounts receivable and ac-
counts payable) are critical to a business’s sur-
vivability during a disaster.22,29 Records of in-
surance policies, bank account numbers, and
supplies of blank checks must be available
during emergency operations to ensure conti-
nuity of operations. Corporation records, stra-
tegic plans, and research-related records must
also be maintained. Redundancy of computer-
ized records should be established by periodi-
cally by backing records up on a duplicate
server. Geographic considerations must be
taken into account in identifying off-site stor-
age locations. Not all records are maintained
electronically, so provisions to safely keep pa-
per copies of records must be considered, es-
pecially if alternate facility operations are im-
plemented.
Insurance
Adequate insurance is essential to the surviv-
ability of any business. The two major types of
insurance coverage for blood collection and
transfusion facilities are property and liability.
Key provisions of these policies include valua-
tions of property; perils covered, such as flood-
ing, wind, and power interruptions; deduct-
ibles; and how to file claims. Business
interruption insurance can cover loss of in-
come. Additional background information on
relevant insurance policies is available from a
variety of resources, including the Small Busi-
ness Administration and insurance compa-
nies.24,30
Cash Reserves
The single most crucial element for the surviv-
ability of a business during a disaster is access
to cash that can provide support until normal
operations can be resumed.31,32 Cash reserves
equal to several months’ worth of operational
expenses should be accrued during normal
operations.
 CHAPTER 4
Emergency Communications Plan
Clear, quick, and effective communication is
vital to protecting life and property during a
disaster, and communication systems are of-
ten the first assets to be strained during an
emergency. Telecommunication systems can
quickly become overloaded or jammed; com-
puter systems may be disrupted because of a
loss of power; and the staff may become sepa-
rated, resulting in general confusion and the
inability to make rapid decisions during and
after a disaster. Organizations should invest
heavily in ensuring that the staff can commu-
nicate both internally and externally and
should develop an Emergency Communica-
tions Plan (ECP). No single definition or all-en-
compassing set of procedures exists for an
ECP, but organizations should consider the fol-
lowing areas when designing one.
IDENTIFYING INTE RNAL AND EXTERNAL
AUDIE NCES FOR THE ECP. Blood collection
and hospital facilities should identify and map
the essential internal departments and per-
sonnel that need to communicate both during
a disaster (response) and after a disaster (re-
covery). All employees need to be included in
the ECP because all employees will need to re-
ceive information regarding the response pro-
cedures and the operational status of the facil-
ity (eg, when to report back to work).
External audiences need to be identified
and assessed for their ability to communicate
with an organization (eg, vendors must have
an ECP for communications with an organiza-
tion). For instance, a hospital blood bank or
transfusion service should identify all essential
departments and personnel at its blood sup-
plier(s). Conversely, blood centers should
identify the key departments and personnel at
hospitals or the testing providers that need to
be contacted during and after an emergency.
KEY CO NTACTS. Key lists should be main-
tained and periodically updated of all of the
critical internal and external personnel, de-
partments, and organizations that need to dis-
seminate or receive information during an
emergency. The contact lists should be readily
available both electronically (eg, through
Disaster Management 䡲 105
smart phone applications) and in print (eg, on
laminated cards) for emergency response per-
sonnel to use during a disaster.
Internal contact lists should include es-
sential personnel and key leaders. External
contact lists should include customers, suppli-
ers, and vendors of critical supplies; the AABB
Disaster Task Force; personnel at state and lo-
cal EMAs; and other key business resources,
such as insurance agents, utility service repre-
sentatives, legal representatives, and banking
personnel. Facilities should consider collect-
ing redundant contact information for each
person in their contact lists (ie, business, mo-
bile, and home telephone numbers; e-mail ad-
dresses; and text messaging number). For or-
ganizational contacts (eg, blood bag suppliers,
delivery services, and electrical utility provid-
ers), facilities should obtain alternate direct
(“back door”) contact information to bypass
standard automated answering systems dur-
ing an emergency.
Human resources staff and legal counsel
should be consulted to ensure that they sup-
port the collection of staff contact informa-
tion. In some cases, the staff may be unwilling
to provide personal contact information due
to privacy concerns.
CHAIN OF COMMAND. Leadership is critical
for maintaining control and direction during a
disaster. Organizations should clearly define
and communicate to all personnel and exter-
nal partners who will be (or who are) in charge
of a given event.4(p6) In addition to a primary
authority, clearly defined lines of decision-
making authority, delegation, and communi-
cation should be established among the lead-
ership team and employees.
STRATEGIES FOR COMMUNICATION RE-
DUNDANCY AND COMPATIBILIT Y. A c r i t i -
cal challenge for organizations during real or
simulated disasters is the ability (or inability)
to communicate using routine methods.
These methods quickly become overloaded,
requiring organizations to use alternate com-
munication methods. It is vital to identify and
test these redundant communication chan-
nels before a disaster because significant
106 䡲 AABB TECHNICAL MANUAL
delays can occur in switching to alternate
methods during an event, resulting in the po-
tential loss of life and property.
No single method is used by organiza-
tions to design communications redundancy.
Local and state jurisdictions use different
types of emergency communications equip-
ment (eg, fire and police personnel use radios)
and have different local telecommunications
infrastructures (eg, cell towers) and capacities.
Organizations should acquire multiple redun-
dant communications technologies and iden-
tify the order in which they should be used
during an emergency. Alternative communica-
tion technologies are described in the AABB
Disaster Handbook.2
COMMUNICATION WITH LOCAL, STATE, AND
NATIONAL E MERGENCY RESPONSE AGEN-
CIES. In addition to communicating with
routine suppliers and vendors, facilities
should establish and maintain relationships
with local, state, tribal, and national emergen-
cy response organizations as shown in Fig 4-1
(see also section on Participating in Disaster
Drills with EMAs).
In particular, blood collection and trans-
fusion facilities should identify agencies that
FIGURE 4-1. Communication with external agencies.
EMAs = emergency management agencies.
can assist with obtaining 1) transportation
support for components, critical supplies, and
staff; 2) power restoration; 3) fuel for backup
generators; 4) fuel for fleet and essential staff
vehicles; 5) communication support; 6) securi-
ty if staff or property is threatened; and 7) oth-
er utilities support (eg, telecommunications
and Internet). Facilities should educate these
agencies on how the blood supply operates
both locally and nationally (eg, the use of
“just-in-time” delivery methods and the need
to deliver blood from regional blood centers to
hospitals) and should request that they be
deemed a critical health entity or critical infra-
structure entity in the emergency response
structure.8
Blood centers and hospitals should work
with the local EMA to ensure their inclusion in
local emergency communications systems.
These systems may include ongoing videocon-
ferences or conference calls, blast e-mail lists,
and 800-MHz radio networks. Furthermore,
blood centers should seek admission to any pri-
ority networks established by local hospitals to
improve communications during an incident.
Periodically, management should pro-
vide informational briefings to members of the
EMA and the public health department about
 CHAPTER 4
the mission of the organization, the scope of
its operations, and the effect on local health-
care facilities if the blood center or hospital is
not operational. Management should also be-
come familiar with the NIMS and NRF docu-
ments and should consider enrolling in FEMA
online educational courses30 or disaster train-
ing courses provided by the state or local com-
munity. Management should request that the
EMA provide informational briefings to key
staff of the blood center or hospital. During ex-
changes with the EMA, management should
discuss resource issues (eg, transportation as-
sistance, fuel support, storage, security, inclu-
sion in EMA notification systems, and assign-
ment of a blood center or hospital liaison to
the EMA). Management should invite the EMA
to participate in its disaster exercises and ac-
tively encourage the EMA to include the blood
center or hospital in its disaster exercise pro-
grams.
National assistance during disasters (eg,
coordination of national media messages and
resupply of blood components to affected fa-
cilities) is provided through the AABB Disaster
Task Force in coordination with other national
and federal organizations. Along with local
planning efforts, blood collection and transfu-
sion facilities are encouraged to integrate the
task force response system into their disaster
plans. The response system is described in the
AABB Disaster Operations Handbook on the
AABB website.2
WORKING WITH PUBLIC MEDIA. Creating
and disseminating a clear and consistent mes-
sage about the status of the local and national
blood supply is a critical component of an
ECP.12-16 Unnecessary donor surges can be pre-
vented, or donations of specific blood compo-
nents (eg, group O red cells or platelets) can be
requested by working with public media out-
lets to effectively convey messages about the
status of the blood supply following a disaster.
The AABB Disaster Task Force has developed
media-related resources for facilities, includ-
ing boilerplate press releases that are available
in the AABB Disaster Operations Handbook.2
Only individuals who have received train-
ing in risk and emergency communication
Disaster Management 䡲 107
should speak with members of the media. Spe-
cific activities should take place before, dur-
ing, and after each interview.33 For instance,
written background information should be
provided to a reporter before an interview, and
a spokesperson should focus the most salient
points of the message into “sound bites” that
can be quoted in a story. Another useful tech-
nique when communicating with the media is
message mapping.34 Message maps help com-
municators develop and synthesize complex
information by identifying three key messages
to be delivered in three short sentences with a
total of 27 words. This process accommodates
both broadcast and print media. Most impor-
tant, a spokesperson should never speak off
the record with a reporter. Many other tips and
techniques can be used to ensure that the cor-
rect messages are conveyed. One resource for
such tips is the Northwest Center for Public
Health Practice in collaboration with Public
Health—Seattle and King County.35
Logistics
During the development of a COOP, facility
planners must place heavy emphasis on the
management of daily operations, including
making available the supplies and equipment
required to perform essential functions related
to 1) recruiting of donors, 2) collecting donated
blood, 3) manufacturing and testing blood
components, and 4) delivering the components
to the hospital for transfusion to patients. Dur-
ing a disaster, the normal logistical systems
may not be available or capable of providing
the necessary support. Key logistical issues to
address in a COOP are described below.
TRANSPORTATI ON. Past disaster and terror-
ist events have clearly demonstrated the vul-
nerability of transportation systems. Blood
centers should coordinate with their local
EMAs to ensure that vehicles engaged in col-
lecting or distributing blood are duly autho-
rized as emergency support vehicles and are
granted the appropriate clearances to operate
on the roads. In addition, memoranda of un-
derstanding, agreements, or statements of un-
derstanding should be established with the
108 䡲 AABB TECHNICAL MANUAL
appropriate EMAs regarding access to the
roads and use of law enforcement, National
Guard, and state militia personnel or vehicles
to deliver blood. Under the provisions of the
NRF, DHHS can ask the Department of Trans-
portation to assist with the movement of blood
by air, rail, water, and motor vehicle.8 Such re-
quests should be directed to the AABB Disaster
Task Force.2
“J U S T- I N - T I M E ” S Y S T E M S . M a n y b l o o d
centers and hospitals have adopted just-in-
time supply systems to minimize expenses as-
sociated with the storage of large quantities of
supplies, maintenance of storage facilities,
staffing, and outdated products. Blood centers
and hospitals should determine the critical
items required for their business operations,
the vendor’s resupply capability (including
manufacturing schedule and transport time-
lines), the shelf life of each supply item, and
any storage requirements (eg, special environ-
mental factors).36
STORAGE . Planners should consider the stor-
age capacity of their customers and identify lo-
cal storage companies that could provide an
emergency storage site. Contingency contracts
or contingency clauses in existing contracts
with customers and ice and dry-ice suppliers
should be considered. Temporary storage
space may be provided by commercial con-
tainers, including prevalidated refrigerated
containers that could be placed at the blood
center, depending on space and electrical
power. The local EMA may permit storage of
consumables at municipal disaster supply fa-
cilities at no cost.
CONTRACTS. All vendor contracts should
identify the process for emergency deliveries
of supplies, equipment, and services. The ven-
dor or supplier should have a disaster plan
that demonstrates the ability to meet a facili-
ty’s supply requirements in a disaster. It is ad-
visable to require vendors to have disaster
plans for their operations. Facilities might also
develop contracts containing noncompete
agreements with local competitor organiza-
tions for implementation during disasters.
BIOHAZ ARDS. In the aftermath of a disaster,
the disposal of biohazardous material may be-
come a serious problem for a blood center or
hospital. Planners should incorporate appro-
priate language in their biohazard material
disposal contracts that will ensure that ven-
dors establish contingency plans to meet the
facility’s requirements. The blood center or
hospital should coordinate with the EMA and
fire or safety agencies and should provide in-
formation on the types of biohazardous wastes
that it generates. Organizations that have irra-
diators should provide local fire or safety agen-
cies with a detailed building plan that includes
photographs of the ingress routes to the device
and of the device as well as the manufacturer’s
specifications and contact information. Affect-
ed facilities should contact the AABB Disaster
Task Force2 if local and state EMA authorities
cannot provide this assistance.
Utilities
Blood centers and hospitals rely on utility ser-
vices (eg, electricity, water, fuel, sewage dis-
posal, telecommunications, and Internet)
from a variety of sources, such as municipali-
ties or publicly or privately owned companies.
Planners should establish contingency sup-
port plans with their service providers. These
plans should identify emergency points of
contact at the utility service provider and in-
clude their telephone numbers and e-mail ad-
dresses. Most utility services have priority lists
for service restoration during emergencies.
During the planning process (and not during
or after an emergency), planners should solicit
the support of the local EMA and hospitals to
ensure that their organization will be designat-
ed to receive priority restoration of services.
Staffing
Blood centers and hospitals depend on their
highly trained, competent, and motivated staff
and volunteers to be successful in their mis-
sion of recruiting blood donors and collecting,
manufacturing, testing, and distributing blood
components. Management should promote
disaster preparedness to staff and volunteers
 CHAPTER 4
and should encourage them to complete fami-
ly preparedness plans.8.22
ESSE NTIAL PERSONNEL. During the emer-
gency planning process, the management
team should identify staff members and vol-
unteers (if applicable) who are responsible for
the vital services required to maintain busi-
ness operations. Each department should de-
velop a “smart book” that describes its essen-
tial functions, essential personnel who
accomplish them, vital information and rec-
ords, contract information, and customer in-
formation.
Employees who are deemed essential be-
cause of their responsibilities must receive ex-
tensive training in the facility’s emergency
plans and participate in disaster training exer-
cises to refine operational procedures under
emergency conditions. Consideration should
be given to establishing alternate work sites
and/or implementing telework programs to
mitigate the effects of the loss of the blood
center or hospital on operations. The human
resources department should work with man-
agement to ensure that employee-training
plans provide for the succession of personnel
into positions held by designated essential
personnel should the incumbent become un-
available. Management should also plan for
housing and feeding essential personnel and
providing them with fuel to help them travel to
and from work when the local infrastructure is
not operational.23
CROSS-FUNCTIONAL TRA INING. Planners,
in close coordination with the human resourc-
es department and, if applicable, any collec-
tive bargaining units, should develop a cross-
functional training program that identifies
personnel whose routine duties do not sup-
port the facility’s essential functions during a
disaster response. These reassigned personnel
may serve as drivers, public affairs representa-
tives, and recruiters or perform other nonreg-
ulated duties. The human resources depart-
ment and management should make the
necessary modifications to the job descrip-
tions of the affected employees or volunteers,
develop training plans, and conduct initial and
Disaster Management 䡲 109
refresher training as required. Management
may need to negotiate with collective bargain-
ing units to modify existing contracts.
HUMAN RESOURCES ISSUES. Of impor-
tance to employees—beyond the safety of
loved ones and availability of food and shel-
ter—are issues such as salary continuation,
flexible work schedules, implementation of
telework procedures, and benefits.8,23 Because
these issues are influenced by many factors,
having a predeveloped decision matrix can be
particularly helpful during times of crisis.
REGULATORY CONSIDERATIONS IN
EMERGENCIES
The blood collection and transfusion commu-
nity is highly regulated, and even slight chang-
es to processes and procedures can have far-
reaching effects on facilities and patients,
especially during an emergency. Blood collec-
tion and transfusion facilities should carefully
consider regulatory issues in their disaster
management plans.
Federal Agencies Involved
The blood community is regulated by numer-
ous agencies. The Center for Biologics Evalua-
tion and Research (CBER) of the Food and
Drug Administration (FDA) has primary re-
sponsibility for ensuring the safety, purity, and
potency of all biologic products, including
blood and blood components. The Center for
Devices and Radiological Health (CDRH), also
part of the FDA, regulates medical devices,
including radiation-emitting devices. The
Department of Transportation regulates the
movement of cargo, including blood, blood
components, and progenitor cells, through the
transportation network. OSHA ensures the
safety of the US workforce. Likewise, the mis-
sion of the Environmental Protection Agency
is to protect human health and the environ-
ment. The Centers for Medicare and Medicaid
Services under the Clinical Laboratory Im-
provement Amendments of 1988 oversees pa-
tient (as distinct from donor) testing at blood
110 䡲 AABB TECHNICAL MANUAL
centers, hospital blood banks, and transfusion
services.
Consideration must be given to the regula-
tions and requirements of each of these agen-
cies when developing emergency response
plans. Such regulatory considerations generally
fall into three categories: effect on available
blood components, effect on donors, and po-
tential consequences for operations.
Effects on Components in Available
Inventory
All blood centers and hospitals must have
written procedures to follow in an emergency.
These procedures should address the provi-
sion of emergency electrical power and con-
tinuous temperature monitoring during stor-
age. These procedures should address the
potential effects of unsuitable storage condi-
tions, such as extreme (high or low) tempera-
tures or exposure to smoke or water.37 Consid-
eration should also be given to nontraditional
emergencies that may affect blood, such as a
radiologic event or exposure to a biologic or
chemical agent.38
Blood components that have potentially
been exposed in an event should be quaran-
tined, and a determination of component suit-
ability should be made before the components
are returned to inventory. When the quality,
safety, purity, or potency of a component is in
question, CBER should be consulted. An ex-
ception or variance may be needed to use
components in such situations.39 In addition,
the AABB Disaster Task Force is available to
assist facilities with questions about a compo-
nent’s safety, purity, or potency during a
disaster.2
All blood released for use during a disas-
ter should be fully tested, including for infec-
tious diseases. An exception to full testing
should be made in the event that blood sup-
plies are exhausted, resupply is not possible,
and blood is needed immediately to save lives.
Blood-testing samples should be retained for
retrospective testing after a disaster. Thor-
ough documentation of the circumstances of
the disaster is essential, and physicians should
be notified regarding what tests have and have
not been completed on the blood.39
In very extreme circumstances, the FDA may
issue emergency guidance to help ensure an
adequate blood supply. For example, on Sep-
tember 11, 2001, the FDA issued the Policy
Statement on Urgent Collection, Shipment,
and Use of Whole Blood and Blood Compo-
nents Intended for Transfusion to Address
Blood Supply Needs in the Current Disaster
Situation.40 Blood collection facilities should
closely follow all developments regarding do-
nor deferrals and all emergency FDA guidance
during disasters.
Effects on Donors
Disasters, particularly emergencies involving
potential infectious diseases or hazardous
chemicals, can affect donor eligibility. Infec-
tious and chemical agents should be evaluated
for 1) transmissibility by blood transfusion, 2)
potential to harm recipients, and 3) potential
for an asymptomatic interval of infectivity or
toxicity after exposure but before or after any
donor illness. If an agent is determined to be
transmissible, a deferral period that ensures
an adequate period of safety from infectivity or
adverse effect must be estimated, and donors
must be deferred appropriately. As with com-
ponent suitability, CBER and the AABB Disas-
ter Task Force should be consulted about po-
tential donor deferral issues.
Potential Consequences on
Operations
Emergencies involving power outages, floods,
or structural damage to facilities disrupt nor-
mal operations. Disasters that do not directly
affect the blood center’s or hospital’s physical
infrastructure can still disrupt operations. For
example, a pandemic influenza outbreak may
cause severe staffing shortages with up to a
40% absenteeism rate.17 Collections should be
performed only by facilities that routinely
collect blood. Facilities that collect only autol-
ogous blood should not attempt to collect allo-
geneic blood during a disaster.
 CHAPTER 4
Adequate staffing can be challenging im-
mediately before, during, and after an emer-
gency while staff members tend to their fami-
lies and personal needs. Only staff trained in
regulated functions should perform these
functions. The FDA recommends that blood
establishments have adequate backup person-
nel in the event of a disaster and that more
than one backup person be trained for each
critical function. This training should be docu-
mented.41 Volunteers can be used to perform
nonregulated functions, such as predonation
education and maintenance of the canteen.
Likewise, appropriate licensure is required to
safely operate fleet vehicles.
Emergency plans should include lists of
staff trained in multiple functions. For exam-
ple, staff members who have transferred from
one area to another (and who have maintained
competency in the prior area) could perform
the initial function with appropriate certifica-
tion and any necessary commercial vehicle li-
censes.
Equipment and supplies should also be
assessed for exposure to water, humidity, and
temperature extremes. CDRH has published
an information guide about the effects of di-
sasters on medical devices; it can be useful in
planning as well as responding to disasters.42
Records Management
Vital records must be secured and maintained.
These documents include records of donors,
donations, manufacturing, testing, quality as-
surance, and product disposition. If these rec-
ords are damaged or lost during a disaster,
blood in inventory at that time may require
quarantine and recall. Efforts should be made
to retrieve and preserve any damaged records.
CBER should be consulted to determine the
disposition of inventory when records are lost.
TE S TI N G TH E DI S AST E R PL A N
Continual improvement of a disaster plan is
critical for maintaining a high state of readi-
ness for future emergencies, and participation
in disaster drills is one way to achieve this goal.
By simulating the conditions of a real disaster,
Disaster Management 䡲 111
a facility can identify and correct deficiencies
and gaps in its disaster plan.
Internal and External Disaster Drills
Drills can be conducted internally (eg, a fire
drill or simulated hazardous spill cleanup) or
in conjunction with other organizations. Mul-
tiple-organization disaster drills are typically
conducted by local or state EMAs. Unfortu-
nately, blood-related issues are often over-
looked in the exercise scenarios developed by
these agencies because many of the planners
are unaware of how blood is collected, stored,
and distributed to hospitals. Blood collection
facilities should develop relationships with
EMAs, as described in the “Major Disaster
Management Factors for Blood and Transfu-
sion” section.
Participating in Disaster Drills with EMAs
The National Strategy for Homeland Security
directed the establishment of a national exer-
cise strategy. Homeland Security Directive 8
directed the DHS to establish a National Exer-
cise Program (NEP).43 In addition to full-scale,
integrated, national-level exercises, the NEP
provides tailored exercise activities that serve
as the primary DHS vehicle for training na-
tional leaders and staff. The NEP enhances
collaborations among partners at all levels of
government for assigned homeland security
missions. National-level exercises provide the
means to conduct “full-scale, full-system
tests” of collective preparedness, interopera-
bility, and collaboration across all levels of
government and the private sector.44 The
program also incorporates elements to allow
identification of any implications of changes
to homeland security strategies, plans, tech-
nologies, policies, and procedures.
Blood centers and hospitals should seek
inclusion in disaster drills and exercises by con-
tacting their local EMAs. Participation in local,
state, and federal exercises increases a facility’s
preparedness. This participation also increases
awareness by the emergency preparedness offi-
cials at all levels of the critical role that blood
centers play in providing the blood compo-
112 䡲 AABB TECHNICAL MANUAL

nents required by victims of a disaster and the
resources that are required to do so.
Including Blood Issues in Drills
Disaster drills range from informal discussions
about improving response methods to full-
scale, real-time exercises that involve hundreds
of organizations. Each method can be used by a
single organization or in conjunction with mul-
tiple organizations. When an organization par-
ticipates in drills with other organizations (ie,
full-scale exercises), it is important for blood
collection and transfusion facilities to “inject”
blood-related scenarios into the drills during
the planning stages. For example, drills should
address 1) the need to transport blood from a
blood center to a hospital (or hospitals) to treat
victims when local roads have been damaged or
destroyed and 2) the effect of an unknown bio-
logic agent release on the local blood supply,
donors (eg, need to defer donors), or both. Ad-
ditional information on the basic types of drills
can be found on the FEMA Emergency Manage-
ment Institute website.45
After-Action Review
Perhaps the most important aspect of a well-
executed drill or actual disaster is the after-ac-
tion review process. During this process, par-
ticipants evaluate what worked well and what
did not work well, with the aim of identifying
lessons learned and implementing corrective
actions to improve future responses.
S U M M A RY O F LE S S O N S
LEAR NED FROM RECENT
D I S A ST E R S
Blood collection facilities and hospitals
around the world have dealt with significant
disasters in recent years. Hurricanes, tsuna-
mis, earthquakes, tornados, fires, industrial
accidents, and terrorism have tested emergen-
cy response systems. Organizations have used
the learning points from these events to fur-
ther sharpen their disaster plans. Many of
these lessons can be found in the AABB Disas-
ter Operations Handbook.2 Blood collection
and transfusion facilities are encouraged to
share the major learning points from real or
simulated disasters that they have experienced
with AABB through the AABB National Blood
Exchange at 1.800.458.9388 or by e-mail to
nbe@aabb.org. AABB is collecting such lessons
and sharing them with facilities around the
world.
CO N CLUS IO N
In the wake of disasters—such as the Septem-
ber 11, 2001, attacks on the World Trade Cen-
ter and Pentagon; the rail transit bombings in
Great Britain, Spain, and Boston; and the dev-
astating natural disasters that occur every
year—organizations around the world have fo-
cused significant attention and resources on
strengthening their disaster plans. Given the
heightened awareness of the need for organi-
zations to be prepared, both the public and
members of the media are holding organiza-
tions to the highest standards of excellence for
ensuring that all necessary planning has been
done to protect both life and property from
known threats. Blood collection and transfu-
sion facilities are not immune to this reality
and should therefore strive to maintain the
highest standards of disaster preparedness.
Above all else, the most important benefit of
careful and continual planning is assurance
that blood and blood components are avail-
able to patients when and where needed.

KEY POINTS

1. Planning is the key to success during a disaster. Disaster planning is not a one-time event; it
requires continual review, exercises and practice, and learning and changing.
2. Continuity of operations planning provides a framework for facilities to maintain or restore the
critical functions necessary to collect, manufacture, store, and distribute blood components.
 CHAPTER 4 Disaster Management 䡲 113

3. The cornerstone of an effective disaster plan is a thorough risk assessment of the probable
hazards and their impact on critical operations. Risks can include internal events, such as a
broken pipe that floods a laboratory, or external events, such as tornadoes and earthquakes.
4. Facilities should use the resources of the AABB Interorganizational Task Force on Domestic
Disasters and Acts of Terrorism in disaster management planning (eg, Disaster Operations
Handbook) and incorporate the task force’s national response procedures into local plans.
5. The ability to communicate during a disaster determines much of the success of a response;
facilities need well-developed and routinely tested communication plans.
6. Hospitals should have emergency plans for triage of blood components in disasters in
which demand exceeds supply (eg, a severe pandemic or nuclear or biologic attack). Blood
centers should work with their hospital customers to ensure that such plans are in place.
7. Adequate cash reserves and insurance are essential for a blood center to survive a major di-
saster.
8. Coordination with local emergency management officials before a disaster is critical to suc-
cess during that disaster.
9. Regulatory considerations during a disaster include the effects on the blood components on
the shelf, the donors in the affected area, and operations.
10. Facilities should routinely reinforce their disaster plan by conducting information sessions
with staff, volunteers and their families, suppliers, and hospital customers. The disaster
plan should be tested through participation in local or regional disaster drills.

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ability and reporting certain changes to an ap-
proved application. Silver Spring, MD: CBER
Office of Communication, Outreach and De-
velopment, 2010. [Available at http://www.fda.
gov/BiologicsBloodVaccines/GuidanceCom
plianceRegulatoryInformation/Guidances/
Blood/default.htm.]
42. FDA offers tips about medical devices and
hurricane disasters. Washington, DC: Food
and Drug Administration, 2013. [Available at
http://www.fda.gov/MedicalDevices/Safety/
EmergencySituations/ucm055987.htm (ac-
cessed May 27, 2013).]
43. Homeland Security Presidential Directive 8:
National preparedness. Washington, DC: De-
partment of Homeland Security, 2011. [Avail-
able at http://www.dhs.gov/xlibrary/assets/
presidential-policy-directive-8-national-pre
paredness.pdf (accessed May 27, 2013).]
44. Exercise. Washington, DC: Federal Emergency
Management Agency, 2013. [Available at http://
www.fema.gov/exercise (accessed May 27,
2013).]
45. IS-120A. An introduction to exercises (FEMA
independent study). Emmitsburg, MD: Feder-
al Emergency Management Agency Emergen-
cy Management Institute, 2008. [Available at
http://training.fema.gov/EMIWeb/IS/course
Overview.aspx?code=is-120.a (accessed May
27, 2013).]
 C h a p t e r 5

Allogeneic and Autologous Blood

Donor Selection

Anne F. Eder, MD, PhD, and

Maria D.L.A. Muniz, MD

THE FOREMOST RESPONSIBILITY OVER VIEW OF BL OOD DONO R

of blood centers is to maintain a safe and
adequate blood supply. The selection of ap-
propriate blood donors is essential to protect
their health during and following donation,
and to ensure the safety, quality, identity, puri-
ty, and potency of the donated blood compo-
nents to protect the transfusion recipient. Key
elements of the selection process, as part of
the overall approach to blood safety, are edu-
cation, the donor health history questionnaire,
a focused physical examination, infectious
disease testing (see Chapter 8), and manage-
ment of postdonation information.
This chapter describes the current feder-
al regulations, accreditation requirements,
and medical considerations related to screen-
ing blood do nors before their bloo d is
collected and tested for various blood-borne
diseases.1-3
S C RE EN I N G
Blood centers provide prospective blood do-
nors with information on the donation pro-
cess and potential donation-related adverse
effects and instruct them not to donate if they
may be infected with blood-borne pathogens.
The screening process includes a focused
physical examination and direct questioning
about specific risk behaviors, medications,
travel, and other factors that potentially affect
recipient or donor safety. The questions ad-
dress risks related to infectious diseases for
which sensitive tests are currently performed
[eg, hepatitis B and C viruses and human im-
munodeficiency virus (HIV)], for which tests
are not universally used (eg, Chagas disease),
and for which licensed screening tests are not
yet available (eg, babesiosis and malaria).

Anne F. Eder, MD, PhD, Executive Medical Officer, American Red Cross, National Headquarters, Biomedical
Services Medical Office, Holland Laboratory, Rockville, Maryland; and Maria D.L.A. Muniz, MD, Clinical
Pathologist, Allegheny Health Network, Pittsburgh, Pennsylvania
The authors have disclosed no conflicts of interest.

117
118 䡲 AABB TECHNICAL MANUAL
If individuals are instructed not to donate
blood for others because of their health histo-
ry, reactive test results, behavioral risks, or
medical reasons, they may be added to a confi-
dential deferral list to prevent future dona-
tions. Deferrals may be for a defined interval of
time or an indefinite period, or they may be
permanent with no potential for reinstate-
ment as a blood donor in the future.1 In addi-
tion, blood centers are required to manage in-
formation received after the donation that
could affect the safety, quality or potency of
the donated blood components and the future
eligibility of the donor, and keep records of de-
ferred individuals.2
The criteria to evaluate individuals who
are donating blood for their own use (ie, autol-
ogous donation) may be less stringent than
those for allogeneic donation. However, the fo-
cus remains on providing the safest possible
blood for transfusion to the donor-patient and
on evaluating the risks that the collection pro-
cedure poses to his or her health.
The blood center must determine donor
eligibility prior to collection and on the day of
collection, in accordance with federal and
state regulations and voluntary accreditation
standards. Specific criteria used to select do-
nors are established by the Food and Drug Ad-
ministration (FDA) through the Code of Feder-
al Regulations (CFR), guidance documents,
and memoranda to industry.2 The AABB also
develops professional standards for donor se-
lection with which accredited blood establish-
ments must comply.1
An industry-wide effort led by AABB to
streamline and standardize donor screening
culminated with the release by the FDA, in Oc-
tober 2006, of a final guidance document rec-
ognizing the donor history questionnaire
(DHQ) as an effective donor screening tool
that provides licensed and unlicensed facilities
with a way to meet all FDA requirements.4 The
DHQ is currently used by most blood centers
in the United States.5,6 The references in this
chapter are to DHQ Version 1.3 (May 2008),
which the FDA officially recognized as accept-
able DHQ documentation in May 2010.4,7
Medical directors of blood collection fa-
cilities are responsible for determining donor
eligibility policies on issues that are not cov-
ered by regulations or standards.3,8-11 Conse-
quently, medical decisions regarding the same
issue may differ among blood centers or even
among physicians at the same blood center.
Considerable variability exists in national and
international practices for determining donor
eligibility, which reveals the inherent uncer-
tainty in risk assessment.8
A precautionary approach attempts to
eliminate the risk of known or potential trans-
fusion-transmitted diseases from the blood
supply but also results in the disqualification
of large segments of the healthy population.
Prospective donors who are deferred from
blood donation may be disappointed, angry,
or confused about the relevance that certain
questions have to their health or ability to do-
nate blood for transfusion to others. Blood
center staff should be able to explain the in-
tended purpose of AABB and FDA require-
ments as well as their center-specific eligibility
screening practices.
The most frequently asked questions
about federal regulations defining blood do-
nor eligibility are available to the public in the
“Questions about Blood” section of the FDA
website.12 Questions about the interpretation
or underlying rationale of AABB Standards for
Blood Banks and Transfusion Services should
be directed to the AABB Standards Depart-
ment (standards@aabb.org); responses and
discussion of selected issues are posted on the
AABB website.
SELECTION OF ALLO GENEIC
B LO OD D ON O R S
Registration and Donor Identification
In the United States, blood components for al-
logeneic transfusion are typically collected
from volunteer, nonremunerated donors; oth-
erwise, they must be labeled as being from
paid donors.2
Prospective blood donors should provide
an acceptable form of identification, and each
donor must be properly identified by the col-
lection staff before each donation. Acceptable
forms of identification include government-
 CHAPTER 5 Allogeneic and Autologous Blood Donor Selection
issued documents, such as a driver’s license or
passport, or a blood-center-issued donor card
with a unique alphanumeric code. Many
blood collectors no longer use social security
numbers for donor identification because of
regulations restricting their use and privacy
concerns.
Accurate records are essential to identify
all prior donations from any given donor, in-
cluding whether the donor has ever given
blood under a different name, so that the link
with all prior donations within the blood sys-
tem is maintained. Accurate records are also
essential to ensure that the donor can be con-
tacted in the weeks following the donation and
informed of test results or other relevant infor-
mation from the current donation, if neces-
sary. Blood centers must make reasonable at-
tempts to notify the donor within 8 weeks of
the donation if any test results disqualify the
individual from continued donation.2 Blood
centers often require donors to provide an ad-
dress and telephone number where they can
be reached during this interval for counseling
or other follow-up, if necessary. Accurate do-
nation records must be kept by the donor cen-
ter for the requisite amount of time, according
to current regulations and standards.1,2
Notably, there is currently no national
registry of deferred blood donors in the United
States, and individuals who are deferred by
one blood center may be eligible in another
blood system. The available evidence suggests
that such donor deferral registries are not nec-
essary because they do not contribute to blood
safety and do not prevent the release of unsuit-
able components.13
Educational Materials and Donor
Consent
US blood centers provide all prospective blood
donors with information about blood dona-
tion during the informed consent process. The
DHQ educational materials incorporate all
necessary elements required by federal regula-
tions and AABB Standards, and blood centers
have added elements to protect both blood
donors and transfusion recipients.1(pp15-16,60-63),4
At each encounter, the blood center staff
䡲 119
should explain the collection procedure to the
donor in terms that the donor understands
and document the donor’s consent, which in-
dicates that the donor has considered all the
educational materials and has had an oppor-
tunity to ask questions.1(p16) The donor should
be informed about possible adverse reactions
to the collection procedure and the infectious
disease tests that will be performed on his or
her donated blood components. The donor
should also be informed of the notification
process for positive test results, any reporting
requirements to federal or state health author-
ities, and the possibility of inclusion in the do-
nor center’s deferral registry and subsequent
deferral from future donation. The individual
should agree not to donate if his or her blood
could pose a risk to the blood supply. Donors
should also be informed if investigational tests
or other research may be performed on sam-
ples or information collected during the blood
donation. Finally, the limitations of the tests to
detect early infections and the possibility that
a test may not be performed if samples are not
adequate should be explained to the donor.
No one should donate blood for the pur-
pose of receiving free infectious disease test-
ing. Information about alternative HIV test
sites or public health options to obtain HIV
tests should be provided to all prospective
donors.
All prospective donors must be able to
give informed consent and provide an accu-
rate health history. They should be given an
opportunity to ask questions and they have
the right to withdraw from the process at any
time. Blood donation centers should docu-
ment donors’ consent to have their blood col-
lected and tested.
Blood centers must comply with applica-
ble state laws to obtain permission from par-
ents or guardians for minors (ie, 16- or 17-year
olds) or legally incompetent adults.1(p16) More-
over, AABB Standard 5.2.2 requires that collec-
tion facilities have a process to provide infor-
mation about the donation process to parents
or legally authorized representatives of donors
when parental permission is required.1(p16)
Donor centers should also establish poli-
cies on accommodating individuals who are
120 䡲 AABB TECHNICAL MANUAL

not fluent in English or are illiterate, are vision
or hearing impaired, or have other physical
disabilities. Many blood centers try to make
reasonable accommodations for donors’ spe-
cial needs. However, centers must also ensure
that the collection procedure does not pose
undue risk to donors or staff members and
that the informed consent process is not com-
promised. The final authority for such deci-
sions rests with the donor center’s physician
who supervises donor qualification and phle-
botomy.1(p1)
Donor Qualification by Focused
Physical Examination and
Hemoglobin or Hematocrit
Measurement
Qualification procedures for blood donation
include a focused physical examination (eg,
taking the donor’s temperature and inspecting
his or her arm) and a hemoglobin or hemato-
crit measurement. These tests have potential
implications for the potency or safety of the
collected component (Table 5-1). Additional
requirements apply to apheresis donors, who
must meet the weight and hemoglobin or he-
matocrit requirements approved by the FDA
for the automated collection device.
Prior to phlebotomy, the collection staff
inspect the donor’s antecubital skin, which
must be free of lesions and evidence of injec-
tion drug use, such as multiple needle punc-
tures (eg, small scars lined up in “tracks”) or
sclerotic veins. Scars or pitting on the forearm
associated with frequent blood donation
should not be mistaken for evidence of injec-
tion drug use. Common and mild skin disor-
ders (eg, poison ivy rash) are not a cause for
deferral unless there are signs of localized bac-
terial superinfection or the condition inter-
feres with proper skin disinfection in the ante-
cubital site before phlebotomy.

TABLE 5-1. Physical Examination and Requirements for Allogeneic and Autologous Whole-Blood Donation

Allogeneic Autologous
AABB Reference Standard 5.4.1A; Title 21, AABB Standard 5.4.4; Title 21, CFR
CFR Part 640.3 Part 640.3

Age Conform to applicable state law or

16 years Alternate requirements defined by
Blood pressure No requirement in AABB standards, systolic and blood center’s medical director (AABB
diastolic blood pressure “within normal limits” Standard 5.4.4).
[Title 21, CFR Part 3(2)].
Pulse No requirement in AABB standards or CFR.
Whole blood vol- Maximum of 10.5 mL/kg of donor weight,
ume collected including samples.
Donation interval 8 weeks after whole blood donation; 16 weeks
after 2-unit red cell collection; 4 weeks after
infrequent plasmapheresis; and 2 days after
plasma-, platelet-, or leukapheresis.
Temperature 37.5 C (99.5 F) if measured orally or equivalent Deferral for conditions presenting risk
if measured by another method. of bacteremia (AABB Standard
5.4.4.4).
Hemoglobin 12.5 g/dL (38%). >11 g/dL (33%).
(hematocrit)
CFR = Code of Federal Regulations.
 CHAPTER 5 Allogeneic and Autologous Blood Donor Selection
The FDA defines the minimum accept-
able hemoglobin concentration for both men
and women as 12.5 g/dL, and the essentially
equivalent hematocrit requirement of 38%.2
This requirement is controversial and is peri-
odically debated because normal hemoglobin
values are influenced by gender, race, nutri-
tional status, and other factors.14,15 In particu-
lar, a single acceptance criterion for men and
women is contentious because normal hemo-
globin values for women are lower than those
for men. However, this requirement has re-
mained unchanged for more than 30 years.
Hemoglobin or hematocrit screening may pre-
vent collection of blood from a donor with sig-
nificant anemia, which could have implica-
tions for the health of the donor as well as the
potency of the collected component.
A hemoglobin level lower than the 12.5 g/dL
cutoff is the most common reason for disqual-
ification of potential blood donors. This single
deferral criterion can be problematic for both
male and female donors. Some female donors
with hemoglobin levels below the 12.5 g/dL
cutoff are actually in the normal range. In con-
trast, some male donors with a hemoglobin
level above 12.5 g/dL may, in fact, be anemic.
Furthermore, hemoglobin screening does not
ensure that the donor has an adequate store of
iron. 16,17 Debate continues on the minimum
hemoglobin requirement for blood donation,
frequent donors’ iron status, and allowable do-
nation intervals.18
Donor hemoglobin screening may ensure
a minimum content of hemoglobin in a unit of
Red Blood Cells (RBCs), but currently neither
the FDA nor the AABB define potency stan-
dards for RBC units prepared from whole-
blood collection. If a donor’s hemoglobin level
is 12.5 g/dL, a 500-mL whole-blood collection
is expected to yield about 62.5 g of hemoglobin
per unit of RBCs, but determining the final
content of hemoglobin in an RBC unit pre-
pared from whole blood is not required. AABB
Standards require apheresis RBC units to be
prepared using a method known to ensure a fi-
nal component containing a mean hemoglo-
bin level of 60 g of hemoglobin, with 95% of
the units sampled containing more than 50 g
of hemoglobin.1(p27)
䡲 121
In general, neither the FDA nor the AABB
specifies the test method, specimen type (cap-
illary, venous blood), or acceptable perfor-
mance characteristics for tests used for hemo-
globin/hematocrit screening. One exception is
that a capillary sample collected from an ear-
lobe puncture is not an acceptable specimen
for hemoglobin/hematocrit screening for allo-
geneic or autologous donors.1(pp19,60) Most US
blood centers use fingerstick capillary samples
for hemoglobin determination. These sam-
ples tend to give slightly higher hemoglobin
values than venous samples.
The methods to measure hemoglobin or
hematocrit are generally selected for their ease
of use in the mobile blood collection setting.
The copper sulfate density method (Method 6-
1) is still an acceptable screening tool in blood
centers in the United States but has been
largely replaced by methods such as spectro-
photometric measurement of hemoglobin
with portable devices (eg, HemoCue Donor Hb
Checker, HemoCue AB, Angelholm, Sweden;
DiaSpect Hemoglobin, DiaSpect Medical AB,
Uppsala, Sweden) or hematology analyzers.
The point-of-care methods that use portable
devices yield quantitative hemoglobin results,
with a coefficient of variation (CV) of 1.5%.15 A
typical automated analyzer measures hemo-
globin levels in a venous sample with a CV
1.2%.15 Most quantitative methods currently
in use reliably measure hemoglobin levels
within approximately 0.2 to 0.5 g/dL, and the
vast majority of deferred donors have hemo-
globin or hematocrit values near the cutoff
value. For capillary-sample-based methods,
the most likely source of preanalytical error is
the sampling technique, and testing must be
performed in compliance with the manufac-
turer’s instructions.
Health History Assessment—DHQ
The AABB DHQ is now used by most blood
centers in the United States, although its use is
not currently mandated by the FDA (Table
5-2).19 Alternative procedures for collecting
required information from blood donors are
subject to FDA review and acceptance. In ad-
dition, the FDA acknowledges that the DHQ
122 䡲 AABB TECHNICAL MANUAL
TABLE 5-2. Donor History Questionnaire and Eligibility Requirements for Allogeneic Blood Donations19
Donor History Questionnaire
1. Are you feeling healthy and well today?
2. Are you currently taking an antibiotic?
3. Are you currently taking any other medica-
tion for an infection?
4. Are you now taking or have you ever taken
any medications on the Medication Deferral
List?
5. Have you read the educational materials?
6. In the past 48 hours have you taken aspirin
or anything that has aspirin in it?
7. Female donors: Have you been pregnant or
are you pregnant now? (Males: Check “I am
male.”)
8. In the past 8 weeks have you donated blood
platelets or plasma?
Brief Description of Eligibility Criteria
The prospective donor shall appear to be in good health and
shall be free of major organ disease (eg, heart, liver, lungs),
cancer, or abnormal bleeding tendency, unless determined
eligible by the medical director.
Variable, temporary deferral criteria as defined by the medi-
cal director.
Variable, temporary deferral if treated for active infections,
as defined by the medical director.
No deferral for prophylactic (preventive) use of antibiotics
(eg, tetracycline for acne).
Variable, temporary deferral if treated for active infections.
Examples:
Potent teratogens (potential for harm to unborn children):
䡲 Finasteride (Proscar, Propecia), isotretinoin (Absorica,
Accutane, Amnesteem, Claravis, Myorisan, Sotret, Zena-
tane): Defer for 1 month after last dose.
䡲 Dutasteride (Avodart, Jalyn): Defer for 6 months after last
dose.
䡲 Acitretin (Soriatane): Defer for 3 years after last dose.
䡲 Etretinate (Tegison): Defer indefinitely.
Potential vCJD risk (but no documented cases of transmis-
sion):
䡲 Bovine insulin manufactured in the United Kingdom: Defer
indefinitely.
N/A
Medications that irreversibly inhibit platelet function pre-
clude use of the donor as sole source of platelets:
䡲 Defer for at least 36 hours after ingestion of aspirin.
䡲 Defer for use of other medications as defined by the facil-
ity’s medical director.
Defer if donor has been pregnant within the last 6 weeks.
Donation interval requirements:
䡲 Defer for 8 weeks after whole blood donation.
䡲 Defer for 16 weeks after 2-unit red cell collection.
䡲 Defer for 4 weeks after infrequent plasmapheresis.
䡲 Defer 2 days after plasmapheresis, plateletpheresis, or
leukapheresis.
 CHAPTER 5 Allogeneic and Autologous Blood Donor Selection
TABLE 5-2. Donor History Questionnaire and Eligibility Requirements for Allogeneic Blood Donations19
(Continued)
Donor History Questionnaire
9. In the past 8 weeks have you had any
vaccinations or other shots?
10. In the past 8 weeks have you had contact
with someone who had a smallpox vaccina-
tion?
11. In the past 16 weeks have you donated
a double unit of red cells using an apheresis
machine?
12. In the past 12 months have you had a
blood transfusion?
13. In the past 12 months have you had a
transplant such as organ, tissue, or bone
marrow?
14. In the past 12 months have you had a
graft such as bone or skin?
䡲 123
Brief Description of Eligibility Criteria
No deferral for receipt of toxoids or synthetic or killed viral,
bacterial, or rickettsial vaccines listed below if the donor is
symptom free and afebrile.
䡲 Anthrax, cholera, diphtheria, hepatitis A, hepatitis B, influ-
enza, Lyme disease, paratyphoid, pertussis, plague, pneu-
mococcal polysaccharide, polio (Salk/injection), Rocky
Mountain spotted fever, tetanus, or typhoid (by injection).
No deferral for receipt of recombinant vaccines (eg. human
papillomavirus vaccine).
No deferral for receipt of intranasal live attenuated flu vac-
cine.
Defer for 2 weeks for receipt of the following live attenuated
viral and bacterial vaccines:
䡲 Measles (rubeola), mumps, polio (Sabin/oral), typhoid
(oral), or yellow fever.
Defer for 4 weeks for receipt of the following live attenuated
viral and bacterial vaccines:
䡲 German measles (rubella) or chicken pox (varicella zos-
ter).
Defer for 12 months unless otherwise indicated by medical
director for receipt of other vaccines, including unlicensed
vaccines.
Refer to current FDA guidance.
Defer for 16 weeks after 2-unit red cell collection.
Defer for 12 months for receipt of blood, components,
human tissue, or plasma-derived clotting factor concen-
trates.
Defer indefinitely for receipt of human (cadaveric) dura
mater.
(Continued)
124 䡲 AABB TECHNICAL MANUAL

TABLE 5-2. Donor History Questionnaire and Eligibility Requirements for Allogeneic Blood Donations19
(Continued)

Donor History Questionnaire Brief Description of Eligibility Criteria

15. In the past 12 months have you come into Defer for 12 months
contact with someone else’s blood? 䡲 From the time of mucous membrane exposure to blood.
16. In the past 12 months have you had an 䡲 For nonsterile skin penetration with instruments or equip-
accidental needle-stick? ment contaminated with blood or body fluids other than
the donor’s own.
17. In the past 12 months have you had sexual
contact with anyone who has HIV/AIDS or has
had a positive test for the HIV/AIDS virus?
18. In the past 12 months have you had sexual
contact with a prostitute or anyone else who
takes money or drugs or other payment for
sex?
19. In the past 12 months have you had sexual Defer for 12 months from the time of sexual contact with an
contact with anyone who has ever used nee- individual with HIV infection or at high risk of HIV infection.
dles to take drugs or steroids, or anything not
prescribed by their doctor?
20. In the past 12 months have you had sexual
contact with anyone who has hemophilia or
has used clotting factor concentrates?
21. Female donors: In the past 12 months have
you had sexual contact with a male who has
ever had sexual contact with another male?
(Males: check “I am male.”)
22. In the past 12 months have you had sexual Defer for 12 months for sexual contact or for having lived
contact with a person who has hepatitis? with an individual who meets any of the following criteria:
23. In the past 12 months have you lived with 䡲 Has acute or chronic hepatitis B (positive hepatitis B sur-
a person who has hepatitis? face antigen test).
䡲 Has symptomatic hepatitis C.
䡲 Is symptomatic for any other viral hepatitis.
24. In the past 12 months have you had a Defer for 12 months from the time of nonsterile skin penetra-
tattoo? tion with instruments or equipment contaminated with blood
or bodily fluids other than the donor’s own, including for tat-
25. In the past 12 months have you had ear
toos or permanent makeup unless, applied by a stage-regu-
or body piercing?
lated entity with sterile needles and ink that is not reused.
26. In the past 12 months have you had or Defer for 12 months or for longest applicable period for the
been treated for syphilis or gonorrhea? following situations:
䡲 Following the completion of treatment for syphilis or gon-
orrhea.
䡲 For a reactive screening test for syphilis where no confir-
matory testing was performed.
䡲 For a confirmed positive test for syphilis (FDA reentry pro-
tocol after 1 year applies).
 CHAPTER 5 Allogeneic and Autologous Blood Donor Selection 䡲 125

TABLE 5-2. Donor History Questionnaire and Eligibility Requirements for Allogeneic Blood Donations19
(Continued)

Donor History Questionnaire Brief Description of Eligibility Criteria

27. In the past 12 months have you been in Defer for 12 months.
juvenile detention, lockup, jail, or prison for
more than 72 hours?
28. In the past three years have you been out- Malaria:
side the United States or Canada? 䡲 Defer for 3 years after departure from malaria-endemic
area(s) for individuals(s) who have lived for longer than
5 consecutive years in a country(ies) with areas consid-
ered endemic by the Malarial Branch, Centers for Disease
Control and Prevention, US Department of Health and
Human Services.
䡲 Defer for 12 months after departure from malaria-
endemic area(s) for other individuals who have traveled to
an area where malaria is endemic.
29. From 1980 through 1996, did you spend
time that adds up to three (3) months or more
in the United Kingdom? (Review list of coun-
tries in the UK.)
30. From 1980 through 1996, were you a
member of the US military, a civilian military
employee, or a dependent of a member of the
U.S. military? Defer indefinitely for risk of vCJD, as defined in most recent
31. From 1980 to the present, did you spend FDA guidance.
time that adds up to five (5) years or more in
Europe? (Review list of countries in Europe.)
32. From 1980 to the present, did you receive
a blood transfusion in the United Kingdom?
(Review list of countries in the UK.)
33. From 1977 to the present, have you received
money, drugs, or other payment for sex? Defer indefinitely donors:
34. Male donor: From 1977 to the present, 䡲 Excluded by current FDA regulations and recommenda-
have you had sexual contact with another tions for the prevention of HIV transmission by blood and
male, even once? (Female donors: Check components.
“I am female.”) 䡲 With present or past clinical or laboratory evidence of
infection with HCV, HTLV, or HIV or as excluded by cur-
35. Have you EVER had a positive test for the rent FDA regulations.
HIV/AIDS virus? 䡲 For use of a needle to administer nonprescription drugs.
36. Have you EVER used needles to take
drugs, steroids, or anything not prescribed by
your doctor?

(Continued)
126 䡲 AABB TECHNICAL MANUAL
TABLE 5-2. Donor History Questionnaire and Eligibility Requirements for Allogeneic Blood Donations19
(Continued)
Donor History Questionnaire
37. Have you EVER used clotting factor
concentrates?
38. Have you EVER had hepatitis?
39. Have you EVER had malaria?
40. Have you EVER had Chagas disease?
41. Have you EVER had babesiosis?
42. Have you EVER received a dura mater
(or brain covering) graft?
43. Have you EVER had any type of cancer,
including leukemia?
44. Have you EVER had any problems with
your heart or lungs?
45. Have you EVER had a bleeding condition or
a blood disease?
46. Have you EVER had sexual contact with
anyone who was born in or lived in Africa?
47. Have you EVER been in Africa?
48. Have any of your relatives had Creutzfeldt-
Jakob disease?
vCJD = variant Creutzfeldt-Jakob disease; N/A = not applicable; HIV = human immunodeficiency virus; AIDS = acquired
immunodeficiency syndrome; FDA = Food and Drug Administration; UK = United Kingdom; HCV = hepatitis C virus; HTLV =
human T-cell lymphotropic virus.
Brief Description of Eligibility Criteria
䡲 Defer as directed by the medical director donors with
abnormal bleeding tendency.
䡲 Defer for 12 months for receipt of blood, components,
human tissues, or plasma-derived clotting factor concen-
trates.
Defer indefinitely for history of viral hepatitis after 11th
birthday.
Defer for 3 years after becoming asymptomatic prospective
donors who have had a diagnosis of malaria.
Defer indefinitely for a history of babesiosis or Chagas
disease.
Defer indefinitely for receipt of human (cadaveric) dura
mater.
Variable deferral criteria, as defined by medical director.
Examples:
䡲 Nonhematologic cancer: Defer for 5 years after comple-
tion of treatment.
䡲 Local skin cancer, in situ cancer: No deferral if treated
completely with excision and healed.
䡲 Hematologic cancer (eg, leukemia): Defer indefinitely.
Variable deferral criteria, as defined by medical director.
Variable deferral criteria, as defined by medical director.
Defer indefinitely, as excluded by current FDA regulations
and recommendations for the prevention of HIV transmis-
sion by blood and blood components.
The questions for risk of HIV group O can be deleted from
the DHQ if the blood center is using an HIV antibody test
that has been approved by FDA to include a claim for detec-
tion of HIV group O.
Defer indefinitely for family history of Creutzfeldt-Jakob
disease.
 CHAPTER 5 Allogeneic and Autologous Blood Donor Selection
documents contain questions related to the
following issues not addressed by any FDA re-
quirement or recommendation: cancer; organ,
tissue, or marrow transplants; bone or skin
grafts; and pregnancy. However, AABB recom-
mends that blood centers implement the DHQ
materials, including the following documents,
in their entirety:
䡲 Blood donor educational materials.
䡲 Full-length DHQ.
䡲 User brochure, including glossary and ref-
erences.
䡲 Medication Deferral List.
The use of the DHQ flow charts is option-
al. All DHQ documents that the FDA has rec-
ognized as acceptable are available on the FDA
website. 20 The most current version is also
available on the AABB website.19
The wording, order, and text of the DHQ
questions should not be changed. A collection
facility may make minor changes to the time
frame of a question on the DHQ to make the
question more restrictive so that its use results
in the deferral of more donors. Blood centers
may choose to include additional questions as
long as they place these questions at the end of
the DHQ. The DHQ documents are intended
to be self-administered by the donor, but
blood centers may choose to use direct oral
questioning, self-administration, or a combi-
nation of both methods to administer the
DHQ.
If AABB standards or FDA regulations do
not address specific medical conditions that a
blood center has chosen to include in the
DHQ, the blood center must develop standard
operating procedures (SOPs) for determining
the criteria for acceptance or deferral of a do-
nor. A rational approach to donor health histo-
ry assessment attempts to balance the need to
take appropriate precautions to protect the
blood supply with avoiding unnecessarily re-
strictive policies that disqualify large segments
of the population without contributing to ei-
ther recipient or donor safety. 3 Decisions
about donor eligibility should be based on
available evidence regarding the risk that the
䡲 127
medical condition or history poses to the
blood donor and transfusion recipients.
If a potential exists for risk to the recipient
or donor, the effectiveness and incremental
benefit of screening donors by questioning
should be evaluated, especially in light of oth-
er safeguards that protect the donor or other
transfusion practices that mitigate potential
risks to the recipient. If the blood center re-
ceives information after the donation that
would have been cause for deferral had it been
reported at the time of donation, then any
subsequent actions, such as product retrieval,
market withdrawal, or consignee notification,
should be commensurate with the potential
hazard and likelihood of possible harm to the
recipient. The center’s approach to develop-
ing donor deferral criteria should take into ac-
count evidence as it becomes available to
modify those decisions. Some of these issues
that allow for medical judgment and for which
questions exist in the DHQ can be explored
further with the donor, but each donor center
must develop and follow its own SOPs.3
B LO OD - C E NT E R - DE F I N ED
D O N O R E L IG IBI L I T Y C R I TE R I A
Unlike questions about potential risks to
transfusion recipients, selection criteria di-
rected primarily at protecting donor safety are
left to the discretion of the blood center’s med-
ical director. Consequently, practice varies at
different blood centers.3,8 AABB Standards re-
quires that prospective donors appear to be in
good health and be free of major organ disease
(eg, diseases of the heart, liver, or lungs), can-
cer, or abnormal bleeding tendency, unless de-
termined eligible by the medical director.1(p61)
The rationale for each deferral for medical
conditions should be carefully considered be-
cause even temporary deferrals adversely af-
fect the likelihood that individuals will return
to donate blood.21 Donor centers have
successfully eliminated some health-related
questions and removed screening require-
ments, such as assessment of pulse, without a
deleterious effect on donor health or dona-
tion-related reactions.4,22 These findings sup-
port the conclusion that some arbitrary and
128 䡲 AABB TECHNICAL MANUAL
rigid selection criteria, ostensibly intended to
protect donors, are unnecessary and can be
safely eliminated.
Cancer
Each year in the United States, blood centers
receive hundreds of reports of cancer in indi-
viduals who had donated blood.
Direct transmission of cancer through
blood transfusion—although biologically
plausible—has not yet been documented to
occur even though people with cancer fre-
quently donate blood before discovering their
diagnosis. 23 A retrospective study examined
the incidence of cancer among patients in
Denmark and Sweden who received blood
from donors with subclinical cancer at the
time of donation.24 Of the 354,094 transfusion
recipients, 12,012 (3%) were exposed to blood
components from precancerous donors, yet
there was no excess risk of cancer among these
recipients compared with recipients of blood
from donors without cancer.24 These data indi-
cate that cancer transmission by blood collect-
ed from blood donors with incident cancer, if
it occurs at all, is so rare that it could not be de-
tected in a large cohort of transfusion recipi-
ents that included the total blood experience
of two countries over several years.
In considering the future eligibility of do-
nors with cancer, some degree of caution is
warranted to allow sufficient time for donors
to recover after chemotherapy or other treat-
ment. There are currently no US federal regu-
lations or professional standards regarding the
criteria that should be used to evaluate donors
with a history of cancer. For this reason, a
blood center’s medical director has consider-
able flexibility in determining donor eligibility
policies.
Almost all licensed blood centers current-
ly accept donors who report localized cancers
after treatment, with no deferral period. These
cancers include skin cancer (eg, basal cell or
squamous cell carcinoma) and carcinoma in
situ (eg, cervical) that have been fully excised
and are considered cured. Most blood centers
defer individuals with a history of a solid organ
or nonhematologic malignancy for a defined
period after completion of treatment provided
that the donor remains symptom free without
relapse. The deferral period following comple-
tion of treatment for nonhematologic cancer
ranges from 1 to 5 years.24 Hematologic malig-
nancy typically results in permanent deferral
from allogeneic blood donation, although
some US blood centers reportedly accept
adults if they have been successfully treated
for childhood leukemia or lymphoma and
have had a long (eg, 10- to 20-year) cancer-free
period following completion of treatment.
These various deferral policies are currently
defensible but should be reevaluated if new in-
formation becomes available about the poten-
tial for cancer transmission through blood
transfusion.
Bleeding Conditions or Blood Diseases
Bleeding conditions and blood diseases have
the potential to affect donor safety as well as
product potency, and blood centers must de-
fine their procedures for handling donors with
hematologic disorders. In general, prospective
donors should be evaluated for bleeding con-
ditions or blood diseases that 1) place the do-
nors at risk of bleeding or thrombosis as a re-
sult of the collection procedure or 2) may
affect the hemostatic efficacy of their blood
and its suitability for transfusion to others.3
Plasma components and Cryoprecipitated
Antihemophilic Factor should contain ade-
quate amounts of functional coagulation fac-
tors and should not contain significant inhibi-
tory or prothrombotic factors. Similarly,
platelet components intended as the sole
source for patients should contain platelets
that have adequate function and are not irre-
versibly impaired by the presence of inhibitors.
Individuals with a history of significant
bleeding complications are usually counseled
to avoid blood donation. However, screening
donors for such a history does not prevent the
rare but serious thrombotic or hemorrhagic
complications in otherwise healthy blood
donors.
Individuals with hemophilia, clotting fac-
tor deficiencies, or clinically significant inhibi-
tors—all of which are manifested by variable
 CHAPTER 5 Allogeneic and Autologous Blood Donor Selection
bleeding tendencies—require deferral for both
donor safety and product potency consider-
ations. The exception is Factor XII deficiency,
which is not associated with either bleeding or
thrombosis.
Carriers of autosomal-recessive or sex-
linked recessive mutations in clotting factors
usually are not at risk of bleeding. They typi-
cally have decreased factor levels but are ac-
cepted by most blood centers because of the
normal, wide variability in clotting factor ac-
tivity levels (50%-150%) compared to the
much lower relative activity that is necessary
to maintain hemostasis (5%-30%).3 Individuals
with von Willebrand disease are typically de-
ferred by most blood centers, although some
centers may allow individuals with mild dis-
ease and no history of bleeding to donate red
cells. Individuals taking anticoagulants to treat
or prevent venous thromboembolism are de-
ferred for both donor safety and product po-
tency considerations.
Heart and Lung Conditions
Cardiovascular disease is common in the Unit-
ed States, affecting an estimated 80 million (1
in 3) adults.25 Prospective blood donors are
asked if they have ever had problems with
their heart or lungs as a donor safety measure,
but the criteria for accepting donors with a
history of heart or lung disease are defined by
each blood center.
The collective, published experience with
autologous donation by patients scheduled for
cardiac procedures has demonstrated that ad-
verse effects are not more frequent than in do-
nors without a history of cardiac disease.26-30
Despite the relative frequency of cardiovascu-
lar disease in the adult population, vasovagal
reactions occur in only about 2% to 5% of
whole-blood donations by healthy donors and
are actually more likely to occur among young,
healthy adolescents than older adults.31
A rational approach to screening donors
with a history of cardiac disease allows the ac-
ceptance of donors who are asymptomatic on
the day of donation, have been medically eval-
uated, and report no functional impairment or
limitations on daily activity after being diag-
䡲 129
nosed or treated for cardiac disease. Some do-
nor centers advise individuals to wait at least 6
months after a cardiac event, procedure, or di-
agnosis. These centers then allow these indi-
viduals to donate if they have been asymptom-
atic and able to perform their usual daily
activities during this interval.
Causes for deferral may include recent
symptoms, limitations on activity or function-
al impairment resulting from unstable angina,
recent myocardial infarction, left main coro-
nary disease, ongoing congestive heart failure,
or severe aortic stenosis.3
Medications
The DHQ and Medication Deferral List con-
tain the requirements for deferrals for specific
medications as stipulated by the AABB and the
FDA. These requisite medication deferrals fall
into five broad categories:
1. Potent teratogens that pose potential harm
to unborn children (although there have
been no documented cases of adverse fetal
outcomes related to transfusions from
donors taking these medications).
2. Anticoagulants and antiplatelet agents that
affect component potency (plasma or
platelet components only).
3. Potential variant Creutzfeldt-Jakob disease
risk from bovine insulin manufactured in the
United Kingdom (although there have been
no documented cases of transfusion trans-
mission from donors taking bovine insulin).
4. Human-pituitary-derived growth hormone,
which theoretically increases the risk of
Creutzfeldt-Jakob disease (although there
have been no documented cases of transfu-
sion transmission from donors taking these
growth hormones).
5. Antibiotics or antimicrobials to treat an
infection that could be transmitted through
blood transfusion (excluding preventive
antibiotics for acne, rosacea, and other
chronic conditions).
Although blood centers may add medica-
tions whose use requires donor deferral to the
130 䡲 AABB TECHNICAL MANUAL
Medication Deferral List, many have chosen to
use the list as developed by the FDA and the
AABB or have added only a few drugs. The
DHQ task force has encouraged blood centers
to fully consider the reasons behind each local
deferral and avoid unnecessary deferral prac-
tices.3
FDA’s pregnancy risk categories, which
are designed to assess the benefit vs risk ratio if
drugs are taken during pregnancy, are often in-
appropriately used for blood donor selection.
For example, categories D and X include some
commonly used drugs (eg, oral contraceptives
and anticholesterol agents) that may be con-
traindicated in pregnancy but pose negligible,
if any risk, to any transfusion recipient.
Local medication deferrals are often
based on concerns about the reason for the
potential donor’s use of the medication and
his or her underlying medical condition rather
than on any inherent threat posed by residual
medication in the collected blood compo-
nent. Most drugs used by donors pose no
harm to recipients, and many factors should
be considered when evaluating the potential
risk of a drug’s use by a donor (eg, the medica-
tion’s half-life, mean and peak plasma concen-
tration, residual concentration in blood com-
ponent, and dilution when transfused to a
recipient).
ABBREVIATE D DHQ FOR
FREQUE N T D O NO R S
Blood centers have recognized for years that
frequent and repeat donors must answer the
same questions at every donation about re-
mote risk factors that are not likely to
change—a situation that leaves many dedicat-
ed donors dissatisfied with the donation expe-
rience. A small number of US blood centers
have received approval from the FDA and im-
plemented an abbreviated DHQ (aDHQ) in
2003 for donors who have successfully com-
pleted the full-length DHQ on at least two sep-
arate occasions and given one or more dona-
tions within the past 6 months.32
The AABB aDHQ, which was developed
and validated by the same task force as the
full-length DHQ, has been officially recog-
nized by the FDA as “acceptable” and can be
implemented by blood establishments using
the corresponding full-length DHQ. 33 Two
“capture questions” about new diagnoses or
treatments since the last donation replace 17
previous questions about remote risks (eg,
blood transfusion, Chagas disease, and babe-
siosis). Although the aDHQ is only somewhat
shorter than the DHQ and its use is restricted
to frequent donors, it may improve donors’ ex-
perience.
RE CIPI ENT-SPE CIF IC
“DESIGNATED” OR “DIRECTED”
B LO OD D ON AT I O N
Exceptional Medical Need
In certain clinical circumstances, a patient
may benefit from blood components collected
from a specific donor, such as 1) a patient with
multiple antibodies or with antibodies to high-
incidence antigens who requires units from
donors whose red cells are negative for the
corresponding antigens or 2) an infant with
neonatal alloimmune thrombocytopenia who
requires antigen-negative platelets from his or
her mother.
Frequent donation by a specific donor for
a specific patient with a medical need requires
that the donor center have a procedure that
typically calls for both a request from the pa-
tient’s physician and approval by the donor
center’s physician. The donor must meet all
allogeneic donor selection requirements, with
the exception of donation frequency, provided
that they are examined and certified by a phy-
sician [21 CFR 640.3(f )]. For frequent blood
donors, the CFR allows centers to qualify a do-
nor by testing the first donation in each 30-day
period. 2 In emergency medical situations,
blood components can be released before in-
fectious disease test results are available pro-
vided that the units are labeled and managed
in accordance with the CFR. Testing on the
units must be completed as soon as possible
after release or shipment, and results must be
promptly reported to the hospital or transfu-
sion service.2
 CHAPTER 5 Allogeneic and Autologous Blood Donor Selection 䡲 131

Directed Blood Donations ing interest in autologous donations may re-

flect the decline in viral risk associated with
The use of directed donations has decreased
allogeneic blood transfusion and, consequent-
over the last 10 years, but the ongoing demand
ly, the minimal medical benefit and increased
from patients for transfusions from specific
cost of autologous blood.35,36
donors during scheduled surgeries likely still
In general, the use of preoperative autolo-
reflects the skewed perception among the gen-
gous blood donation alone provides only a rel-
eral public of the risk for HIV associated with
atively small benefit in reducing the probabili-
blood transfusion. Most blood centers and
ty of allogeneic transfusion and may actually
hospitals surmount the associated collection,
increase the risk of lower postoperative he-
storage, and tracking logistical difficulties to
matocrits, with enhanced risk of ischemic
provide this service.
complications after surgery. Preoperative au-
Directed donations have higher viral
tologous donations may still be used in
marker rates than volunteer donations, most-
conjunction with other blood conservation
ly but not entirely reflecting the higher preva-
methods, such as acute normovolemic hemo-
lence of first-time donors among the former
dilution, perioperative blood recovery, and
group.34 There is no evidence that directed do-
pharmacologic strategies. (See further discus-
nations are safer to use than donations from
sion in Chapter 24.)
volunteer community donors. On the contrary,
Patients identified as candidates for au-
some concerns persist that directed donors
tologous donation are evaluated by the donor
may feel unduly pressured to give blood,
center. The following criteria for autologous
which could compromise blood safety.
donations are specified by the FDA, AABB, or
Directed donors must meet the same cri-
both:
teria as voluntary donors, and their blood can
be used for other patients if not needed by the
䡲 A prescription or order from the patient’s
individual for whom the donations were ini-
physician.
tially intended. If collection of whole blood is
䡲 Minimum hemoglobin concentration of
required from a directed donor more than
11 g/dL or hematocrit of 33%.
once in an 8-week period, the CFR requires
䡲 Collection at least 72 hours before the an-
that the donor be examined and certified to be
ticipated surgery or transfusion.
in good health by a physician on the day of do-
䡲 Deferral for conditions presenting a risk of
nation [21 CFR 640.3(f)].
bacteremia.
The donor center should clearly commu-
䡲 Use only for the donor-patient if labeled
nicate its directed-donation procedures so
“autologous use only.”
that the expectations regarding availability of
directed-donor units are known to the order-
Contraindications to autologous blood
ing physician and patient. The communica-
donation should be defined by the blood cen-
tion required includes defining the mandated
ter and may include medical conditions asso-
interval between collection of the blood and
ciated with the greatest risk from blood dona-
its availability to the patient, mentioning the
tion, such as 1) unstable angina, 2) recent
possibility that the patient will identify donors
myocardial infarction or cerebrovascular
who are not ABO compatible or not otherwise
accident, 3) significant cardiac or pulmonary
acceptable blood donors, and defining the
disease with ongoing symptoms but without
policy for release of donor-directed units for
an evaluation by the treating physician, or 4)
transfusion to other patients.
untreated aortic stenosis.37 Both the ordering
physician and the donor center physician
Autologous Blood Donations
need to carefully balance the risks of the col-
Autologous donations have declined dramati- lection procedure against any perceived bene-
cally in the United States since the 1990s. Wan- fit to the patient-donor.
132 䡲 AABB TECHNICAL MANUAL

KEY POINTS

1. The DHQ and associated documents were developed by an AABB task force, and their use is
recognized by the FDA as an adequate process to determine the eligibility of volunteers for
allogeneic blood donation.
2. The most current versions of the DHQ and associated documents are available on the AABB
website, and all DHQ documents that the FDA has recognized as acceptable are available on
the FDA website.19,20
3. Prospective blood donors are informed of the risks of blood donation, clinical signs and
symptoms associated with HIV infection, behavioral risk factors for transmission of blood-
borne pathogens, and importance of refraining from blood donation if they are at an in-
creased risk of being infected.
4. To be accepted for allogeneic blood donation, individuals must feel healthy and well on the
day of donation and must meet all AABB and FDA requirements as well as medical criteria
defined by the donor center.
5. In certain clinical circumstances (eg, rare phenotype or antigen-negative Red Blood Cell
units needed), a patient may benefit from blood components collected from a specific do-
nor who may not otherwise meet all the requirements for a volunteer donor. The blood cen-
ter must have procedures and follow federal regulations for frequent donation by a specific
donor for a designated patient with a medical need.
6. The ongoing demand from patients to choose specific donors to provide blood for their
transfusions during scheduled surgeries in the absence of a defined medical need has dra-
matically decreased in the last 10 years but persists despite the lack of evidence of improved
safety with directed donations.

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ican Red Cross during 2005 to 2010. Transfu-
sion 2013;53:1250-6.
35. Brecher ME, Goodnough LT. The rise and fall
of preoperative autologous blood donation.
Transfusion 2002;42:1618-22.
36. Schved JF. Preoperative autologous blood do-
nation: A therapy that needs to be scientifical-
ly evaluated. Transfus Clin Biol 2005;12:365-9.
37. Goodnough LT. Autologous blood donation.
Anesthesiol Clin North Am 2005;23:263-70.
 C h a p t e r 6

Whole-Blood Collection and

Component Processing

Larry J. Dumont, MBA, PhD; Mona Papari, MD;

Colleen A. Aronson, MT(ASCP)SBB; and
Deborah F. Dumont, MT(ASCP)SBB

T H E C O L L E C T I O N O F whole blood
(WB) from the donor and subsequent
processing of the donation into components
requires careful attention to proper tech-
niques to ensure the optimal care of both the
donor and the recipient. The classical, manu-
ally intense component-preparation methods
are undergoing significant changes as more
automation is incorporated by developers,
from hands-off expression of components to
completely hands-off methods for the entire
process. Differences are also recognized in
methods for platelet preparation by the “buffy
coat” intermediary process and the “platelet-
rich plasma” (PRP) intermediary process.
This chapter describes WB collection and
component preparation and processing that
may take place at the blood center or the hos-
pital-based collection facility.
W B CO L L E C T I ON
The phlebotomy staff involved in WB collec-
tion must be trained in proper collection tech-
niques to minimize contamination of donated
units and phlebotomy-related local complica-
tions, such as hematoma or nerve injury, using
locally approved standard operating proce-
dures. Phlebotomy must be performed only af-
ter the donor has been found to be eligible for
blood donation and the following items have
been properly labeled using a unique dona-
tion identification number (DIN) label: the
blood donor record, primary and satellite con-
tainers, and sample tubes. A final check of ap-
propriate labeling before phlebotomy helps
ensure that the donor history data, laboratory
data, and other manufacturing data are associ-
ated with the correct blood components. The

Larry J. Dumont, MBA, PhD, Associate Professor, Geisel School of Medicine at Dartmouth, Lebanon, New
Hampshire; Colleen A. Aronson, MT(ASCP)SBB, Regional Director, Transfusion Services, ACL Laboratories,
Rosemont, Illinois; Mona Papari, MD, Medical Director, LifeSource, Rosemont, Illinois; and Deborah F.
Dumont, MT(ASCP)SBB, Research Technologist, Center for Transfusion Medicine Research, Dartmouth
Hitchcock, Lebanon, New Hampshire
L. Dumont has disclosed financial relationships with Advanced Preservation Technologies, New Health Sci-
ences, Fenwal, Terumo BCT, Haemonetics/Hemerus, Advanced Cell Technology, BioMed/CitraLab, and
EryDel. M. Papani, C. Aronson, and D. Dumont have disclosed no conflicts of interest.

135
136 䡲 AABB TECHNICAL MANUAL
final check may be documented by having the
staff initial the donor record.
Blood Containers
Desirable Properties
WB collection containers must be approved by
the appropriate regulatory authority [eg, the
Food and Drug Administration (FDA); Japa-
nese Ministry of Health, Labor and Welfare; or
Health Canada] and must be CE marked for
use in Europe. The containers should be ster-
ile, pyrogen free, and identified by a lot num-
ber. They should also list an expiry date and
include other label data required by regulatory
authorities.
The desired properties of storage contain-
ers include being easily formed and processed;
flexible; kink resistant; transparent; and resis-
tant to damage throughout the anticipated
use, including component processing, storage,
and transfusion. In addition, the container’s
material must be compatible with sterilization
by gamma irradiation, ethylene oxide, electron
beam, and/or steam.
The gas (oxygen, carbon dioxide, and wa-
ter) permeability properties should be appro-
priate for the intended use. Gas exchange and
water loss before use is often limited by an
overwrap or pouch. After removal from a
pouch, the collection set should be used with-
in the time prescribed by the manufacturer.
Pouches should be labeled with the date and
time that they are opened, and unused
containers should be stored within closed
pouches.
Blood containers are usually made of
thermoplastics, such as polyvinylchloride
(PVC), ethylvinylacetate, polyolefins (polyeth-
ylene or polypropylene), or fluoropolymers.
The material formulation determines the
physical properties that are to be matched to
the use requirements, such as high gas perme-
ability for platelet storage or low glass transi-
tion temperature for freezing.
The glass transition temperature of PVC
bags is about –25 C to –30 C. At and below
these temperatures, the container is brittle
and should be treated as if it were made of
glass and fragile enough to break during trans-
port and handling. PVC is rigid at room tem-
perature and requires the addition of a plasti-
cizer such as di-(2-ethylhexyl) phthalate
(DEHP). DEHP not only imparts flexibility to
the PVC but also has been shown to protect
red cells against hemolysis during storage. Be-
cause of concerns over possible toxicity of
DEHP, alternative plasticizers, such as butyryl-
trihexyl-citrate, have been made available in
some collection sets. Because of the protective
effect of DEHP on red cells, finding an equally
effective substitute for DEHP has been chal-
lenging. This has recently been reviewed by
Simmchen.1
Blood collection and processing sets are
generally latex free. The manufacturer’s label
should be consulted to verify the absence of
latex before use in patients with a latex allergy.
Configurations, Anticoagulants, and
Additive Solutions
Collection and storage systems come in differ-
ent configurations based on the intended pro-
cessing methods (manual or automated) and
the number and types of final components to
be prepared from a unit of WB. Blood collec-
tion systems may be intended for final
processing with a classical combination of
manual and mechanical methods (eg, centrif-
ugation, manual bag transfers, and manual ex-
pression of contents) or may be designed for
use in a fully automated system of blood com-
ponent preparation. There will be differences
in the configurations based on the method of
choice for preparing platelets (buffy coat or
PRP processes). Blood collection systems may
also include in-line filters for removal of leuko-
cytes from WB, Red Blood Cell (RBC) units,
and/or platelets. Approved anticoagulants in-
clude anticoagulant citrate dextrose solution
Formula A (ACD-A) or Formula B (ACD-B), an-
ticoagulant citrate phosphate dextrose solu-
tion (CPD), anticoagulant citrate phosphate
double-dextrose solution (CP2D), and citrate
phosphate dextrose adenine solution (CPDA-
1) (see Tables 6-1 and 6-2). RBCs anticoagulat-
ed with CPDA-1 have a shelf life of 35 days in
the United States. The use of additive solutions
(ASs) enables extension of RBC shelf life to
 CHAPTER 6 Whole-Blood Collection and Component Processing 䡲 137

TABLE 6-1. Anticoagulant-Preservative Solutions for Collection of 450-mL Whole Blood*

Variable CPD CP2D CPDA-1

pH 5.0-6.0 5.3-5.9 5.0-6.0

Ratio (mL solution to blood) 1.4:10 1.4:10 1.4:10
FDA-approved shelf life (days) 21 21 35
Content (mg in 63 mL)
Sodium citrate, dehydrate 1660 1660 1660
Citric acid, anhydrous 188 206 188
Dextrose, monohydrate 1610 3220 2010
Monobasic sodium phosphate, 140 140 140
monohydrate
Adenine 0 0 17.3
*Data were supplied and verified by manufacturers.
CPD = citrate-phosphate-dextrose; CP2D = citrate-phosphate-dextrose-dextrose; CPDA-1 = citrate-phosphate-dextrose-
adenine; FDA = Food and Drug Administration.

42 days in the United States and to 56 days in
some other jurisdictions. Four ASs are current-
ly approved in the United States, and others
are approved in other regions (Table 6-3).2
Containers have integrally attached tub-
ing that permits aseptic/closed transfers be-
tween bags. This tubing is marked with repeat-
ing serial numbers and may be sealed at
several locations with either a dielectric (heat)
sealer or a metal clip (grommet) to prepare ap-
proximately 13 to 15 segments. These seg-
ments may be used later for ABO/Rh typing,

TABLE 6-2. Anticoagulant-Preservative Solutions for Collection of 500-mL Whole Blood*

Variable CPD CP2D CPDA-1

pH 5.0-6.0 5.3-5.9 5.0-6.0

Ratio (mL solution to blood) 1.4:10 1.4:10 1.4:10
FDA-approved shelf life (days) 21 21 35
Content (mg in 70 mL)
Sodium citrate, dehydrate 1840 1840 1840
Citric acid, anhydrous 209 229 300
Dextrose, monohydrate 1780 3570 2230
Monobasic sodium phosphate, phosphate, 155 155 155
monohydrate
Adenine 0 0 19.3
*Data were supplied and verified by manufacturers.
CPD = citrate-phosphate-dextrose; CP2D = citrate-phosphate-dextrose-dextrose; CPDA-1 = citrate-phosphate-dextrose-
adenine; FDA = Food and Drug Administration.
138 䡲 AABB TECHNICAL MANUAL

TABLE 6-3. RBC Additive Solutions Currently in Routine Use around the World*†

AS-1
Adsol AS-3 AS-5 AS-7
Fresensius- Nutricel Optisol SOLX
Constituents Kabi Haemon- Terumo Haemon- PAGGSM
(mM) SAGM (Fenwal) etics BCT etics MAP MacoPharma

NaCl 150 154 70 150 - 85 72

NaHCO3 - - - - 26 - -

Na2HPO4 - - - - 12 - 16

NaH2PO4 - - 23 - - 6 8

Citric acid 2 - - 1

Na3-citrate - - 23 - - 5 -

Adenine 1.25 2 2 2.2 2 1.5 1.4

Guanosine - - - - - - 1.4

Dextrose 45 111 55 45 80 40 47
(glucose)

Mannitol 30 41 - 45.5 55 80 55

pH 5.7 4.6 - 7.2 5.8 5.5 8.5 5.7 5.7

‡ ‡
Density (g/mL) 1.02 1.012 1.005 1.01 1.02
‡
Osmolality 347 - 383 462 418 393 237 270 - 310
(mOsm)

Anticoagulant CPD CPD CP2D CPD CPD ACD CPD

FDA-licensed No Yes Yes Yes Yes No No

Countries used Europe United United United United Japan Germany

United States States States States
Kingdom Canada Europe
Australia
Canada
New
Zealand
Adapted from Sparrow.2
*Data were supplied and verified by manufacturers.
†
Brand names and primary sources are listed for each solution. Formulations may be available through other sources.
‡
Not reported.
SAGM = saline, adenine, glucose, and mannitol; AS = additive solution; MAP = mitogen-activated protein; PAGGSM = phos-
phate-adenine-glucose-guanosine-saline-mannitol; NaCl = sodium chloride; NaHCO3 = sodium bicarbonate; Na2HPO4 =
sodium hydrogen phosphate; NaH2PO4 = monosodium phosphate; Na3-citrate = trisodium citrate; FDA = Food and Drug
Administration.
 CHAPTER 6 Whole-Blood Collection and Component Processing
crossmatching, antigen typing, investigation
of adverse events of transfusion, or other ap-
propriate laboratory tests. A unique number is
imprinted on each segment. A printed DIN is
wrapped around the segment and is archived
for investigative purposes in case any transfu-
sion reactions occur.
Containers also contain integral access
ports for open connection of infusion sets or
other spike entry. Such open access limits the
outdate from the time of entry to reduce the
risk of septic transfusion reactions caused by
bacteria. This period may range from 24 hours
for RBCs held at 1 to 6 C to 4 hours for RBCs
held at room temperature, cryoprecipitate,
plasma, and platelets.
Base labels and any additional labels that
are placed directly on the container must use
approved adhesives. In accordance with the
FDA guideline issued in 1985 for the uniform
labeling of blood and blood components, only
those substances that are FDA approved as
“indirect food additives” may be used in adhe-
sives and coating components for labels
placed over the base label.3 The FDA has addi-
tional standards for labels that are applied di-
rectly on plastic blood containers. Because of a
lack of sufficient space for attaching labels di-
rectly to the container, tie tags may be used as
an appropriate alternative, particularly for
items that do not have to be directly affixed to
the container. National regulatory require-
ments should be verified when selecting labels
and establishing labeling policies.
Diversion Pouch
Collection systems intended for the prepara-
tion of platelets must include a diversion
pouch immediately after the access nee-
dle.4(p22) Diversion of the first few milliliters of
blood into a pouch has been shown to reduce
the risk of bacterial contamination of the
blood unit by capturing bacteria from the skin
(also known as a skin plug).5 The diversion
pouch should be isolated by a heat seal or met-
al clip prior to any collection into the primary
collection container. After the pouch is isolat-
ed, blood in the pouch may be used for donor
testing.
䡲 139
Container Modification by Sterile
Connecting Device
Blood containers may be modified by using a
sterile connecting device cleared or approved
by the appropriate regulatory body for that
purpose.6 FDA-approved indications for the
use of a sterile connecting device are present-
ed in Table 6-4. Use of such a device is increas-
ing, primarily to obtain a sample from aphere-
sis platelets for bacterial contamination
testing, attaching a leukocyte reduction filter
to prepare prestorage leukocyte-reduced (LR)
RBCs and platelets, and preparing prestorage
pooled platelets and pooled cryoprecipitate.
Recently available computer software permits
tracking of lot numbers (eg, for wafers, bags,
and filters) and product codes, as well as other
steps that involve the scanning of bar codes.
Donor and Blood Component
Identification
Identification of blood components and test
results to maintain association and linkage to
the blood donor is critical to ensure the trans-
fusion recipient’s safety by permitting look-
back investigations and product withdrawals if
indicated. Before phlebotomy, the donor is
asked to present appropriate identification.
Donor-identifying information commonly in-
cludes the donor’s first name, middle name or
initial, last name, and birth date. A donor’s So-
cial Security number is less commonly used as
an identifier because of concerns about identi-
ty theft. Contact information is necessary for
future recruitment and notification of the do-
nor about abnormal test results.
The blood component identity (ID) pro-
cess uniformly uses both a bar-coded and an
eye-readable, unique DIN that is assigned to
each sample tube and each component pre-
pared from the donation. The donor history
form and blood sample tubes are similarly la-
beled with the unique number for each dona-
tion. Electronic records of the donation are
also assigned the same number. The DIN
should be verified on the donation record, col-
lection set primary and secondary containers,
and sample tubes before blood collection can
140 䡲 AABB TECHNICAL MANUAL
TABLE 6-4. Use of Sterile Connecting Devices for Modification of Collection Containers6
Use
To add a new or smaller needle to a blood collection
set
To prepare components
To pool blood products
To prepare an aliquot for pediatric use and divided
units
To connect additional saline or anticoagulant lines
during an automated plasmapheresis procedure
To attach processing solutions
To add an FDA-cleared leukocyte reduction filter
To remove samples from blood product containers
for testing
RBC = Red Blood Cell; FDA = Food and Drug Administration; SOPs = standard operating procedures.
proceed as well as during and after the collec-
tion.
Phlebotomy
Vein Selection
The phlebotomist selects one arm for phlebot-
omy after inspecting both of the donor’s arms.
The phlebotomist checks for the presence of a
prominent, large, firm vein in the antecubital
fossa to permit a single, readily accessible
phlebotomy site that is devoid of scarring or
skin lesions. A blood pressure cuff inflated at a
pressure of between 40 and 60 mm Hg or a
tourniquet is applied to enlarge the vein. Pal-
pating the vein is also recommended before
the phlebotomy site is prepared.
The first choice for the phlebotomy site is
the well-anchored median vein, which is cen-
trally located in the antecubital fossa. The sec-
ond choice is the cephalic vein that lies laterally
Comment
If a needle is added during the procedure, an
approved device to weld liquid-filled tubing should be
used.
Examples include adding a fourth bag to make cryo-
precipitate, adding solution to the RBC unit, or adding
an in-line filter.
Appropriate use of a sterile connecting device to pool
platelets prepared from whole blood collection may
obviate potential contamination from the spike and
port entries commonly used.
FDA provides specific guidance if this activity is con-
sidered to involve manufacturing of new products.
SOPs are required, although prior approval from the
FDA is not required.
Examples include washing and freezing RBCs.
The purpose is to prepare prestorage leukocyte-
reduced RBCs.
The label may need revision if the product’s cell count
is affected.
(shoulder side) and is often superficial. The
third choice is the basilic vein, which lies on the
underside of the antecubital fossa; the basilic
vein is not well anchored and may roll during
phlebotomy. Excessive probing with the needle
should be avoided to prevent nerve injury.
Although the Occupational Safety and
Health Administration provides an exemption
from the requirements to wear gloves during
phlebotomy of volunteer blood donors, some
blood collection centers require their employ-
ees to wear gloves for phlebotomy of all alloge-
neic and autologous donors.
Disinfection Methods for Venipuncture
Site
Specific instructions in the product insert for
the use of approved agents should be followed
for arm and phlebotomy site preparation
(Method 6-2). These methods provide surgical
 CHAPTER 6 Whole-Blood Collection and Component Processing
cleanliness, but none of the methods can
achieve an absolutely aseptic site.
Approximately 50% of donors have no
bacterial colonies on culture of the venipunc-
ture site after disinfection with povidone io-
dine or isopropyl alcohol plus iodine tincture.
Bacterial growth with low colony numbers (1
to 100) occurs in about half of the donors.
More than 100 colonies is rare (1%) in such
cultures.7 Bacteria residing deep within skin
layers are not accessible to disinfectants.
Therefore, if skin tissue enters the collection
bag, it can result in contamination. In one
study, pigskin epidermal cells were detect-
able in the lavage fluid in 1 out of 150 punc-
tures.8 Whether human skin fragments or epi-
dermal cells are carried into the bag during
routine blood donation has not been studied
in detail. Nonetheless, AABB Standards for
Blood Banks and Transfusion Services (Stan-
dards)4(p22) requires collection containers that
divert the first few milliliters of blood into a
special pouch that retains skin plugs to pre-
vent contamination when platelet compo-
nents will be prepared from the collection.
Diversion of the first aliquot of blood passing
through the needle has been shown to reduce
the proportion of blood products containing
viable bacteria.9
Blood Collection Process
Method 6-3 describes steps for blood collec-
tion and sample collection for processing and
compatibility tests. The average time to collect
500 mL of blood is less than 10 minutes. A
draw time longer than 15 to 20 minutes may
not be suitable for collecting platelets or plas-
ma for transfusions.
The collection bag should be periodically
mixed during the collection to ensure uniform
distribution of anticoagulant. Devices are
available to automatically mix the unit during
collection.
Samples for donor testing may be collect-
ed from the diversion pouch after the pouch is
isolated either by clamping or sealing the entry
line. Samples for testing may also be aseptical-
ly obtained from the collection bag with either
an integrally attached sample container or a
䡲 141
sampler that is sterile and connected to the
bag. If insufficient volume is available for test
samples, a second phlebotomy may be per-
formed immediately after the blood collection.
Care should be taken to avoid diluting sample
tubes.
Blood collection containers now include
safety devices, such as a sliding sheath needle
guard, to prevent accidental needle-stick inju-
ries. These devices allow retraction of the nee-
dle into a safety guard at the completion of
blood collection. Capping of needles should
be avoided to prevent injuries.
Volume of Blood Collected
AABB Standards permits collection of 10.5 mL
of blood per kilogram of the donor’s weight for
each donation, including the blood unit and
all samples for testing.4(p60) In North America
and Europe, the volume of blood collected
during routine phlebotomy is typically either
450 ± 10% (405-495 mL) or 500 mL ± 10% (450-
550 mL). The volume may be different in other
regions and may be as low as 200 to 250 mL.
For general blood banking applications, the
volume of blood collected can be determined
from the net weight in grams collected divided
by the density of WB (1.053 g/mL).
Empirical estimates may be used for the
density of RBC components as reported by
Burstain et al.10 The theoretical density may
also be calculated from the densities of the red
cells (DRBC), density of the suspending medium
(DS), and measured hematocrit (H, L/L): den-
sity of RBC component = (DRBC – DS ) × H + DS ×
(1 – H).
Collection volumes must be within the
manufacturer’s specified range to ensure the
correct anticoagulant-to-WB ratio. High-vol-
ume allogeneic collections should be discard-
ed. Low-volume allogeneic collections should
be relabeled as “RBCs Low Volume” (Table 6-5).
Plasma and platelets from low-volume units
should be discarded.
Low-volume autologous collections may
be retained if they are approved by a physi-
cian. Plasma from low-volume units has a
higher citrate concentration than plasma from
high-volume units and should be discarded.
142 䡲 AABB TECHNICAL MANUAL
TABLE 6-5. Weight and Volumes of Routine-Volume, Low-Volume, and Overweight Whole Blood
Collections*
450 mL Collection Bag
Low-Volume Collection Volume
Weight
Routine-Volume Collection Volume
Weight
Overweight Collection Volume
Weight
*A density of 1.053 g/mL was used for conversion calculations. The volume and weight of anticoagulant and bag(s) are not
accounted for in this table. Low-volume Red Blood Cell products must be labeled as low-volume products.
The evidence indicates that the volume
collected does not affect in-vivo red cell sur-
vival. Red cell recovery (average) after 21 days
of storage in CPD was reported to be accept-
able (approximately 80%) when only 300 g
(285 mL) of blood was collected.11 Recovery
was more than 90% when 400 g (380 mL) of
blood was collected and stored for 21 days.
Similarly, as much as 600 g (570 mL) of blood
collected in a standard 450-mL CPD container
and stored for 21 days had normal in-vivo red
cell recovery. Undercollected units (275 mL or
290 g) in CPDA-1 stored for 35 days had a
mean 24-hour survival rate of 88%.12 These
data show that for undercollected and overcol-
lected units, variability in the amount collect-
ed does not adversely affect red cell storage for
21 to 35 days.
Volumes of other components may also
be determined gravimetrically by dividing the
net weight in grams by the appropriate density
in g/mL. Table 6-6 provides generally accepted
densities and, for some, the applicable regula-
tory documents provide a specific value.13,16
Often, the specific gravity (ratio of density
component to density water) is used for this
purpose, assuming that the density of water is
1.00 g/mL.
Donor Care After Phlebotomy
Immediately after collection, the needle is
withdrawn into a protective sleeve to prevent
accidental injuries. Local pressure is applied
500 mL Collection Bag
300-404 mL Volume 333-449 mL
316-425 g Weight 351-473 g
405-495 mL Volume 450-550 mL
427-521 g Weight 474-579 g
>495 mL Volume >550 mL
>521 g Weight >579 g
by hand to the gauze placed directly over the
venipuncture site while the donor’s arm is kept
elevated. Pressure is applied until hemostasis
is achieved, which may require more time for
donors who are taking anticoagulants or anti-
platelet drugs. Once hemostasis is evident, a
bandage or tape may be applied.
Postphlebotomy care includes observing
the donor for signs or symptoms of reactions.
If the donor tolerates a sitting position without
problems, he or she may proceed to the can-
teen area and is encouraged to drink fluids and
wait until being released. Local or state laws
may specify the amount of time that the donor
must wait after the donation and before leav-
ing the collection area. The donor is encour-
aged to drink more fluids and refrain from
heavy exercise for the next 4 hours, avoid alco-
hol ingestion for several hours, and refrain
from smoking for 30 minutes. The donor is
also instructed to apply local pressure to the
phlebotomy site if any bleeding recurs and to
call the blood center if the bleeding does not
stop with pressure. A telephone number is
provided so that the donor can report if he or
she feels that the donated unit should not be
used, has any reactions, or experiences any
signs or symptoms of infection.
Adverse Donor Reactions
In donor follow-up surveys, minor reactions,
such as dime-sized hematomas at the phlebot-
omy site, are reported by as many as one-third
 CHAPTER 6 Whole-Blood Collection and Component Processing 䡲 143

TABLE 6-6. Density of Principal Blood Cells and Components

Blood Cell or Component Specific Gravity Source*

Cell Council of Europe13

Platelet 1.058
Monocyte 1.062
Lymphocyte 1.070
Neutrophil 1.082
Red cell 1.100
Components
Red cells collected by automated 1.06† FDA guidance14
methods with additive solution
Red cells collected by automated 1.08† FDA guidance14
methods without additive solution
Apheresis Platelets 1.03 FDA guidance15
Plasma Dependent on manufacturer of
plasma or plasma derivatives
Octapharma AG‡ 1.026
CSL Plasma§ 1.027
Fenwal (Amicus Cell Separator) II
1.027
Whole blood 1.053† FDA guide for inspection of blood
banks16
*Specific gravity (SG) is the density relative to water. Assuming a density of water of 1000 g/mL, the values of SG and
density are equal.
†
See text for alternate methods to determine density of whole blood and Red Blood Cell components.
‡
Octapharma AG, Lachen, Switzerland.
§
CSL (previously ZLB) Plasma, Boca Raton, FL.
II
Fenwal, Lake Zurich, IL.
FDA = Food and Drug Administration.

of all donors.17 Adverse reactions occur at the
time of donation or are reported later in about
3.5% of donations. The American Red Cross
observed a similar rate of 4.35% (435 per
10,000 donations) in 4.3 million donations in
2007 (see Table 6-7).18 Reactions that need
medical care after the donor has left the dona-
tion site occur in 1 in 3400 donors. A popula-
tion-based European study found the rate of
complications leading to long-term morbidity
or disablement to be 5/100,000 donations and
2.3/100,000, respectively.19 Donor hemovigi-
lance programs instituted by the American
Red Cross also recorded a lower rate of compli-
cation rates for WB collections than with
apheresis methods for platelet and 2-unit RBC
collection, with minor presyncopal reactions
and small hematomas representing most of
the reactions. Major reactions were slightly
more common for WB collection (7.4/10,000)
compared with plateletpheresis (5.2/10,000)
and 2-unit red cell apheresis (3.3/10,000).20
Systemic Reactions
Vasovagal reaction complex includes dizzi-
ness, sweating, nausea, vomiting, weakness,
144 䡲 AABB TECHNICAL MANUAL

TABLE 6-7. Postcollection Adverse Event Rates in Calendar Year 2007 Based on 4,348,686 Whole
Blood Collections in the American Red Cross System*

Adverse Event Number Rate per 10,000 Collections

Minor reactions
Presyncope 120,561 277.2
Hematoma (small) 61,029 140.3
†
Citrate (minor) 31 0.1
Other (minor) 165 0.4
Allergic (minor) 30 0.1
Subtotal 181,816 418.1
LOC and major reactions
LOC (<1 minute) 4,269 9.8
LOC (>1 minute) 591 1.4
Prolonged recovery 842 1.9
LOC with injury 438 1.0
Other (major) 82 0.2
Citrate (major)† 3 0.0
Allergic (major) 2 0.0
Subtotal 6,227 14.3
Major phlebotomy-related reactions
Hematoma (large) 387 0.9
Nerve irritation 314 0.7
Arterial puncture 449 1.0
Subtotal 1,150 2.6
All adverse events 189,193 435.1
Outside medical care 1,814 4.2
18
Adapted with permission from Benjamin et al.
*Citrate reactions after WB donations represent errors in classification.
†
95% confidence interval includes 1.0.
LOC = loss of consciousness; ND = not determined.

apprehension, pallor, hypotension, and brady-
cardia. In severe cases, syncope and convul-
sions may be observed. The pulse rate is often
low during vasovagal reactions, but the rate is
often high during volume depletion. Some do-
nors with severe reactions or with prolonged
recovery times may need short-term observa-
tion, intravenous fluid administration in the
emergency room, or both. A protocol for tele-
phone follow-up of donors who have experi-
enced severe reactions is helpful to assess the
donors for any residual symptoms. Donor re-
actions after WB donation do not predict accu-
rately the possibility of recurrent syncope in
 CHAPTER 6 Whole-Blood Collection and Component Processing
returning donors, although they reduce the
likelihood of future donations.21
Approximately 60% of systemic reactions
occur in the canteen area.17 The main predic-
tors of immediate and delayed vasovagal and
presyncopal reactions are young age, low esti-
mated blood volume, and first-time donation
status.22,23 Ingestion of antihypertensive medi-
cations does not appear to be a risk factor.24
Approximately 15% of reactions occur away
from the donation site, usually within 1 hour
of donation.17 In donors experiencing a reac-
tion, an injury to head, face, or extremity may
occur. The staff should be vigilant to detect re-
actions early and prevent injuries as much as
possible. Deferral strategies for young donors
with estimated low blood volume (<3.5 liters)
and physiologic strategies to minimize donor
reactions in young donors are aimed at im-
proving donor safety.21 Donor education, envi-
ronmental controls, instructions to donors to
drink water right before donation, dietary in-
terventions, and muscle tension are all effec-
tive in reducing the rate of adverse reactions,
especially in young donors, in whom a 20% re-
duction has been observed.25,26
In case of a vasovagal reaction, phleboto-
my should be stopped, and the donor should
be placed in a recumbent position as soon as
the reaction is suspected. Applying cold wet
towels to the donor’s neck and shoulder area
and loosening the donor’s clothes can assist in
symptom management.
Oral fluid intake before and soon after
nonautomated WB donation appears to re-
duce the frequency of systemic reactions.17
Coffee ingestion can reduce the frequency of
reactions, but it may also reduce blood flow
during collection because of its vasoconstric-
tive effect.
Bruise or Hematoma
Both symptoms are common after phleboto-
my but generally do not prevent donors from
donating again.
Fatigue
First-time donors and females are more likely
to report fatigue after donation. Donor return
䡲 145
is reduced by one-third among donors experi-
encing this symptom.17
Local Nerve Injury
Nerve injuries may be unavoidable because
nerves cannot be palpated. In 40% of cases of
nerve injuries, phlebotomy is performed with-
out difficulty.17 Donors may complain of sen-
sory changes away from the phlebotomy site,
such as in the forearm, wrist, hand, upper arm,
or shoulder. These injuries are usually tran-
sient, and recovery almost always occurs; how-
ever, in 7% of injured donors, recovery may
take 3 to 9 months.17 In severe cases, referral to
a neurologist may be indicated.
Arterial Puncture
Presence of bright red blood, rapid collection
(within 4 minutes), and a pulsating needle
suggest arterial puncture. The hematoma rate
is higher with arterial puncture. When punc-
ture is recognized early, the needle should be
pulled out immediately, and local pressure
should be applied for an extended period.
Most donors recover quickly and completely,
but some might present with waxing and wan-
ing hematomas and should be evaluated for
pseudoaneurysm by ultrasound studies.
Upper-Extremity Deep Vein Thrombosis
Only one case of this thrombosis has been re-
ported in the literature.17 Symptoms include
pain; antecubital fossa tenderness; swelling of
the arm; and a prominent, palpable, cord-like
thickening of the thrombosed vein. Medical
referral of donors who are experiencing deep
vein thrombosis should not be delayed so that
treatment can begin promptly.
Postdonation Mortality
The FDA requires blood establishments to re-
port deaths caused by blood donation. For fis-
cal years 2008-2012, the FDA received 50 re-
ports of postdonation fatalities for automated
and manual collections, of which only 11 fol-
lowed WB donation. After medical review, one
case was ruled out, and in the remaining
146 䡲 AABB TECHNICAL MANUAL
10 cases, there was no evidence of a causal re-
lationship between blood donation and the
donors’ demise.27 Most deaths that take place
after blood donation are coincidentally related
to the donation rather than caused by it.
B LO O D CO M P O N E N T
PREPARATION AND
P RO C E S S I N G
Improvements and innovations continue to be
made to WB collection and its processing into
components. Apheresis is growing in impor-
tance for collection of all major components,
as discussed in Chapter 7. Single WB units are
collected in plastic containers containing anti-
coagulant; typically CDP, CP2D, or CPDA-1
(Methods 6-3, 6-4, and 6-5). Innovations in this
area include scales for monitoring the collec-
tion volume plus automatic mixing and devic-
es to add anticoagulant at a fixed ratio as the
blood is withdrawn from the vein.
Methods and devices are available to pro-
cess WB in a variety of manners for WB leuko-
cyte reduction, separation of plasma, and sep-
aration of red cells and platelets. Important
secondary processing includes leukocyte re-
duction, irradiation to prevent graft-vs-host
disease (GVHD), and pathogen reduction.
For preparation of platelets from WB, two
primary methods are available (Method 6-12):
preparation from buffy coats and preparation
from PRP. The buffy coat method is employed
in many regions but is not currently cleared in
the United States. In short, non-leukocyte-
reduced WB units are first centrifuged under a
high g-force (ie, hard or heavy spin) and plas-
ma, and red cells and buffy coats are removed
for further processing. Buffy coats from 4 or 5
units are pooled with 1 unit of plasma and
then centrifuged under a low g-force (ie, soft or
light spin) to separate the platelet concentrate
for additional processing, such as leukocyte
reduction. These processes are often associat-
ed with extended holding of the WB and buffy
coats for 8 to 24 hours at 20 to 24 C.28
Preparation of platelets from PRP begins
with a soft spin of the WB, followed by separa-
tion and hard spin of the PRP. Plasma for fur-
ther processing is removed from the platelet
pellet, which is held undisturbed for 30 to 60
minutes before being resuspended.29 Leuko-
cyte reduction may occur at the PRP or final
platelet concentrate stage, depending on the
device system used. Platelet concentrates are
labeled and stored as individual units or, if
cleared by regulatory authorities, pooled and
stored as transfusable doses.30
Compared to the PRP preparation meth-
od, the buffy coat method yields more plasma,
greater red cell loss, better initial white blood
cell (WBC) reduction before filtration, and
moderate reduction in viable bacteria in the
platelets.
Plastic collection containers, satellite
bags, and integrally attached tubing that are
hermetically sealed allow component manu-
facturing to take place in a closed system. The
blood container should not be entered before
issue except for the purposes of blood collec-
tion or transfer of components to a different
container. The container material should not
have any adverse effect on the safety, purity, or
potency of the blood. Components prepared
with an open system require a reduction in
their expiration time from the time that the
system was opened. The use of approved ster-
ile connection devices maintains a functional-
ly closed system when various connections are
performed, such as pooling or sampling,
thereby maintaining the product’s original ex-
piry date.
WB Handling Before Component
Processing—Timing and Temperatures
The temperature conditioning and handling of
WB immediately after collection is determined
by downstream processing requirements for
component preparation. In some cases, this
may mean that collections at mobile blood
drives or fixed collection sites should be trans-
ported as soon as possible to the central com-
ponent preparation laboratory. With other
processing methods, transportation may not
be as urgent.
Requirements for cooling and transporta-
tion methods are quite variable, and the speci-
fications of the device manufacturer should be
 CHAPTER 6 Whole-Blood Collection and Component Processing
carefully followed. By the time phlebotomy is
completed, the blood has air cooled to about
30 C.31 If such units are left at the ambient tem-
perature, the cooling rate is quite slow and
about 6 hours more are needed for units to
reach 25 C.31 To cool blood more rapidly, units
are typically placed in specific storage envi-
ronments. Some centers use cooling plates
that provide rate-controlled cooling toward
20 C. These cooling plates contain 1, 4-butane-
diol that has a melting temperature of 20 C and
serves as a heat absorber. With the cooling
plates, about 2 hours are needed for the col-
lected blood to reach 20 C.31
Many WB leukocyte reduction systems al-
low filtration at ambient temperature begin-
ning soon after collection and lasting up to 24
hours. WB filtration may also be started and/
or completed at refrigerator temperatures. WB
destined for platelet preparation should not be
cooled to less than 20 C. Platelets may need to
be separated from WB within 8 hours in some
regions or with some collection systems. Other
methods are approved or are under consider-
ation for up to a 24-hour hold of WB at 20 to 24
C before separation of plasma and platelets.
All processes for preparing platelets by the
buffy coat methods have an extended hold of
WB at 20 to 24 C for approximately 2 to 24
hours following collection and before the sep-
aration of the buffy coat and plasma.13 The
pooled buffy coat is often held undisturbed for
approximately 2 to 18 hours at 20 to 24 C be-
fore separation of the platelet concentrate.
The details of these hold times are gener-
ally adjusted to ease the operational logistics
for the blood center. For example, morning
collections are held until the afternoon when
plasma is separated and buffy coats prepared.
The buffy coats are held overnight as either in-
dividual units or pools, and the platelets are
separated the following morning. Likewise, af-
ternoon collections may be held overnight, the
buffy coats are prepared the next morning,
and platelets are prepared in the afternoon.
WB that will not be used to prepare plate-
lets should be cooled to refrigerator tempera-
ture as soon as possible; this is often accom-
plished by placing the unit on wet ice or other
appropriate cooling media. The allowable time
䡲 147
window for separation of plasma from WB is
quite variable from region to region. FFP
should be separated from the WB and frozen
within the time frame specified in the direc-
tions for use of the blood collection, process-
ing, and storage system. In the United States,
plasma that is separated from WB collection
and placed in the freezer within 8 hours can be
labeled “FFP”; if it is within 8 to 24 hours after
collection of WB (manual or apheresis meth-
od), it can be labeled as “Plasma Frozen Within
24 Hours After Phlebotomy.” In Europe, if the
WB is refrigerated, plasma may be separated
within 18 hours; if the WB is held at 20 to 24 C
for platelet production, plasma can be separat-
ed within 24 hours, frozen, and labeled as FFP.
Shipping containers and cooling methods
used to transport and hold WB and compo-
nents must be sufficiently validated according
to local and national regulations and guide-
lines. Validation is generally performed with
various quantities of units in the transport
container and a fixed amount of ice or gel-
pack to determine the number of units that
can be stored and transported while maintain-
ing the desired temperature. Portable temper-
ature monitors are available to record temper-
ature continuously.
Separation by Centrifugation
All current methods for separation and prepa-
ration of the three major blood components—
RBCs, platelets, and plasma—rely on one or
more centrifugation steps. As mentioned
above, the first step may be a high g-force cen-
trifugation followed by a low g-force step for
the buffy coat platelet preparation method
(the opposite of the PRP platelet separation
method) or an automated sequence of steps
in recently developed systems. Centrifuges
should be properly validated, maintained, and
calibrated in a way that is consistent with local
and regional guidelines. Centrifuge methods
for blood component preparation should be
calibrated or checked in a systematic manner
to verify the processing conditions.
The major variables that affect the recov-
ery of cells from WB by differential centrifuga-
tion are rotor size, centrifuge speed, duration
148 䡲 AABB TECHNICAL MANUAL
of centrifugation, and acceleration/decelera-
tion protocol. Published papers often refer to
relative centrifugal force (g-force) that is de-
rived from the radius of the centrifuge rotor
and its revolutions. For a given centrifuge, the
rotor size is generally not variable. Therefore,
the other two variables (centrifuge speed and
duration) can be altered in a stepwise fashion
in a simplex strategy to determine the optimal
conditions for preparing PRP.32 The simplex
strategy can also be used to identify the opti-
mal conditions of centrifugation for platelet
concentrates when quality control (QC) data
show that the platelet counts in the platelet
concentrates are not satisfactory. Method 8-4
describes a process of functional calibration of
the centrifuge to maximize platelet yield. The
device manufacturer should be consulted for
recommendations on validating the perfor-
mance of automated systems.
On the day of preparation, some platelet
units may contain clumps composed of plate-
let aggregates.33 In routine practice, visual in-
spection is adequate to determine the degree
of clumping subjectively and ensure that units
with excessive clumping are not released for
labeling. Most of the clumps seen on day 0 dis-
appear on day 1 of storage with continuous ag-
itation, particularly those showing light to
moderate clumping.33 The temperature at
which platelets are prepared may influence
clumping; platelets prepared at 24 C appear to
show the least amount of clumping compared
to those prepared at less than 24 C.33 Visual in-
spections after the platelet concentrates are
prepared have shown an absence of visible red
cells in the vast majority of units, which im-
plies that the units contain fewer than 0.4 × 109
red cells. Generally, the number of red cells in
a unit of platelets does not exceed 1.0 × 109.34
Blood Component Division
Following separation by centrifugation, com-
ponents must be carefully divided into sepa-
rate containers for further processing. Many
laboratories use manual expressers for this
purpose. Automated and semiautomated de-
vices, available in some regions, control the
rate of expression, monitor component
weights, add storage solutions, clamp and seal
tubing, and perform other useful functions
that assist in the consistent preparation of
blood components. Some systems also com-
bine nearly all of the functions, including cen-
trifugation, component expression, filtration,
sealing, and additive addition, without relying
on operator interventions. The systems and
methods used for these steps, whether
predominantly manual or fully automated,
should be validated by the user.
Blood component extractors are avail-
able for making components in a semiauto-
mated manner. After primary centrifugation,
WB is placed in the extractor, and a pressure
plate creates an outflow of components from
the container. Outflow can occur from the top
or bottom of the container, depending on the
device.
When a cellular interface is detected by
an optical sensing device, outflow tubing is au-
tomatically clamped at an appropriate level.
Such devices may improve the standardization
of components, but they are not widely used in
the United States. An automated system is
available in Europe that performs multiple
functions to prepare pooled platelet concen-
trates derived from WB by the buffy-coat
method. Steps that are automated include
pooling, rinsing, centrifugation, transfer, filtra-
tion, and sealing.
DESCRIPTIONS OF MAJOR
B LO OD CO M P O N E NTS
WB
The minimum hematocrit of WB units in anti-
coagulant-preservative, such as CPD, is usual-
ly approximately 33%. WB is most often sepa-
rated into components and is rarely used for
transfusion directly. Severe hemorrhage cases,
such as those resulting from trauma, may ben-
efit from fresh WB transfusions when platelets
are not available.35 Labile coagulation factors
diminish and platelets may activate and devel-
op a storage lesion as the storage interval in-
creases.
WB in ACD, CPD, or CP2D has an expira-
tion date of 21 days when stored at 1 to 6 C; the
 CHAPTER 6 Whole-Blood Collection and Component Processing
maximum storage time for units in CPDA-1 is
35 days. WB may be reconstituted by combin-
ing RBCs with thawed plasma to achieve a de-
sired hematocrit level—for example, when
used for neonatal exchange transfusions. Ad-
ditional information about the preparation of
reconstituted WB is provided in Chapter 9.
RBCs
RBCs in anticoagulant-preservative CPD or
CPD2 have a shelf life of 21 days at 1 to 6 C with
a hematocrit of 65% to 85%, or 35 days in
CPDA-1 with a hematocrit of <80%. The addi-
tion of additive solution (Table 6-3) reduces
the hematocrit to approximately 55% to 65%.
Additive solutions maintain the red cells dur-
ing storage (for example, reduce hemolysis)
and extend the expiry date to 42 days or 56
days, depending on regulatory approvals. He-
molysis at the end of storage should be less
than 1% in the United States or less than 0.8%
in the European Union (EU).
Hemoglobin content per unit varies be-
cause of differences between donors and pro-
cessing specifics. For example, more hemo-
globin is generally lost when buffy coat
preparation methods are used than with PRP-
type methods.
Hemoglobin content per unit may be
more precisely controlled with automated col-
lections. Total hemoglobin content is not di-
rectly regulated in the United States but has a
lower limit of 45 g per unit or 40 g per unit for
leukoreduced RBCs in the EU.36 Some experts
have advocated for standardizing the amount
of hemoglobin per RBC unit at 50 g.37 Depend-
ing on local practice, RBCs stored for less than
7 to 10 days are issued for neonatal or pediatric
transfusions, and some neonatologists may
prefer RBCs without additive solutions (see
Chapter 23).
RBCs that are labeled as low-volume units
are made available for transfusion when 300 to
404 mL of WB is collected into an anticoagu-
lant volume calculated for 450 ± 45 mL, or
when 333 to 449 mL of WB is collected into an
anticoagulant volume calculated for 500 ± 50
mL. Although the resulting RBCs may be trans-
䡲 149
fused, other components, such as platelets,
plasma, and cryoprecipitate, should not be
prepared from low-volume units.
RBCs may be subject to several secondary
processing steps, for example, leukocyte re-
duction, gamma or x-ray irradiation to prevent
GVHD, and pathogen reduction. The FDA re-
quires leukoreduced units to contain <5 × 106
WBCs per unit and retain >85% of the preleu-
koreduced RBC content. The EU regulations
call for <1 × 106 WBCs per unit.
Retention segments are held at the blood
center for 7 days after the expiration date of
the unit containing red cells and at the hospi-
tal transfusion service for 7 days after the
transfusion. Hemolysis identified in a seg-
ment by visual inspection often does not cor-
relate with the presence of hemolysis in the
unit. In one study, approximately three-
fourths of the visual assessments did not agree
with the hemoglobin levels measured by
chemical methods, indicating a high false-
positive rate with visual assessment.38
Visual inspection of RBC units can detect
white particulate matter, abnormal color
caused by bacterial contamination, hemoly-
sis, and clots. Abnormal color caused by bacte-
rial contamination may be observed in the
bag, where some segments appear to be darker
than others because of oxygen consumption in
the bag. The bag content may look purple with
or without hemolysis, and large clots may be
evident. The unit can be centrifuged to facili-
tate inspection of the supernatant in the case
of suspected bacterial contamination, and vi-
sual inspection of the supernatant may reveal
murky, brown, or red fluid.39 However, visual
inspection will not detect all contaminated
units.
Blood clots in RBC units are often too
small to be detected by visual inspection. Clots
are sometimes revealed during transfusion
when they clog the filter or in the component
laboratory when the units are filtered through
a leukocyte reduction filter. Units known to
have clots should not be released for transfu-
sion.
150 䡲 AABB TECHNICAL MANUAL
Platelets
The two major methods used in preparing
platelets from WB, buffy coat, and PRP, are de-
scribed above and in Method 6-12. Single units
of platelets are normally suspended in 40 mL
to 70 mL of plasma, although studies have
shown good recovery and survival rates when
platelets are stored in plasma volumes of 35 to
40 mL.40,41 Buffy-coat-derived platelets are
nearly always pooled with the addition of
1 unit of plasma from one of the donor units or
the addition of a platelet additive solution. In
the United States, no platelet additive solu-
tions are approved for use with platelets pre-
pared from WB. Platelets prepared by the PRP
or the buffy-coat methods can be further pro-
cessed to reduce leukocytes by using a leuko-
cyte reduction filter as described in Method 6-
12.
Platelets have demonstrated superior
in-vivo recovery when stored between 20 and
24 C.42 However, at this storage temperature,
contaminating bacteria can proliferate during
storage and result in septic transfusion reac-
tions, some of which are fatal. Therefore,
platelet shelf life is limited to 5 days in the
United States, 3 days in Japan, 4 days in Ger-
many, and up to 7 days in most EU and other
countries. These limits are primarily deter-
mined by regulatory risk assessments of the
likelihood of septic transfusion reactions.
Practices to prevent and detect bacterial
contamination have been effectively imple-
mented for apheresis platelets and prestorage
pooled platelets from WB. However, develop-
ing effective and affordable detection methods
for single-unit platelets from WB has proved
challenging, although several manufacturers
are working to develop an effective testing so-
lution. Pathogen-reduction technologies are
anticipated by many to be the best general
protection but have been slow to gain regula-
tory approval and be implemented in the field.
Platelets are metabolically active
throughout the storage period. They use both
glycolysis and oxidative phosphorylation to
produce adenosine triphosphate (ATP), a re-
quirement to maintain cell integrity and func-
tion. The product of glycolysis in platelets, lac-
tic acid, is buffered by bicarbonate and
exogenous buffers in additive solutions (eg,
phosphate). These buffer systems are limited,
and lactic acid can cause the pH of the stored
platelets to decline to less than 6.2 at 22 C, a
level that results in unacceptably low in-vivo
recoveries.43 Use of oxidative phosphorylation
does not result in acid production, and it is a
much more efficient pathway for ATP produc-
tion that, in turn, reduces the need for glycoly-
sis to run at a high rate—again limiting acid
production. Modern platelet storage contain-
ers permit oxygen to enter the storage bag to
support oxidative phosphorylation and car-
bon dioxide escape; the latter maintains the
bicarbonate buffer function. This advance in
storage containers has allowed the extended
storage of platelets beyond 3 days. The use of
acetate, a component of many platelet addi-
tive solutions, as a metabolic fuel also contrib-
utes positively to pH control because it con-
sumes protons and is metabolized through
oxidative phosphorylation.44
Because of their metabolic requirements,
platelets must be continuously agitated during
storage. This action keeps platelets suspended
in the storage media, ensuring effective ex-
change of oxygen, carbon dioxide, and lactic
acid between the platelets and the suspending
media. Long periods of static storage of plate-
lets interrupts this dynamic and can result in
lactic acid production with a decrease in pH.45
During transport to hospitals from the blood
center or long-distance air transport, when
components are exchanged between blood
centers, platelets are not agitated. Instead,
they are double wrapped and secured in a
shipping system. In-vitro studies have shown
that platelets are not damaged when they are
stored without agitation for 24 hours.
Platelets must be stored and shipped at
20 to 24 C. Shipping, temporary equipment
failures, and power outages present challenges
to meeting this requirement. Platelets main-
tained in-vitro function when they are stored at
37 C for 6 hours followed by room-temperature
storage without agitation for an additional 18
hours.46 Several studies have demonstrated
negative effects on in-vivo platelet recovery
and survival when platelets are stored at tem-
 CHAPTER 6 Whole-Blood Collection and Component Processing 䡲 151

peratures <20 C.47,48 Thus, proper steps should stored at –18 C or colder. FFP that is stored at
be taken to maintain the required range of –65 C may be stored for longer than 12
temperatures during storage at the blood cen- months, but such storage requires FDA ap-
ter and during transport. proval.4(pp56-57) Rapid freezing of plasma can be
accomplished using a blast freezer, dry ice, or a
Plasma mixture of dry ice with either ethanol or anti-
freeze. Plasma should be thawed at 30 to 37 C
Plasma preparations are defined and regulated
in a waterbath or by using an FDA-cleared de-
through a dizzyingly broad combination of
vice. When a waterbath is used, the compo-
collection methods, storage temperatures,
nent should be placed in a protective plastic
freezing methods, secondary processing, tim-
overwrap. Thawing of larger units of FFP col-
ing, and storage after thawing. These specifi-
lected by apheresis may require more time.
cations are covered in an array of standards,
FFP, once thawed, has a shelf life of 24 hours at
rules, and guidelines overlaid with various re-
1 to 6 C. Thawed plasma held longer than 24
quirements of the country where the plasma is
hours must be relabeled as Thawed Plasma,
prepared and/or used. Major, although not ex-
and it can be stored for an additional 4 days at
haustive, sources of this information include
1 to 6 C.
the US Code of Federal Regulations, FDA guid-
In the past several years, many blood cen-
ance documents, the US Circular of Informa-
ters in the United States have increased the
tion,49 the AABB Standards, and EU directives.
amount of WB collected from 450 mL to 500
The definitions and requirements of the coun-
mL, resulting in a larger amount of plasma per
try where the plasma is prepared should al-
unit. A unit can contain 500 mL to 800 mL of
ways be consulted.
plasma when collection is performed by auto-
Plasma is prepared from WB collections
mated (single-donor) plasmapheresis.
and by apheresis. Plasma is generally frozen to
The Council of Europe defines “plasma,
maintain factor activity and provide an ex-
fresh frozen” as prepared from either WB or
tended shelf life. Frozen plasma is thawed for
collected by apheresis. Plasma freezing must
clinical use and may be maintained at 1 to 6 C
be initiated within 6 hours of collection, within
for some time prior to use. Frozen plasma is
18 hours if the WB is held at 1 to 6 C, or within
also the source of cryoprecipitate and cryopre-
24 hours if WB or apheresis plasma is rapidly
cipitate-reduced plasma. There are several
conditioned to 20 to 22 C following collection.
methods available for pathogen reduction of
Freezing must be completed within 1 hour to
plasma that may be applied depending on na-
less than –30 C. Plasma, fresh frozen has an ex-
tional regulatory approvals. Plasma may be
piry time of 36 months if held at less than –25 C
used for preparation of specific plasma protein
or 3 months if held at –18 C to –25 C.13 The
products through fractionation processes. The
Council of Europe does not address specific
descriptions below are based on the prevailing
thawing methods or treatment of the plasma
guidance documents and regulations of the
following thawing, including expiry dating.
FDA and Council of Europe.
AABB requires4(p17) interventions to mini-
mize the preparation of high-plasma volume
FFP
components (apheresis platelets, plasma prod-
In the United States, FFP is plasma collected ucts, and WB) for transfusion from donors who
either from a single unit WB collection or by are at risk of developing HLA antibodies.50
apheresis. FFP must be placed in the freezer These products should be collected from
within 8 hours of collection; within 6 hours if males, never-pregnant females, or parous fe-
anticoagulated with ACD; or as directed by the male donors who test negative for HLA anti-
manufacturer’s instructions for use of the bodies.
blood collection, processing, and storage sys- FFP contains normal amounts of all coag-
tem. FFP has a shelf life of 12 months when ulation factors, antithrombin, and ADAMTS13.
152 䡲 AABB TECHNICAL MANUAL
Quarantine FFP
Quarantine plasma was introduced to increase
the viral safety of plasma. The Council of Eu-
rope notes that quarantine FFP can be re-
leased from quarantine after the donor returns
to the blood center and has repeatedly nega-
tive test results for, at a minimum, hepatitis B
and C viruses, HIV-1, and HIV-2 beyond a min-
imum quarantine period that is greater than
the diagnostic window period for viral infec-
tion, typically 6 months. With the use of nucle-
ic acid tests for viral screening, this window
period for quarantine FFP may be reduced.51
Plasma Frozen within 24 Hours After
Phlebotomy
The FDA defines plasma that is frozen within
24 hours of collection as Plasma Frozen within
24 Hours After Phlebotomy (PF24). Once
thawed, PF24 has a shelf life of 24 hours at 1 to
6 C. Thawed plasma held longer than 24 hours
must be relabeled as Thawed Plasma, which
can be stored for an additional 4 days at 1 to
6 C.
Plasma Frozen within 24 Hours After
Phlebotomy Held at Room Temperature
up to 24 Hours After Phlebotomy
The FDA defines Plasma Frozen within 24
Hours After Phlebotomy Held at Room Tem-
perature up to 24 Hours After Phlebotomy
(PF24RT24) as apheresis plasma that is held
for up to 24 hours after collection at room tem-
perature and then stored at less than –18 C.
Once thawed, PF24 and PF24RT24 have a shelf
life of 24 hours at 1 to 6 C. Thawed plasma held
for longer than 24 hours must be relabeled as
Thawed Plasma and can be stored for an addi-
tional 4 days at 1 to 6 C. A similar product pre-
pared from WB held at room temperature for
more than 8 hours (ie, overnight hold) may be
defined in the future if FDA approves an over-
night-hold WB process.
Thawed Plasma
The FDA defines Thawed Plasma (which is not
licensed by the FDA) as FFP, PF24, or
PF24RT24 that has been thawed and held at
1 to 6 C for >24 hours. Thawed Plasma may be
held at 1 to 6 C for up to 5 days after thawing.
Stable Factor II, fibrinogen, and reduced
amounts of other factors have been observed
in Thawed Plasma. Thawed Plasma prepared
from FFP and stored for 5 days contains re-
duced levels of Factor V (>60%) and Factor VIII
(>40%). ADAMTS13 levels are well maintained
for 5 days at 1 to 6 C in Thawed Plasma.
Plasma Cryoprecipitate Reduced
Plasma cryoprecipitate reduced (United States)
or plasma, fresh frozen cryoprecipitate deplet-
ed (Europe) is a byproduct of cryoprecipitate
preparation. In the United States, plasma cryo-
precipitate reduced must be refrozen within
24 hours at less than –18 C. The storage tem-
peratures and expiration that apply to FFP ap-
ply to this component in both the United
States and Europe. The product contains a
normal level of Factor V (85%). Even after the
removal of cryoprecipitate, the product has a
fibrinogen level of about 200 mg/dL.52 The lev-
els of the following coagulation factors have
been demonstrated to be normal in plasma
cryoprecipitate reduced: Factor I, Factor VII,
Factor X, antiplasmin, antithrombin, protein
C, and protein S. Levels of Factor VIII, the von
Willebrand factor (vWF) antigen, vWF activity,
fibrinogen, and Factor XIII are decreased.53
Liquid Plasma
In the United States, liquid plasma for transfu-
sion can be separated from WB at any time
during storage and stored at 1 to 6 C for up to 5
days after the WB’s expiration date.
Recovered Plasma
Blood centers often convert plasma and liquid
plasma to an unlicensed component, “recov-
ered plasma (plasma for manufacture),” which
is usually shipped to a fractionator and pro-
cessed into derivatives, such as albumin and/
or immune globulins. To ship recovered plas-
ma, the collecting facility must have a “short
supply agreement” with the manufacturer. Be-
cause recovered plasma has no expiration
 CHAPTER 6 Whole-Blood Collection and Component Processing
date, records for this component should be re-
tained indefinitely. Storage conditions for re-
covered plasma are established by the frac-
tionator. FFP used as human plasma for
fractionation in Europe must comply with the
applicable European Pharmacopoeia guide-
lines.
Pathogen-Reduced Plasma
Plasma can be treated to inactivate microbial
agents for pathogen reduction. Four such
methods are available and in use in Europe:
the methylene blue, psoralen (amotosalen), ri-
boflavin, and solvent/detergent treatments.
Methylene blue (approximately 0.085 mg/
unit of plasma) can be added to thawed FFP,
followed by activation using white light. After
removal of methylene blue with a filter (resid-
ual concentration: 0.3 µmol), plasma can be
frozen. Methylene-blue-treated plasma con-
tains approximately 15% to 20% less Factor
VIII and fibrinogen than untreated plasma.
Plasma prepared from WB or by automat-
ed methods can be treated with 15 mL of amo-
tosalen per 250 mL of plasma, followed by illu-
mination with ultraviolet A light (320-400 nm)
with 3.0 J/cm2. After amotosalen is removed by
exposing treated plasma to an adsorption de-
vice, the unit is frozen for storage at –18 C. Av-
erage activity values for coagulation and anti-
thrombotic factors are reported to be within
reference ranges for untreated plasma.
Plasma prepared by apheresis or from
single WB units (volume range 170-360 mL)
can be treated with the Mirasol system by add-
ing 35 mL of riboflavin (vitamin B2) followed
by illumination for 6 to 10 minutes. Immedi-
ately after illumination, the plasma can be re-
leased or frozen below –30 C for 2 years. Resid-
ual RBC levels up to 15 × 109 per liter have been
qualified to result in successful pathogen re-
duction and leukocyte inactivation. Coagula-
tion and anticoagulation proteins are well pre-
served in plasma treated with the Mirasol
system.54,55
Solvent/detergent-treated plasma (SD
plasma) is prepared from a pool of plasma
from many donors (no more than 2500) that
undergoes treatment with 1% tri-n-butyl
䡲 153
phosphate and 1% Triton X-100 for pathogen
reduction. This treatment has been shown to
significantly inactivate lipid-enveloped virus-
es. SD plasma is manufactured in facilities that
can manage large-scale production rather
than in blood centers. Each unit contains 200
mL of plasma that is stored frozen at –18 C
with an expiration date of 12 months.56 All co-
agulation factors are reduced by 10% in SD
plasma, except for Factor VIII, which is re-
duced by 20%.57 Also, SD plasma contains 50%
less functional protein S in comparison to the
nontreated FFP.58 The product is labeled with
the ABO blood group and, once thawed,
should be used within 24 hours. SD plasma is
available in Europe and has been recently ap-
proved in the United States.59
Cryoprecipitated Antihemophilic
Factor
Cryoprecipitated antihemophilic factor (AHF),
or simply “cryoprecipitate” in Europe, is pre-
pared from FFP. Cold-insoluble protein that
precipitates when FFP is thawed to 1 to 6 C is
collected by centrifugation; supernatant plas-
ma is transferred into a satellite container; the
precipitate is resuspended in a small amount
of residual plasma, generally 15 mL; and the
precipitate is refrozen as described in Method
6-11. FFP can be thawed to prepare cryopre-
cipitated AHF by placing the FFP in a refrigera-
tor (at 1 to 6 C) overnight or in a circulating
waterbath at 1 to 6 C. An alternate method that
uses microwaves for thawing has been de-
scribed. The cryoprecipitated AHF is placed in
a freezer within an hour of removal from the
refrigerated centrifuge and can be stored at
–18 C for 12 months from the original collec-
tion date. In Europe, thawing is to be per-
formed at 2 to 6 C, and the product can be
stored for up to 36 months below –25 C and for
3 months at –18 to –25 C.
AABB Standards4(pp28-29) requires that cryo-
precipitated AHF contain at least 80 interna-
tional units (IU) of Factor VIII and 150 mg of
fibrinogen per unit, although the average fi-
brinogen content is generally 250 mg.60 Euro-
pean standards are at least 70 IU/unit of Factor
VIII, 140 mg/unit of fibrinogen, and 100 IU/unit
154 䡲 AABB TECHNICAL MANUAL
of vWF. More current preparations are report-
ed to have much higher amounts of fibrinogen
(median, 388 mg/unit).61 Cryoprecipitated
AHF also contains the vWF ristocetin cofactor
activity (approximately 170 units/bag), Factor
XIII (approximately 60 units/bag), and fibro-
nectin. Rapid freezing of FFP is found to in-
crease the Factor VIII yield in cryoprecipitated
AHF.62 ADAMTS13 levels are normal in cryo-
precipitated AHF.63 Anti-A and anti-B are known
to be present in cryoprecipitated AHF, but the
combined amount of these antibodies from
the unit of plasma is only 1.15% of the total.64
Thawed cryoprecipitated AHF should be
used as soon as possible but may be held at
room temperature (20-24 C) for 6 hours as sin-
gle units or a pool prepared as a closed system
using an approved sterile connecting device or
for 4 hours if pooling was with an open system.
Pooling may be accomplished with the aid of a
diluent, such as 0.9% sodium chloride (USP), to
facilitate removal of material from individual bags.
At room temperature, the mean declines
of Factor VIII levels at 2, 4, and 6 hours are ap-
proximately 10%, 20%, and 30%, respectively.65
Cryoprecipitated AHF from blood groups A and
B has higher levels of Factor VIII compared to
that derived from blood group O donors (about
120 vs 80 IU per bag, respectively).66 Thawed
cryoprecipitate should not be refrozen.
Granulocytes
Although it is possible to prepare granulocytes
from fresh WB, current practice is to collect
granulocytes by apheresis. Refer to Chapter 7
for information on automated collections.
B LO O D CO M P O N E N T
MODIFICATION
Prestorage Leukocyte Reduction by
Filtration
For RBCs that are leukocyte reduced, the FDA
requires a residual number of <5.0 × 106 leuko-
cytes per unit. The Council of Europe requires
that the residual number be <1 × 106 per unit.
In the United States, leukocyte reduction by
filtration of RBCs should result in a compo-
nent that contains at least 85% of the original
red cell content. The Council of Europe’s stan-
dards require a minimum of 40 g of hemoglo-
bin to be present in each unit after leukocyte
reduction.13
For WB-derived platelets, AABB Stan-
dards requires that the leukocyte reduction
process ensures that 95% of the platelet units
sampled contain <8.3 × 105 leukocytes per
unit, at least 75% of the units sampled contain
5.5 × 1010 platelets, and at least 90% of the units
sampled have a pH of 6.2 at the end of the al-
lowable storage period.4(p29) The number in the
Council of Europe’s standards is <0.2 × 106 re-
sidual leukocytes per unit of platelets from
WB.13
In practice, approximately 1% of RBC
components do not achieve the levels of <1 ×
106 residual leukocytes in the component.
Sickle cell trait of red cells is the most common
cause of filter failure. Approximately 50% of
the RBC units with sickle cell trait fail to filter.
Although the other 50% pass through the filter,
the residual leukocyte content may be higher
than the allowable limits.67
Prestorage leukocyte reduction is general-
ly performed soon after WB collection and is
always performed within 5 days of collection.
In-line WB filters are available in collection
sets that permit preparation of LR RBCs and
FFP. WB filters that spare platelets are now
available as well. If WB is collected without the
in-line leukocyte reduction filter, a filter can be
attached to the tubing by an FDA-cleared ster-
ile connecting device. The number of platelets
in LR platelet concentrates is generally lower
than in non-LR platelet concentrates.
Methods that measure residual leuko-
cytes include Nageotte hemocytometry and
flow cytometry (see Method 8-11). In a multi-
center study, flow cytometry gave better re-
sults than Nageotte hemocytometry when
freshly prepared samples (within 24 hours)
were tested. For instance, at a concentration of
5 white cells per µL for RBC units, the intersite
coefficient of variation was 4.9% for flow cy-
tometry and 54% for Nageotte hemocytome-
try. In general, Nageotte hemocytometry tends
to underestimate the number of white cells
compared to flow cytometry.68 A new semiau-
 CHAPTER 6 Whole-Blood Collection and Component Processing
tomated methodology offers to reduce the
technical burden associated with both Na-
geotte and flow cytometry methods.69
Cryopreservation
RBCs
Glycerol is the most commonly used cryo-
preservative agent and is added in either high
or low concentration to RBCs within 6 days of
collection. Two commonly used protocols for
the high-glycerol method are described in
Methods 6-6 and 6-7.
Frozen RBCs must be stored at tempera-
tures colder than –65 C and expire after 10
years. Rare frozen units may be used beyond
the expiration date, but only after medical re-
view and approval based on the patient’s
needs and availability of other rare compatible
units. The units should be handled with care
because the containers may crack if they are
bumped or handled roughly. The containers
can also crack during transportation.
The units should be thawed at 37 C and
generally take about 10 minutes to thaw com-
pletely. Glycerol must be removed after thaw-
ing and before transfusion. This removal is
generally accomplished by instruments that
allow the addition and removal of sodium
chloride solutions. In most cases, addition of
the glycerol and its removal (deglycerolization)
require the system to be opened; for this rea-
son, thawed and deglycerolized units can be
stored only for 24 hours at 1 to 6 C. The final
solution in which cells are suspended is 0.9%
sodium chloride and 0.2% dextrose. Dextrose
provides nutrients and has been shown to sup-
port satisfactory posttransfusion viability for 4
days of storage after deglycerolization.70 For
QC, determining the volume of red cells in the
unit after deglycerolization and examining the
last wash for hemolysis are recommended (see
Method 6-8).
Recently, automated addition of glycerol
to RBCs and removal from RBCs in a closed
system has become possible. With this sys-
tem, glycerol is added within 6 days of WB col-
lection. Postthaw red cells prepared in this
manner are suspended in additive solution 3
(AS-3) and can be stored for 14 days at 4 C.70
䡲 155
Postwash units have a hematocrit of 51% to
53% and contain a mean of about 9.0 × 106 leu-
kocytes per unit.71
Platelets
Cryopreservation of platelets is not widely
available because the procedures for cryo-
preservation are complex and not routinely
practiced at most blood centers. Several cryo-
protectants have been described for platelet
cryopreservation72,73 However, 5% or 6% di-
methyl sulfoxide (DMSO) is most commonly
used, mainly for autologous platelet transfu-
sions in patients who are refractory to alloge-
neic platelets. Cryopreserved platelets can be
stored for at least 2 years. After thawing, the
platelet recovery rate in vitro is about 75%,
which may be reduced further if thawed plate-
lets are centrifuged to remove the DMSO be-
fore transfusion. The in-vivo recovery rate af-
ter transfusion of thawed, DMSO-reduced
platelets is about 35% to 42%.74 In vivo, the
platelets that survive after transfusion are he-
mostatically effective.75
Irradiation
Cellular blood components can be irradiated
for prevention of GVHD. Frozen plasma com-
ponents, such as FFP and cryoprecipitated
AHF, are generally not irradiated because they
are considered noncellular components. In
addition, the small number of T lymphocytes
present in the components may not survive
the freeze-thaw cycle.
The irradiation sources in use include
gamma rays—from either cesium-137 or co-
balt-60 sources—and x-rays produced by radi-
ation therapy linear accelerators or stand-
alone units. Both sources achieve satisfactory
results in rendering T lymphocytes inactive.
Freestanding instruments that allow the use of
each of these sources are commercially avail-
able for blood bank use. The US Nuclear Regu-
latory Commission requires an increased num-
ber of controls for the radioactive material
license that is needed for a gamma irradiator
and its source onsite.76 The increased controls
are designed to reduce the risk of unauthor-
ized use of radioactive materials. The licensee
156 䡲 AABB TECHNICAL MANUAL
is required to secure any area where radioac-
tive source is present, and only approved indi-
viduals may access the site to perform their
duties.
In the United States, the radiation dose to
the center of the irradiation field must be at
least 25 gray (Gy) [2500 centigray (cGy)] and no
more than 50 Gy (5000 cGy).77 During dosime-
try, the delivered dose of 25 Gy is targeted to
the internal midplane of the container. More-
over, the minimum delivered dose to any por-
tion of the blood components must be at least
15 Gy in a fully loaded canister.4(p25) The Euro-
pean standard requires a higher dose, with no
part of the component receiving less than 25
Gy and a maximum dose to any part of the
component of 50 Gy.13
Each instrument must be routinely moni-
tored to ensure that an adequate dose is deliv-
ered to the container that houses the blood
components during irradiation. Dose map-
ping is used to monitor the instrument’s func-
tion. For this purpose, irradiation-sensitive
films or badges that monitor the delivered
dose are used for QC of the irradiators. Several
systems consisting of irradiation films or
badges are commercially available.77 Verifica-
tion of the delivered dose must be performed
annually for the cesium-137 source and semi-
annually for the cobalt-60 source. For x-ray ir-
radiators, the dosimetry should be performed
in accordance with the manufacturer’s recom-
mendations. Dose verification is also required
after major repairs or relocation of the irradia-
tor. For gamma irradiators, the turntable oper-
ation, timing device, and lengthening of irradi-
ation time caused by source decay should also
be monitored periodically.
Another important quality assurance step
is the demonstration that the product that was
irradiated received the desired amount of irra-
diation. For that reason, irradiation-sensitive
labels are used to demonstrate that irradiation
of each batch of units was accomplished; this
is a requirement in Europe.13
In the United States, RBCs may be irradi-
ated up to the end of their storage shelf life.
The postirradiation expiration date is 28 days
or the original expiration date, whichever is
earlier. In Europe, RBCs may be irradiated only
up to day 28 following collection. Irradiated
cells may not be stored longer than the earlier
of 14 days postirradiation or 28 days postcol-
lection. Platelets may be irradiated until their
expiration date, and their postirradiation expi-
ration date is the same as the original expira-
tion date.
Irradiation of RBCs followed by storage
does result in a decrease in the percentage of
recovery after transfusion. In addition, an in-
creased efflux of potassium from red cells
causes the potassium levels to rise approxi-
mately twofold compared to nonirradiated
units. Platelets are not damaged by an irradia-
tion dose as high as 50 Gy.78
Pooling
Platelets that are pooled have an expiration
time of 4 hours from when the system was
opened for pooling. A closed system for
prestorage pooling of platelets has been li-
censed by the FDA. This system permits the
storage of pooled platelets for up to 5 days
from the time of WB collection. Four to six LR
or non-LR platelet units that are ABO identical
can be pooled using a set consisting of a multi-
lead tubing manifold for sterile connection. If
non-LR units are pooled, they are then filtered
as part of the pooling process. The shortest ex-
piration date of the pooled units determines
the expiration date of the pool.
In the United States, each pool prepared
from LR platelets must have <5.0 × 106 residual
leukocytes. The approved pooling set also al-
lows sampling of the pool for detection of bac-
terial contamination. A record of the unique
DIN for each individual member of the pool
must be available. The pool must be labeled
with the approximate total volume, ABO/Rh
type of the units in the pool, and number of
units in the pool.
Many countries in Europe prepare
prestorage pools of buffy-coat platelets that
are preserved in platelet additive solution or in
the plasma from one of the units from which
platelets are prepared.79 Instruments that au-
tomate the pooling process are increasingly
used in Europe. In a few countries in Europe,
systems for prestorage pooling of buffy-coat-
 CHAPTER 6 Whole-Blood Collection and Component Processing
derived platelets followed by pathogen-reduc-
tion treatment have become available. The lat-
ter systems are not yet licensed by the FDA.
Cryoprecipitated AHF units may be
pooled immediately before transfusion in an
“open” system; the pool has an expiration time
of 4 hours at 20 to 24 C storage. Prestorage
pools can also be prepared in an “open” sys-
tem and stored for 12 months at –18 C as de-
scribed in Method 6-11. After thawing, the
product expires in 4 hours. Prestorage pools
prepared with the use of an FDA-cleared ster-
ile connecting device are stored for 12 months
at –18 C; postthaw expiration time is 6 hours.
The number of units pooled may vary and can
consist of 4, 5, 6, 8, or 10 units. Prestorage
pools must be placed in a freezer within
1 hour. The potency of the pool is calculated
by assuming that each unit in the pool con-
tains 80 IU of coagulation Factor VIII and 150
mg of fibrinogen multiplied by the number of
units in the pool. If normal saline is used to
rinse the bags during preparation of the pool,
the amount of saline in the pool must be stat-
ed on the label.
Volume Reduction (Platelets)
Volume-reduced platelets may be needed for
patients in whom a reduced amount of plasma
is desired to prevent cardiac overload, to mini-
mize ABO antibody infusion, or for intrauter-
ine transfusion. Method 6-13, describes vol-
ume reduction by centrifugation. Platelet
concentrate volume can be reduced to 10 to
15 mL/unit. In-vitro properties such as platelet
morphology, mean platelet volume, hypotonic
shock response, synergistic aggregation, and
platelet factor 3 activity, appear to be main-
tained in the volume-reduced platelets stored
for 5 days.80 The in-vitro recovery rate of plate-
lets is about 85% after the volume-reduction
step. Platelets from WB that are volume re-
duced by centrifugation (580 × g for 20 min-
utes) from an approximate volume of 60 mL to
between 35 and 40 mL yield a high platelet
count (>2.3 × 109/L).81 Lowering the pH of
platelets prepared from WB avoids platelet ag-
gregates visible to the unaided eye (macroag-
gregates).82 The addition of 10% ACD-A to
䡲 157
platelets to lower the pH before centrifugation
can help with resuspension of high-concentra-
tion platelets and avoid aggregation.
Apheresis platelets can also be volume re-
duced during the collection process. In early
trials, collection of apheresis platelets in ap-
proximately 60 mL showed good in-vitro plate-
let characteristics and function. In-vivo autol-
ogous recovery was at least equivalent to that
of control apheresis platelets at standard con-
centrations when the storage time of 1, 2, or
5 days was adjusted based on the platelet con-
centration.83,84 For apheresis platelets, the vol-
ume reduction from 250 mL to 90 mL by cen-
trifugation has been shown to cause a mild
increase in platelet activation and an im-
paired aggregation response to adenosine di-
phosphate but not to collagen. In these experi-
ments, the platelet count before the volume
reduction was 1.0 × 109/mL; after the reduc-
tion, the count was 1.9 × 109/mL.85
Recent developments include refine-
ments in collection protocols for apheresis in-
struments that result in the collection of plate-
lets in a high concentration that may,
therefore, obviate the need for volume reduc-
tion. Platelet collections were at concentra-
tions as high as 3.0 to 4.0 × 109/L.83,86 Further-
more, the highly concentrated platelets may
be suspended in a platelet additive solution
with the autologous plasma at a ratio of 5:1 to
3:1. Platelet units prepared in this manner
contain much lower amounts of plasma com-
pared to standard apheresis platelets.80 Re-
cently, the FDA has approved additive solu-
tions for apheresis platelets.
Posttransfusion platelet increments have
been satisfactory after transfusion of volume-
reduced platelets.80 However, the overall in-
vivo survival data for volume-reduced plate-
lets are limited. If an open system is used, the
maximum allowable storage time is 4 hours.
The maximum allowable storage time has not
been established for closed systems.
QUARANTINE
All units of blood collected should be immedi-
ately placed in quarantine in a designated area
until donor information and donation records
158 䡲 AABB TECHNICAL MANUAL
have been reviewed, the current donor infor-
mation has been compared to the previous in-
formation, the donor’s previous deferrals have
been examined, and all laboratory testing has
been completed.87 Because of the limited
amount of time after collection that is avail-
able for component separation, WB units may
be separated into components before all of the
earlier processes have been completed. Sepa-
rated components are quarantined at the ap-
propriate temperature until all of the suitabili-
ty steps have been completed and reviewed.
Often, physical and electronic quarantine are
used simultaneously.
Certain blood components from previous
donations by donors whose more recent dona-
tions test positive for infectious disease also
require quarantine and appropriate disposi-
tion, as do units identified as unsuitable for
transfusion because of postdonation informa-
tion. Other components may need to be quar-
antined so that the QC samples can be taken
and analyzed. For instance, if a sample is ob-
tained for bacterial contamination testing, the
component is held in quarantine for some pre-
set amount of time and then released if the test
results are negative.
A thorough understanding of the quaran-
tine process is needed to prevent erroneous
release of unsuitable blood components.
Components may be removed from the quar-
antine area, labeled, and released for distribu-
tion if all of the donor information, previous
donor records, and current test results are sat-
isfactory. Some nonconforming autologous
blood components may be released for autolo-
gous use only.
Some blood components require emer-
gency release because they have a very short
storage time. Such is the case for granulocytes.
Emergency release requires physician approv-
al and a label or tie tag to indicate that testing
was incomplete at the time of release.
Despite the widespread use of software to
control manufacturing processes, instances of
failure of quarantine and release of unsuitable
products continue to be reported to the FDA.
For instance, during fiscal year 2011, the FDA
received 54,947 reports of blood and plasma
product deviations. Of these reports, 4258
(7.7%) involved QC and distribution errors.88
L ABE L IN G
Blood component labeling is a highly regulat-
ed activity, and the documents that describe
the requirements are listed below. Readers are
advised to consult these documents and spe-
cific national regulations for details.
The FDA requirements for labeling of
blood and components are detailed in the
Guidelines for Uniform Labeling of Blood and
Blood Components published in 1985.3 The
FDA approved the International Society of
Blood Transfusion (ISBT) 128 symbology, Ver-
sion 1.2.0, in 2000 and Version 2.0.0 in 2006.89
Detailed requirements are described in the
Code of Federal Regulations (Title 21, CFR
Parts 606.120, 606.121, and 606.122). AABB
Standards requires that accredited facilities
use ISBT 128 labels.4(p12)
The FDA rule that requires all blood com-
ponents to be labeled with a bar-coded label
became effective on April 26, 2006. The rule re-
quires that, at a minimum, the label contain
the following bar-coded information: 1) the
unique facility identifier (eg, registration num-
ber), 2) lot number relating to the donor, 3)
product code, and 4) ABO group and Rh type
of the donor. These pieces of information must
be present in eye-readable and machine-read-
able format. The rule applies to blood centers
that collect and prepare blood components.
The rule also applies to hospital transfusion
services that prepare pooled cryoprecipitate
and/or prepare divided units or aliquots of
RBCs, platelets, and plasma for pediatric use.
Another major part of labeling in the
United States is the information circular. The
circular must be made available to everyone
involved in the transfusion of blood compo-
nents. The Circular of Information for the Use
of Human Blood and Blood Components is pro-
duced by AABB, America’s Blood Centers, the
American Red Cross, and the Armed Services
Blood Program and is recognized as accept-
able by the FDA.49 The circular provides im-
portant information about each blood compo-
 CHAPTER 6 Whole-Blood Collection and Component Processing
nent and should be consulted for information
not included in this chapter.
Special message labels may also be af-
fixed to blood component containers. The la-
bels may include one or more of the following
indications: 1) hold for further manufactur-
ing, 2) for emergency use only, 3) for autolo-
gous use only, 4) not for transfusion, 5) irradi-
ated, 6) biohazard, 7) from a therapeutic
phlebotomy, and 8) screened for special fac-
tors [eg, HLA type or cytomegalovirus (CMV)
antibody status]. ISBT 128 allows incorpora-
tion of special attributes of the component,
such as CMV antibody status.
Additional information on the container
can be conveyed using a tie tag. Tie tags are es-
pecially useful for autologous and directed do-
nations. Tie tags include the patient’s identify-
ing information, name of the hospital where
the patient will be admitted for surgery, date of
surgery, and other information that may be
helpful to the hospital transfusion service.
Each component must also bear a unique
DIN that can be traced back to the blood do-
nor. If components are pooled, a pool number
must allow tracing to the individual units with-
in a pool.
For groups planning to implement ISBT
128, an important source of information is
ICCBBA (formerly known as the International
Council for Commonality in Blood Banking
Automation). This organization’s website fea-
tures updates and a revised list of product
codes.90
Immediate benefits of ISBT 128 include
the following:
䡲 Uniform labels applied on blood compo-
nents manufactured by different collection
centers.
䡲 Better traceability of components.
䡲 Improved self-checking features per char-
acter.
䡲 Encoding of entire ASCII character set that
includes alphanumeric and special charac-
ters.
䡲 Improved accuracy through reduction in
the number of misreads during scanning of
the bar-coded information.
䡲 159
䡲 Double-density coding of numeric charac-
ters, which permits encoding of more infor-
mation in a given space.
䡲 Larger number of product codes, which al-
lows more detailed descriptions of blood
components.
䡲 Enhanced scanning, which permits more
facile auditing of movements of blood com-
ponents from one location to another.
䡲 Ability to add information for autologous
donations.
䡲 Ability to read more than one bar code with
a single sweep (concatenation).
䡲 Less laborious importation of “foreign” in-
ventory into “own” inventory via the avail-
ability of uniform systems for DINs and
product codes.
In the future, ISBT 128 is expected to per-
mit information transfer by radiofrequency ID
tags or other means of electronic data trans-
mission.
QC OF BLOOD CO MPONENTS
A quality management system is critical for es-
tablishing a current good manufacturing prac-
tice-compliant operation (see Chapter 1). Test-
ing of blood components is necessary to
ensure the safety, purity, and potency of the
product and verify compliance with national
regulatory requirements, such as those of FDA.
These requirements are minimum standards,
and an individual manufacturer may establish
more stringent ones. QC failures can serve as
an indicator of unexpected suboptimal re-
agents or materials. Furthermore, QC data can
reveal previously unrecognized variations
from validated procedures and processes. A
timely detection system provides a proactive
approach to early identification and resolution
of a manufacturing problem.
Equipment QC is necessary to ensure that
blood components achieve desired properties
in a consistent manner. Suggested QC steps for
critical equipment in component laboratories
are described in Chapter 1 and are listed in Ap-
pendix 1-3.
Limitations of QC are demonstrated, for
example, in QC failures that are due to poor
160 䡲 AABB TECHNICAL MANUAL

sampling techniques and failures that may be
ascribed to donor-related variables that can-
not be controlled. Examples of donor-associ-
ated variables include occult donor bactere-
mia or viremia and leukocyte reduction filter
failure resulting from the presence of sickled
red cells.
National regulatory authorities provide
specific minimum requirements for QC testing
of blood products. These may differ from
country to country, and the most recent guid-
ance documents and regulations should be re-
viewed. For certain blood components, it may
be impractical to perform the QC steps. For ex-
ample, the FDA and Council of Europe do not
require QC of bedside leukocyte reduction fil-
ters.
A statistical process control approach has
been suggested for QC of blood compo-
nents.15,91,92 Such an approach is expected to
provide a definition of product conformance
to a standard with a given probability. This ap-
proach also allows a limit to be established for
nonconformance, facilitates implementation
of corrective actions, and permits QC to be in-
dividualized for different blood components.

KEY POINTS

1. Modern blood containers are composed of soft plastic and identified by a lot number. They
should be pyrogen-free and flexible yet tough and both kink- and scratch-resistant. During
frozen storage, each plastic container has a glass transition temperature below which it be-
comes brittle and susceptible to breakage during transportation.
2. The initial 35 to 45 mL of blood drawn is allowed to collect into a diversion pouch at the col-
lection tubing. The pouch reduces bacterial contamination of collected blood by diverting
bacteria from the skin that may have otherwise entered the collection. Blood in this pouch
may be used for laboratory tests.
3. The average rate of adverse donor reactions after donation is 3.5% to 4.5%. Most reactions
are mild and require no further medical care. These reactions can be systemic (eg, fainting)
or local (eg, hematoma). About 1 in 3400 donors experience a reaction after leaving the do-
nation site and may need medical care. Deferral of low-blood-volume (less than 3.5 L) do-
nors may be helpful in reducing the risk of reactions, especially in young donors.
4. During centrifugation for component preparation, primary variables that affect cell separa-
tion and cell recovery are rotor size, centrifuge speed, and duration. The platelet-rich plas-
ma method is used in the United States for platelet concentrate preparation, and the buffy-
coat method is more common in Canada and Europe.
5. LR RBCs and platelets are not to exceed 5.0 × 106 residual WBCs per transfusion dose in the
United States or 1.0 × 106 residual WBCs per transfusion dose in Europe.
6. In plasma prepared from WB (by manual or apheresis methods) and labeled as “Plasma Fro-
zen Within 24 Hours After Phlebotomy,” the levels of all the coagulation factors are similar
to those of FFP except for some decrease in coagulation Factor VIII.
7. The radiation dose must be 25 to 50 Gy with a minimum delivered dose to any portion of the
blood components of 15 Gy in the United States. RBCs may be irradiated up until the end of
their storage shelf life; the postirradiation expiration date is 28 days after collection or the
original expiration date, whichever is sooner. The European standard requires that no part
of the component receive less than 25 Gy, a maximum dose to any part of the component of
50 Gy, and irradiation of RBCs only until day 28, with storage for no longer than 14 days after
irradiation or 28 days after collection, whichever is earliest. Platelet shelf life is not reduced
after irradiation.
8. Bar-coded and eye-readable container labels are increasingly using the ISBT symbology
(ISBT 128). ISBT 128 offers a number of advantages over the Codabar symbology, including
identification of the manufacturer throughout the world, more product codes, better accu-
 CHAPTER 6 Whole-Blood Collection and Component Processing
racy as a result of reduced misreads during scanning, and enhanced conveyance of other la-
beling information.
9. Various approaches for QC of blood components have been promulgated by the AABB,
Council of Europe, and FDA.
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 C h a p t e r
Blood Component Collection
by Apheresis
James W. Smith, MD, PhD
“A P H E R E S I S , ” “ P H E R E S I S , ” “hema-
pheresis,” and all of the various terms
used to refer to automated blood component
collection procedures are derived from the
Greek word “aphairos,” meaning “to take from.”
Specifically, whole blood is separated into
components during collection, the desired
component is removed/modified, and the re-
maining components are returned to the do-
nor or patient. Centrifugal and membrane-
based apheresis techniques were under
development in the late 19th and early 20th
centuries. By the 1970s, multiple improved
technologies had emerged and apheresis pro-
cesses advanced rapidly.
Today, centrifugal technique is primarily
used in the United States, whereas membrane
filtration is used in other parts of the world (pri-
marily Europe and Japan) for donor apheresis.
Early versions of automated, computer-
ized, and centrifugal techniques facilitated
large-scale donations of platelets, plasma, and
granulocytes. As the technology continued to
evolve, equipment, disposables, and software
became increasingly sophisticated, and now
James W. Smith, MD, PhD, Medical Director, Oklahoma Blood Institute, Oklahoma City, Oklahoma
The author has disclosed no conflicts of interest.
7
the collection of various combinations of com-
ponents is possible (see Table 7-1). This chap-
ter provides a discussion of the technology
and instrumentation used during donor
apheresis, with special consideration given to
the associated regulatory requirements.
CO M P O N E N T CO L L E C T I O N
The collection of components by apheresis fol-
lows many of the same rules and guidelines
that apply to whole-blood donation. Like
whole-blood donors, apheresis donors must
be given sufficient information to enable them
to give informed consent to donate blood. Al-
though the apheresis collection and prepara-
tion processes differ from those used for
whole-blood-derived components, the storage
and transportation requirements and several
quality-control steps are the same for both
processes. Another similarity is that the facility
must maintain written procedures and proto-
cols for all types of collections used and must
keep records of each procedure as required by
AABB Standards for Blood Banks and Transfu-
167
168 䡲 AABB TECHNICAL MANUAL

TABLE 7-1. Components that Can Be Collected with Various Instruments

Instrument GRAN PLT cRBC 2-RBC PLASMA cPLASMA

Fenwal ALYX X X X
Fenwal Amicus X X X
Fenwal Autopheresis C X
Fresenius AS104 X
TerumoBCT (COBE) X X X
Spectra
TerumoBCT Spectra Optia X
TerumoBCT Trima V-4 X X X X
TerumoBCT Trima Accel X X X X
Haemonetics Cymbal X
Haemonetics MCS+ X X X
LN9000
Haemonetics MCS+ X X X
LN8150
Haemonetics PCS-2 X
*Concurrent collection refers to the ability to collect more than one type of product.
GRAN = granulocytes; PLT = plateletpheresis (single, double, triple); cRBC = concurrent* 1 unit of Red Blood Cells (RBCs);
2-RBC = double unit of RBCs; PLASMA = 1 unit of plasma; cPLASMA = concurrent plasma; V-4 = software version 4.

sion Services.1(pp62-67) The circumstances that each of which must meet minimum standards.
are unique to apheresis collection are ad- Some instruments are programmed to calcu-
dressed in the sections that follow. late the platelet yield based on the donor’s he-
matocrit, platelet count, height, and weight.
Platelets For alloimmunized patients who do not
respond to random allogeneic platelets, trans-
Apheresis is used to obtain platelets from vol-
fusions of platelets from an apheresis donor
unteer donors, patients’ family members, or
selected on the basis of a compatible platelet
donors with HLA or platelet-antigen-compati-
crossmatch or that are matched for HLA anti-
ble phenotypes. By design, apheresis proce-
gens may be the only way to achieve a satisfac-
dures are intended to collect large numbers of
tory posttransfusion platelet increment. In the
platelets from an individual, thereby providing
United States, the use of apheresis platelets
a more potent product with fewer donor expo-
has been steadily increasing over the past 25
sures for the patient. AABB Standards requires
years. It is estimated that 90% of platelets
that an apheresis platelet component contain
transfused in the United States are apheresis
at least 3 × 1011 platelets in 90% of sampled
platelets.2
units.1(pp28-29)
With newer technology and more effi-
Donor Selection and Monitoring
cient processes, higher yields of platelets may
be obtained from one donor, and the original Plateletpheresis donors may donate more fre-
apheresis unit may be split into multiple units, quently than whole-blood donors but must
 CHAPTER 7 Blood Component Collection by Apheresis
meet all of the other criteria for whole-blood
donation. The interval between donations
should be at least 2 days, and donors should
not undergo plateletpheresis more than twice
in a week or 24 times in a rolling 12-month pe-
riod.1(p20),3 If the donor donates a unit of whole
blood or if it becomes impossible to return the
donor’s red cells during plateletpheresis, at
least 8 weeks should elapse before a subse-
quent plateletpheresis procedure unless the
extracorporeal red cell volume (ECV) is less
than 100 mL. Platelets may be collected from
donors who do not meet these requirements
only if the component is expected to be of par-
ticular value to a specific intended recipient
and a physician certifies in writing that the do-
nor’s health will not be compromised by the
donation. Donors who have taken antiplatelet
medications that irreversibly inhibit platelet
function are deferred for specific intervals be-
fore donation (48 hours for aspirin/aspirin-
containing medications and piroxicam, 14
days for clopidogrel and ticlopidine) because
apheresis platelets are often the sole source of
platelets given to a patient.1(p61),3
A platelet count is not required before the
first apheresis collection; however, triple col-
lections of platelets may not be drawn from
first-time donors unless a qualifying platelet
count is obtained on a sample collected before
the procedure.3 If the donation interval is less
than 4 weeks, many facilities prefer that the
donor’s platelet count be above 150,000/µL be-
fore plateletpheresis occurs to prevent a post-
donation count of less than 100,000/µL. AABB
Standards permits qualification of a donor
with a platelet count from a sample collected
immediately before the procedure or one ob-
tained either before or after the previous pro-
cedure.1(p21) Exceptions to these laboratory cri-
teria should be approved in writing by the
apheresis program physician based on docu-
mented medical need.
The Food and Drug Administration (FDA)
specifies that the total volume of plasma col-
lected should be no more than 500 mL (or 600
mL for donors weighing more than 175 lb) or
the volume described in the labeling of the au-
tomated blood cell separator device (which
may be more or less than the 500-mL or 600-
䡲 169
mL volume previously mentioned). The plate-
let count of each unit should be kept on record
but need not be written on the component la-
bel. Units containing less than 3.0 × 1011 plate-
lets should be labeled with the actual platelet
count.3
It is possible to collect plasma concur-
rently with platelets. Such collection is dis-
cussed in more detail in the “Plasma” section
below.
Vasovagal and hypovolemic reactions are
rare in apheresis donors but may occur. Pares-
thesias (tingling sensations) and other reac-
tions to citrate anticoagulant are not uncom-
mon. (Similar citrate toxicity reactions in
recipients are discussed along with whole
blood transfusions in Chapter 27.) Serious re-
actions occur less often among apheresis do-
nors than whole blood donors.4
Laboratory Testing
Tests for ABO group, Rh type, unexpected allo-
antibodies, and transfusion-transmitted dis-
eases must be performed by the collecting fa-
cility in the same manner as for other blood
components. Each unit must be tested unless
the donor is undergoing repeated procedures
to support a specific patient, in which case
testing for infectious disease markers needs to
be repeated only at 30-day intervals.5
If red cells are visible in a product, the he-
matocrit should be determined. AABB Stan-
dards state that if the component contains
more than 2 mL of red cells, the red cells must
be ABO compatible with the recipient’s plasma
and be crossmatched.1(pp37-38) In such cases, a
sample of donor blood is attached to the con-
tainer for compatibility testing. In some in-
stances, it may be desirable for the donor plas-
ma to be ABO compatible with the recipient’s
red cells (eg, if the recipient is a child or an
ABO-mismatched allogeneic progenitor cell
transplant recipient). In the United States, to
be considered leukocyte reduced, apheresis
platelets must contain less than 5 × 106 leuko-
cytes per unit and platelets must meet the
specifications of the apheresis device
manufacturer.3 In Europe, the guideline for
170 䡲 AABB TECHNICAL MANUAL
leukocyte-reduced components is fewer than
1 × 106 leukocytes per unit.
Record-Keeping
Complete records must be kept on each proce-
dure. All adverse reactions occurring during
collection procedures (or transfusion) must be
documented along with the results of thor-
ough investigations. Records of all laboratory
findings and collection data must be periodi-
cally reviewed by a knowledgeable physician
and must be found to be within acceptable
limits. FDA guidelines require a periodic re-
view of donor records to monitor platelet
counts.3 Facilities must have policies and pro-
cedures in place to ensure that donor red cell
loss during each procedure does not exceed
acceptable limits.1(p18)
Plasma
Apheresis devices can be used to collect plas-
ma for transfusion or as Source Plasma for
subsequent manufacturing. Recently, the FDA
approved apheresis devices for the collection
of plasma held at 1 to 6 C within 8 hours and
frozen within 24 hours after phlebotomy and
plasma held at room temperature for up to 24
hours and frozen within 24 hours after phle-
botomy.
The FDA has provided guidance with re-
gard to the volume of plasma that may be col-
lected using automated devices. A distinction
is made between infrequent plasmapheresis,
in which the donor undergoes plasmapheresis
no more frequently than once every 4 weeks,
and serial plasmapheresis (or Source Plasma
collection, the process to collect plasma for
fractionation into plasma components), in
which the donation is more frequent than
once every 4 weeks. For donors in infrequent
plasmapheresis programs, donor selection
and monitoring requirements are the same as
those for whole-blood donation. Plasma ob-
tained by these processes is intended for direct
transfusion.
For serial plasma (Source Plasma) collec-
tion using either automated instruments or
manual techniques, the following principles
apply6:
1. Donors must give consent for the proce-
dure, and they must be observed closely
during the procedure. Emergency medical
care must always be available.
2. Red-cell losses related to the procedure,
including samples collected for testing,
should be monitored so that no more than
200 mL of red cells are removed in 8 weeks.
If the donor’s red cells cannot be returned
during an apheresis procedure, hemapher-
esis or whole-blood donation should be
deferred for 8 weeks.
3. For manual collection systems, a mecha-
nism must exist to ensure safe reinfusion of
the autologous red cells.
4. In manual procedures for donors weighing
110 to 175 lb, no more than 500 mL of whole
blood should be removed at one time or no
more than 1000 mL during a session or
within a 48-hour period. The limits for
donors who weigh  175 lb are 600 mL and
1200 mL, respectively. For automated pro-
cedures, the allowable volume has been
determined for each instrument by the
FDA.
5. At least 48 hours should elapse between
successive procedures. Donors should not
undergo more than two procedures within
a 7-day period.
6. At the time of initial plasmapheresis and at
4-month intervals for donors undergoing
serial (large-volume) plasmapheresis (donors
undergoing plasmapheresis more often
than once every 4 weeks), serum or plasma
must be tested for total protein and for
serum protein electrophoresis or for quan-
titative immunoglobulins. Results must be
within normal limits.
7. A qualified licensed physician, knowledge-
able about all aspects of hemapheresis,
must be responsible for the program.
For manual collection systems, a process
that is rarely used in the United States at pres-
ent, requirements are outlined in the Code of
Federal Regulations6 and have been summa-
rized in previous editions of this Technical
Manual.
 CHAPTER 7 Blood Component Collection by Apheresis
Red Cells and Multicomponent
Donations
Both AABB Standards and FDA guidance doc-
uments address the removal of red cells by au-
tomated apheresis methods. A guidance docu-
ment issued in 2001 by the FDA finalized
recommendations for the use of automated
apheresis equipment to collect the following7:
䡲 Single units of RBCs and plasma.
䡲 Single units of RBCs and platelets.
䡲 Single units of RBCs, platelets, and plasma.
䡲 Double units of RBCs only.
The guidance document includes FDA
regulations requiring that equipment perform
and be used in the manner for which it was de-
signed to collect or process blood and compo-
nents. Standard operating procedures, includ-
ing device manufacturers’ instructions for use
and maintenance of current records, are de-
scribed. The sections below summarize the in-
formation in the guidance document.
Donor Selection and Monitoring
The FDA requires that an adequate hemoglo-
bin level be determined by a quantitative
method for predonation hemoglobin or the
hematocrit of donors undergoing double-RBC
collection. The procedure is limited to persons
who are larger and have a higher hematocrit
than the minimum standards for whole-blood
donations. For males, the minimum weight is
130 lb, and the minimum height is 51. For fe-
males, the minimum weight is 150 lb, and the
minimum height is 5 5. The minimum hema-
tocrit is 40% for both genders. Donors who are
shorter than the minimum height or who
weigh less than the minimum weight, as estab-
lished by the FDA in device operator manuals,
should be further evaluated. These donors
must also meet all relevant FDA criteria for al-
logeneic or autologous whole-blood donation.
Donors who have given a single unit of
RBCs with platelets, plasma, or both should be
deferred for at least 8 weeks. The exception is
when a donor serves as a plateletpheresis do-
nor or a donor of platelets with plasma by-
䡲 171
products within 8 weeks and the ECV of the
procedure is <100 mL. Donors should be de-
ferred for at least 16 weeks after a double-RBC
donation. If an apheresis procedure is discon-
tinued before completion and absolute red cell
loss is <200 mL, the donor may donate again
within 8 weeks if he or she meets all donor eli-
gibility criteria.
If a donor has a second red cell loss of
<100 mL during a subsequent donation within
8 weeks of a previous donation, the donor
should be deferred for 8 weeks. If the total ab-
solute red cell loss within 8 weeks is >300 mL,
the donor should be deferred for 16 weeks
from the date of the last red cell loss. If an
apheresis procedure is discontinued and the
absolute red cell loss is >200 mL but <300 mL,
the donor should be deferred for 8 weeks. If an
apheresis procedure is discontinued and total
absolute red cell loss is >300 mL, the donor
should be deferred for 16 weeks.
Saline infusion is used to minimize vol-
ume depletion.
Quality-Control Issues
The FDA has promulgated quality-control
(QC) programs for RBC unit collection by
apheresis. There are two phases:
䡲 In Phase I QC, 100 consecutive RBC units
are tested to determine the expected or tar-
get red cell volume in accordance with the
specifications in the device operator’s man-
ual. Target values are compared with the
actual values to determine product accept-
ability. If the QC results are satisfactory,
which includes that at least 95% of the units
meet product specifications, the establish-
ment may proceed to Phase II.
䡲 Phase II QC consists of monthly testing of a
representative sample of 50 units of the
manufactured product from each collec-
tion center. At least 1 unit from a single
RBC protocol or both units from a double-
RBC protocol device used at the center
should be included in the testing. At least
95% of the products tested should meet
product specifications as described in the
device operator’s manual.
172 䡲 AABB TECHNICAL MANUAL
Record Requirements
US blood establishments must update their
blood establishment registrations and product
listing forms with the FDA to collect RBCs us-
ing automated methods. Automated RBC col-
lection has substantial potential to have an ad-
verse effect on the identity, strength, quality,
purity, or potency of a product. Blood estab-
lishments that are approved to manufacture
RBCs with one manufacturer’s device and that
want to use another manufacturer’s device in-
stead must submit a prior approval supple-
ment and must receive FDA approval before
distribution of the product manufactured on
the new device. The FDA requires these estab-
lishments to make available a number of rec-
ords and forms regarding RBC or multicompo-
nent unit collection for FDA inspection. These
records and forms include documents ad-
dressing donor consent, donor eligibility,
product collection, and product QC.7
Granulocytes
The use of granulocyte transfusions has been
controversial for a number of years. Analysis of
randomized controlled trials of granulocyte
transfusions in adults has indicated that an ac-
ceptable minimum dose (>1 × 1010 granulo-
cytes/day) and crossmatch compatibility (no
recipient antibodies to granulocyte antigens
have a major impact on the effectiveness of
such transfusions.8 Recently, there has been
renewed interest in granulocyte transfusion
therapy because much larger cell doses may be
obtained from donors who have received re-
combinant colony-stimulating factors.
Agents Administered to Increase Yields
AABB Standards requires that 75% of granulo-
cyte components contain at least 1 × 1010 gran-
ulocytes,1(p30) although the optimal therapeutic
dose in adult patients is unknown. For infants
and children, a dose of 10 to 15 mL/kg may
provide an adequate number of granulocytes
per dose. To collect this number of cells in a
unit of granulocytes, one must administer
drugs or sedimenting materials to the donor.
Sedimenting agents enhance granulocyte har-
vest by increasing sedimentation of the RBCs,
thereby enhancing the interface in the collec-
tion device and resulting in minimal RBC con-
tent in the final product. The donor’s consent
to the procedure must provide permission for
any of these drugs or sedimenting agents to be
used.
HYDROXYETHYL STARCH. A common sedi-
menting agent, hydroxyethyl starch (HES)
causes red cells to aggregate, thereby sedi-
menting them more completely. Because HES
can be detected in donors as long as a year af-
ter infusion, AABB Standards requires facilities
performing granulocyte collections to have a
process to control the maximum cumulative
dose of any sedimenting agent administered
to a donor within a given interval.1(p23) HES is a
colloid that acts as a volume expander. Donors
who receive HES may experience headaches
or peripheral edema because of expanded cir-
culatory volume. A boxed warning from the
FDA exists for its use due to increased mortali-
ty and severe renal injury in certain patient
populations.9
C O RT I C O S T E R O I D S . Corticosteroids can
double the number of circulating granulo-
cytes by mobilizing granulocytes from the
marginal pool. The common protocol is to use
60 mg of oral prednisone in a single or divided
dose before donation to collect large numbers
of granulocytes with minimal systemic steroid
activity. Another protocol uses 8 mg of oral
dexamethasone. Donors should be questioned
about their relevant medical history before
they use systemic corticosteroids. Hyperten-
sion, diabetes, cataracts, or peptic ulcer can be
relative or absolute contraindications to corti-
costeroid use.
GROW TH FACTORS. Although not a licensed
indication, granulocyte colony-stimulating
factor (G-CSF) can effectively increase granu-
locyte yields. Hematopoietic growth factors
given alone can result in the collection of up to
4 to 8 × 1010 granulocytes per apheresis proce-
dure. Typical doses of G-CSF are 5 to 10 µg/kg
given 8 to 12 hours before granulocyte collec-
tion. Preliminary evidence suggests that in-
vivo recovery and survival of these granulo-
 CHAPTER 7 Blood Component Collection by Apheresis
cytes are excellent and that growth factors are
well tolerated by donors.
Laboratory Testing
Testing for ABO and Rh group, red cell anti-
bodies, and infectious disease markers is re-
quired on a sample drawn at the time of phle-
botomy. Red cell content in granulocyte
products is inevitable; the red cells should be
ABO compatible with the recipient’s plasma. If
>2 mL of red cells are present, the component
should be crossmatched for Rh compatibility
and HLA compatibility. 1(pp37-38)
Storage and Infusion
Granulocyte function deteriorates rapidly dur-
ing storage, and concentrates should be trans-
fused as soon as possible after preparation.
AABB Standards mandates a storage tem-
perature of 20 to 24 C for no longer than 24
hours.1(p55) Agitation during storage is undesir-
able. Irradiation is required for products to be
administered to immunodeficient recipients
and is indicated for nearly all recipients be-
cause their primary diseases are likely to in-
volve deficiencies in their immune systems.
Use of a microaggregate or leukocyte reduc-
tion filter is contraindicated because it re-
moves the collected granulocytes.
IN S T RUM EN TS A N D S Y S T EMS
FOR DONOR APHERESIS
CO L LE CT IO N S
This section provides descriptions of equip-
ment that is available for use in the United
States for the collection of blood components
by automated techniques. A brief description
is given of each instrument; more detailed
information can be found in other resourc-
es.10,11 This section does not include the
CS3000 and CS3000+ (Fenwal, Lake Zurich, IL)
because the company has discontinued the
sale of these devices in the United States (al-
though disposables for the devices are still
available at the time of this writing). These two
devices are capable of collecting RBCs, plate-
lets, concurrent plasma, and granulocytes.
䡲 173
Plasma
Fenwal Autopheresis C
The Autopheresis C (Fenwal) is an instrument
designed to collect plasma only.11 It uses a
rotating cylindrical filter to separate the plas-
ma from the cellular elements of blood. Be-
cause of the high efficiency of the rotating fil-
ter, the filter is small and the system’s ECV is
approximately 200 mL. The Autopheresis C is a
single-access system, and saline replacement
can be administered. It is considered an open
system and can collect several units of plasma.
According to the FDA definition, an open sys-
tem requires that plasma outdates 4 hours af-
ter thawing. Variances can be granted that al-
low 24-hour outdates, but these units cannot
be relabeled as Thawed Plasma.
Haemonetics PCS-2
The PCS-2 (Haemonetics, Braintree, MA), a
simplified version of the MCS Plus, is designed
for plasma collection.12 The PCS-2 uses a blow-
molded (grenade-shaped) centrifuge bowl to
separate plasma from cellular elements. De-
pending on the degree of cell reduction re-
quired, one of three versions of the PCS-2 bowl
can be used: standard, filter core, or high sepa-
ration core. The standard bowl uses centrifu-
gal force to remove the plasma from the top of
the bowl. To increase cell reduction, the filter
core and high separation core bowls allow
plasma to pass through the core, which is cov-
ered with a filter membrane.13-15 Use of this de-
vice results in the greatest cell reduction, but it
has not been released in the United States at
the time of this writing. The ECV of the PCS-2
is variable depending on the hematocrit of the
donor and ranges from 491 mL (38% hemato-
crit) to 385 mL (50% hematocrit). The PCS-2 is
a single-access system, and saline replace-
ment can be administered. It is considered an
open system. As noted for the Auto-C, a vari-
ance can be granted allowing 24-hour out-
dates of plasma processed with this device, but
these units may not be relabeled as Thawed
Plasma. The PCS-2 can collect several units of
plasma at a time.
174 䡲 AABB TECHNICAL MANUAL
Equipment for Concurrent Plasma
Collection
Plasma can be collected as a concurrent prod-
uct during collection of apheresis platelets or
automated RBCs. The amount that can be col-
lected is determined by the volume of plate-
lets, red cells, or both, that are being collected
and by the maximum volume that can be re-
moved from the donor. Equipment capable of
concurrent plasma collection includes Hae-
monetics MCS+ LN9000, Haemonetics MCS+
LN8150, Fenwal Amicus, Fenwal ALYX, Teru-
moBCT (COBE) Spectra (TerumoBCT, Lake-
wood, CO), TerumoBCT Trima, and Teru-
moBCT Trima Accel.
Platelets
TerumoBCT (COBE) Spectra
The TerumoBCT (COBE) Spectra is capable of
collecting single, double, or triple apheresis
platelet units as well as concurrent plasma, de-
pending on the size and platelet count of the
donor.10,16-19 This device uses a dual-stage (dif-
ferent radius) channel to collect leukocyte-
reduced platelets. Leukocyte reduction to <5 ×
106 White Blood Cells (WBCs) can be obtained
in approximately 85% of the collections for
software versions that are lower than 5.0.20 Use
of software versions 5.0 and 7.0 can more con-
sistently result in the collection of products
containing <1.0 × 106 WBCs with the leukocyte-
reduction system (LRS), which uses a cone in
the centrifuge and saturated, fluidized, particle-
bed filter technology to remove residual WBCs
leaving the second stage of the channel.10,16-19
The Spectra is capable of single- or double-
access procedures. The ECV of the single-
access kit is 361 mL and of the double-access
kit is 272 mL. The Spectra is being replaced by
the more efficient Trima or Trima Accel.11
TerumoBCT Trima and Trima Accel
The TerumoBCT Trimas were designed as
automated donor collection machines for
platelets, plasma, and RBCs only. The Teru-
moBCT Trima (Version 4) uses a smaller, mod-
ified, dual-stage channel and LRS cone to con-
sistently collect leukocyte-reduced platelets
(<1.0 × 106 WBCs). To increase platelet yields,
the Trima Accel (Versions 5.0, 5.01, and 5.1)
uses a single-stage, doughnut-shaped channel
and larger LRS cone to consistently collect leu-
kocyte-reduced platelets.21 The Trimas use sin-
gle-access kits only. The ECV of the Trimas is
182 to 196 mL. They are capable of collecting
single, double, or triple units of apheresis
platelets as well as concurrent plasma and
RBCs, depending on donor size, platelet count,
and hematocrit.21-23
Fenwal Amicus
The Amicus is capable of collecting single,
double, or triple apheresis platelets as well as
concurrent plasma and RBCs (single-access kit
only), depending on the donor’s size, platelet
count, and hematocrit.10,16,22,24 The Amicus
uses centrifugal force and a double compart-
ment belt wrapped around a spool to separate
the platelets. The platelets accumulate in the
collection chamber and are transferred to the
final collection bags at the end of the proce-
dure. The Amicus is capable of single- or
double-access procedures. The ECV of the
double-access kit is 210 mL, and that of the
single-access kit is 209 mL. The ECV of the
whole-blood bag is adjustable. Consistent leu-
kocyte reduction is accomplished without ex-
ternal filtration, and this process is approved
by the FDA for submission of a prior approval
supplement.
Haemonetics MCS+ LN9000
The Haemonetics MCS+ LN9000 system is ca-
pable of conducting several different apheresis
procedures, including apheresis platelet col-
lection. It uses the Latham conical bowl, plas-
ma-controlled hematocrit, and plasma surge
technique to build up the platelets and float
them off the bowl with rapid plasma infusion.
Although this technique results in leukocyte-
reduced platelets, the use of an inline leuko-
cyte reduction filter ensures consistency in
leukocyte reduction.10,25-28 The LN9000 uses a
single-access kit, and the ECV ranges from 480
mL (38% hematocrit) to 359 mL (52% he-
 CHAPTER 7 Blood Component Collection by Apheresis
matocrit). The LN9000 is capable of collecting
single, double, or triple units of apheresis
platelets as well as concurrent plasma, de-
pending on donor size and platelet count.10,25-28
RBCs
TerumoBCT Trima and Trima Accel
As previously mentioned, the Trima can col-
lect a single unit of RBCs concurrently with
platelets.11,23,29 A kit is available to collect a
double unit of RBCs with or without concur-
rent plasma, depending on the donor’s size
and hematocrit. The Trima uses the single-
stage channel for the double-RBC collection,
and saline is returned to the donor during the
collection. The addition of the preservative
solution and leukocyte reduction by filtration
are performed manually and off-line after the
collection is complete for both the single and
double units of RBCs.
Fenwal ALYX
The Fenwal ALYX was developed as an auto-
mated, double-RBC collection system only.
Recently, it has been approved to collect con-
current plasma as well. The ALYX uses a rigid,
cylinder-shaped chamber in the centrifuge to
separate the plasma from the cells. The plasma
is collected in one bag, and the red cells are
collected in a separate bag. During reinfusion,
the plasma and saline are returned to the do-
nor. When the collection is complete, the ALYX
automatically adds the preservative solution
and pumps the red cells through an inline leu-
kocyte reduction filter into the final storage
bags. The ALYX uses a single-access kit only,
and its ECV is approximately 110 mL. The
ALYX is capable of collecting 2 units of RBCs or
1 unit of RBCs and concurrent plasma at a
time, depending on donor size and hemato-
crit.11,28-30
Fenwal Amicus
As mentioned previously, the Fenwal Amicus
can collect a single unit of RBCs concurrently
with plateletpheresis, but only with the single-
䡲 175
access kit.11,31 The addition of the preservative
solution and leukocyte reduction by filtration
are performed manually and off-line after the
collection is complete.
Haemonetics Cymbal
The Cymbal is a collection system that uses an
expanding, variable-volume bowl. The system
has a relatively small ECV when compared to
the Haemonetics MCS+ LN8150 and collects a
double-RBC unit.32
Haemonetics MCS+ LN8150
The Haemonetics MCS+ LN8150 was designed
as a donor instrument only for the collection
of RBCs and concurrent plasma. The LN8150
uses the blow-molded bowl that is also used
for plasma collection. It uses a single-access
kit and its ECV varies, depending on the do-
nor’s hematocrit, from 542 mL (38% hemat-
ocrit) to 391 mL (54% hematocrit). The LN8150
is capable of collecting a single or double unit
of RBCs with or without concurrent plasma,
depending on the donor’s size and hemat-
ocrit.11,29,33 The addition of preservative and
leukocyte reduction by filtration are per-
formed manually and off-line after the collec-
tion is complete.
Granulocytes
TerumoBCT (COBE) Spectra
The Spectra is capable of collecting granulo-
cytes.11,34,35 It uses a doughnut-shaped, single-
stage channel in the centrifuge to isolate the
granulocytes that are continuously collected
in the final storage bag. It uses a double-access
kit only, and its ECV is 285 mL.
TerumoBCT Spectra Optia
The Spectra Optia system (not to be confused
with the Spectra system above) used mainly
for therapeutic apheresis procedures has re-
cently been approved for granulocyte collec-
tions.36 Its ECV is 191 mL, as it is modified from
the MNC protocol.
176 䡲 AABB TECHNICAL MANUAL

Fresenius AS 104 Haemonetics MCS+ LN9000

The Fresenius AS 104 is capable of collecting
several components, including granulo-
cytes.11,37 It uses a doughnut-shaped, single-
stage channel to separate the granulocytes.
The granulocytes build up in the centrifuge
and are harvested into the final collection bag
intermittently. The AS 104 uses a double-
access kit, and its ECV is 175 mL.
The LN9000 can also be used to collect granu-
locytes. It uses the conical Latham bowl for
separation, and the buffy coat is then trans-
ferred to one of two bags. The red cells settle to
the bottom of the bag before being returned to
the donor. The LN9000 switches from one bag
to another to return the red cells. A single- or
double-access kit can be used for granulocyte
collection, and the ECV ranges, based on the
hematocrit of the donor, from 480 mL (38%
hematocrit) to 359 mL (52% hematocrit).

KEY POINTS

1. Apheresis components must meet many of the same basic regulatory requirements (eg, do-
nor consent, storage conditions, and transportation requirements) as whole-blood-derived
components, although more specific requirements also apply to each type of apheresis-
component collection.
2. The majority of platelets collected and transfused in the United States are apheresis-derived
platelets.
3. Plasma can be collected by apheresis for transfusion or as Source Plasma for subsequent
manufacturing. The FDA provides guidance regarding the volume of plasma that may be
collected using automated devices.
4. In multicomponent donations, a variety of components or combinations of components
may be collected with apheresis technology. Regulations specific to this practice apply to
donor selection and monitoring, QC, and records.
5. Red cells can be removed concurrently with other components, or a double RBC unit may
be collected.
6. Several instruments and systems have been developed and/or adapted for apheresis collec-
tion of blood components that use different technologies. Some are appropriate for the col-
lection of only one type of component, and others can collect multiple types of compo-
nents.
7. Granulocyte collection differs from that of other components. Specific techniques should
be used and certain factors must be taken into consideration for the optimal collection of
granulocytes by apheresis.

REFER ENCES

1. Levitt J, ed. Standards for blood banks and
transfusion services. 29th ed. Bethesda, MD:
AABB, 2014.
2. US Department of Health and Human Servic-
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utilization survey report. Washington, DC:
DHHS, 2013:19.
3. Food and Drug Administration. Guidance for
industry and FDA review staff: Collection of
platelets by automated methods. (December
17, 2007) Silver Spring, MD: CBER Office of
Communication, Outreach, and Development,
2012. [Available at http://www.fda.gov/Biolog
icsBloodVaccines/GuidanceComplianceRegu
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073382.htm (accessed December 10, 2013).]
4. Wiltbank TB, Giordano GE. The safety profile
of automated collections: An analysis of more
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than 1 million collections. Transfusion 2007;
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5. Code of federal regulations. Title 21, CFR Part
610.40. Washington, DC: US Government
Printing Office, 2014 (revised annually).
6. Code of federal regulations. Title 21, CFR Part
640, Subpart G. Washington, DC: US Govern-
ment Printing Office, 2014 (revised annually).
7. Food and Drug Administration. Guidance for
industry: Recommendations for collecting red
blood cells by automated apheresis methods.
(January 30, 2001) Silver Spring, MD: CBER Of-
fice of Communication, Outreach, and Devel-
opment, 2001. [Available at http://www.fda.
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GuidanceComplianceRegulatoryInformation/
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Granulocyte transfusions for preventing infec-
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2009;1:CD005341.
9. Food and Drug Administration. Safety com-
munication: Boxed warning on increased mor-
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Rockville, MD: Office of Community Outreach
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30, 2014).]
10. Burgstaler EA. Current instrumentation for
apheresis. In: McLeod BC, Szczepiorkowski
ZM, Weinstein R, Winters JL, eds. Apheresis:
Principles and practice. 3rd ed. Bethesda, MD:
AABB Press, 2010:71-110.
11. Burgstaler EA. Blood component collection by
apheresis. J Clin Apher 2006;21:142-51.
12. Hood M, Mynderup N, Doxon L. Evaluation of
Haemonetics PCS-2 and Fenwal Auto-C plas-
mapheresis collection systems (abstract).
J Clin Apher 1996;11:99.
13. Burkhardt T, Kappelsberger C, Karl M. Evalua-
tion of a new combined centrifugation/filtra-
tion method for the collection of plasma via
plasmapheresis (abstract). Transfusion 2001;
41(Suppl):50S.
14. Burnouf T, Kappelsberger C, Frank K, Bur-
khardt T. Protein composition and activation
markers in plasma collected by three apheresis
procedures. Transfusion 2003;43:1223-30.
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15. Burnouf T, Kappelsberger C, Frank K, Bur-
khardt T. Residual cell content in plasma pro-
duced by three apheresis procedures. Transfu-
sion 2003;43:1522-6.
16. Burgstaler EA, Pineda AA, Bryant SC. Prospec-
tive comparison of plateletapheresis using
four apheresis systems on the same donors.
J Clin Apher 1999;14:163-70.
17. Perseghin P, Mascaretti L, Riva M, et al. Com-
parison of plateletapheresis concentrates pro-
duced with Spectra LRS version 5.1 and LRS
Turbo version 7.0 cell separators. Transfusion
2000;40:789-93.
18. Zingsem J, Glaser A, Weisbach V. Evaluation of
a platelet apheresis technique for the prepara-
tion of leukocyte-reduced platelet concen-
trates. Vox Sang 1998;74:189-92.
19. Zingsem J, Zimmermann R, Weisbach V, et al.
Comparison of COBE white cell-reduction and
standard plateletapheresis protocols in the
same donors. Transfusion 1997;37:1045-9.
20. Maresh S, Randels M, Strauss R, et al. Compar-
ison of plateletapheresis with a standard and
an improved collection device. Transfusion
1993;33:835-7.
21. McAteer M, Kagen L, Graminske S, et al. Trima
Accel improved platelet collection efficiency
with the merging of single stage separation
technology with leukoreduction performance
of the LRS chamber (abstract). Transfusion
2002;42(Suppl):37S.
22. Burgstaler EA, Winters JL, Pineda AA. Paired
comparison of Gambro Trima Accel vs Baxter
Amicus single-needle plateletapheresis. Trans-
fusion 2004;44:1612-20.
23. Elfath MD, Whitley P, Jacobson MS, et al. Eval-
uation of an automated system for the collec-
tion of packed RBCs, platelets, and plasma.
Transfusion 2000;40:1214-22.
24. Yockey C, Murphy S, Eggers L, et al. Evaluation
of the Amicus separator in the collection of
apheresis platelets. Transfusion 1999;38:848
25. Valbonesi M, Florio G, Ruzzenenti MR, et al.
Multicomponent collection (MCC) with the
latest hemapheresis apparatuses. Int J Artif Or-
gans 1999;22:511-15.
26. Paciorek L, Holme S, Andres M, et al. Evalua-
tion of the continuous filtration method with
double platelet products collected on the
MCS+ (abstract). J Clin Apher 1998;13:87.
27. Ford K, Thompson C, McWhorter R, et al. Eval-
uation of the Haemonetics MCS+ LN9000 to
produce leukoreduced platelet products (ab-
stract). J Clin Apher 1996;11:104.
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28. Rose C, Ragusa M, Andres M, et al. Evaluation
of the MCS + LN9000 in-line leukoreduction
filter (abstract). Transfusion 1996;36(Suppl):85.
29. Picker SM, Radojska SM, Gathof BS. Prospec-
tive evaluation of double RBC collection using
three different apheresis systems. Transfus
Apher Sci 2006;35:197-205.
30. Snyder EL, Elfath MD, Taylor H, et al. Collec-
tion of two units of leukoreduced RBCs from a
single donation with a portable multiple-com-
ponent collection system. Transfusion 2003;
43:1695-705.
31. Moog R, Frank V, Müller N. Evaluation of a
concurrent multicomponent collection sys-
tem for the collection and storage of WBC-
reduced RBC apheresis concentrates. Transfu-
sion 2001;41:1159-64.
32. Nussbaumer W, Grabmer C, Maurer M, et al.
Evaluation of a new mobile two unit red cell
apheresis system (abstract). J Clin Apher 2006;
21:20.
33. Smith JW. Automated donations: Plasma, red
cells, and multicomponent donor procedures.
In: McLeod BC, Szczepiorkowski ZM, Wein-
stein R, Winters JL, eds. Apheresis: Principles
and practice. 3rd ed. Bethesda, MD: AABB
Press, 2010:125-40.
34. Worel N, Kurz M, Peters C, Höcker P. Serial
granulocyte apheresis under daily administra-
tion of rHuG-CSF: Effects on peripheral blood
counts, collection efficiency, and yield. Trans-
fusion 2001;41:390-5.
35. Dale DC, Lises WC, Llewellyn C, et al. Neutro-
phil transfusions: Kinetics and functions of
neutrophils mobilized with granulocyte colo-
ny-stimulating factor and dexamethasone.
Transfusion 1998;38:713-21.
36. Food and Drug Administration. Substantially
equiva lent 510 (k) device infor m ation,
BK130065 summary. TerumoBCT Spectra Op-
tia. Silver Spring, MD: FDA, 2013. [Available
at:// http://www.fda.gov/BiologicsBloodVac
cines/BloodBloodProducts/ApprovedProd
ucts/SubstantiallyEquivalent510kDeviceIn
formation/ucm390957.htm (accessed March
31, 2014).]
37. Kretschmer V, Biehl M, Coffe C, et al. New fea-
tures of the Fresenius blood cell separator
AS104. In: Agishi T, Kawamura A, Mineshima
M, eds. Therapeutic plasmapheresis (XII): Pro-
ceedings of the 4th International Congress of
Apheresis of the World Apheresis Association
and the 12th Annual Symposium of the Japa-
nese Society for Apheresis, 3-5 June 1992, Sap-
poro, Japan. Utrecht, the Netherlands: VSP BV,
1993:851-5.
 C h a p t e r 8

Infectious Disease Screening

Susan A. Galel, MD

BLOOD COMPONENTS, LIKE all
other medications in the United States,
are regulated by the Food and Drug Adminis-
tration (FDA). The FDA requires medication
manufacturers to verify the suitability of every
raw material in their products.1 For biologic
pharmaceuticals, the donor is the key ingredi-
ent whose suitability must be scrutinized.
Blood banks test a sample of blood from
each donation to identify donors and donated
components that might harbor infectious
agents. This screening process is critically im-
portant because many blood components (eg,
red cells, platelets, plasma, and cryoprecipi-
tate) are administered intravenously to recipi-
ents without pasteurization, sterilization, or
other treatments to inactivate infectious
agents. Thus, infectious agents in a donor’s
blood at the time of donation that are not de-
tected by the screening process can be trans-
mitted directly to recipients.
H I S TO R I C A L OVE RV I EW O F
BLOOD DONOR SCREENING
Table 8-1 shows the progression over time of
donor testing for infectious diseases in the
United States. Initially, donors were screened
only for syphilis. In the 1960s, studies showed
that more than 30% of patients who received
multiple transfusions developed posttransfu-
sion hepatitis (PTH).2 Studies in the early
1970s found that hepatitis B virus (HBV) ac-
counted for only 25% of PTH cases.2 Both HBV
and non-A, non-B (NANB) hepatitis occurred
more frequently in recipients of blood from
commercial (paid) blood donors than in recip-
ients of blood from volunteer donors. By the
mid-1970s, implementation of sensitive tests
for hepatitis B surface antigen (HBsAg) and a
nearly universal changeover to a volunteer do-
nor supply resulted in a dramatic reduction in
the incidence of both HBV and NANB PTH.
Still, NANB PTH continued to occur in approx-
imately 6% to 10% of multitransfused pa-
tients.2,3
In the absence of a specific test for the
causative agent of NANB PTH, investigators
searched for surrogate markers that could be
used to identify donations associated with
NANB hepatitis. The presence of antibody to
hepatitis B core antigen (anti-HBc) and/or the
presence of elevated alanine aminotransfer-
ase in blood donors were shown to be associ-

Susan A. Galel, MD, Associate Professor of Pathology, Stanford University School of Medicine, and Medical
Director of Clinical Services, Stanford Blood Center, Palo Alto, California
The author has disclosed a financial relationship with Roche Molecular Systems.

179
 180
䡲

TABLE 8-1. Changes in US Donor Testing for Infectious Diseases

Year First
Implemented Screening Test Comments

1940s-1950s Syphilis The syphilis test was mandated by FDA in 1950s.

1970s HBsAg The first-generation test was available in 1970, and a higher-sensitivity test was required in 1973.
1985 Antibody to HIV (anti-HTLV-III) The initial name for HIV, the virus that causes AIDS, was HTLV-III. The first test for antibody to
HIV was called “anti-HTLV-III.”
1986-1987 ALT and anti-HBc ALT and anti-HBc were recommended by AABB as surrogate tests for NANB hepatitis. These tests
were initially not licensed by FDA for donor screening. AABB’s recommendation for donor ALT
testing was dropped in 1995 after antibody testing for HCV was in place. Anti-HBc was licensed
and required by the FDA in 1991.
1988 Anti-HTLV-I Although HTLV-I infection is usually asymptomatic, a small percentage of infected individuals
AABB TECHNICAL MANUAL

develop leukemia, lymphoma, or a neurologic disease.

1990 Antibody to HCV, Version 1 (anti-HCV 1.0) HCV was identified as the cause of most cases of NANB hepatitis.
1991 Anti-HBc Anti-HBc was previously recommended by AABB as a surrogate screen for NANB hepatitis. It was
required by the FDA in 1991 as an additional screen for HBV.
1992 Anti-HCV 2.0 This version had improved ability to detect antibody to HCV.
1992 Anti-HIV-1/2 The new HIV antibody tests had improved ability to detect early infection and an expanded range
of detection that included HIV-2 in addition to HIV-1.
1996 HIV-1 p24 antigen test This test was found to detect HIV-1 infection 6 days earlier than the antibody test. FDA permitted
discontinuation of HIV-1 p24 antigen testing with the implementation of a licensed HIV-1 nucleic
acid test.
1997-1998 Anti-HTLV-I/II The new HTLV antibody tests detected HTLV-II in addition to HTLV-I.
1999 HIV-1 and HCV nucleic acid tests to detect These tests were implemented initially as investigational assays and were licensed by FDA in
HIV and HCV RNA 2002. They detect infection earlier than antibody or antigen assays.
2003 West Nile virus nucleic acid test to detect This test was implemented initially as an investigational assay and was licensed by FDA during
WNV RNA 2005-2007. Testing of individual donations, rather than minipools, at times of increased WNV
activity in the region was recommended by the AABB in 2004 and the FDA in 2009.
CHAPTER 8

2004 Sampling of platelet components to detect Testing was recommended by the AABB in 2004. Some tests are approved by FDA as quality-
bacterial contamination control tests. Since 2011, AABB has accepted only FDA-approved tests or those validated to have
equivalent sensitivity.
2006-2007 Antibody to Trypanosoma cruzi This test was approved by FDA as a donor screen late in 2006, and widespread testing was imple-
mented in 2007. The rarity of seroconversion in US residents led to endorsement in FDA 2010
guidance of one-time donor screening.
2007-2008 HBV nucleic acid test (detects HBV DNA) This test was initially implemented as part of automated multiplex assays that detect HIV RNA,
HCV RNA, and HBV DNA simultaneously. HBV DNA screening was explicitly recommended by
FDA guidance issued in October 2012.
Infectious Disease Screening

HBsAg = hepatitis B surface antigen; AIDS = acquired immune deficiency syndrome; FDA = Food and Drug Administration; HIV = human immunodeficiency virus; HTLV = human T-cell lym-
photropic virus; ALT = alanine aminotransferase; HBc = hepatitis B core antigen; HCV = hepatitis C virus; NANB = non-A, non-B; HBV = hepatitis B virus; RNA = ribonucleic acid; WNV = West
Nile virus; DNA = deoxyribonucleic acid.
䡲
181
182 䡲 AABB TECHNICAL MANUAL
ated with an increased risk of NANB PTH.4-7
However, concerns about the nonspecific na-
ture of these tests led to a delay in their imple-
mentation for donor screening.
The concept of surrogate testing was re-
visited in the early 1980s when concerns arose
about the transmission of AIDS by transfu-
sions before the identification of its causative
agent. In an effort to reduce the potential
transmission of AIDS by transfusion, some
blood banks implemented donor testing for
anti-HBc (because this antibody was highly
prevalent in populations at increased risk of
AIDS) or donor screening for inverted CD4/
CD8 T-cell ratio (an immune abnormality
found in both AIDS patients and those with
the pre-AIDS lymphadenopathy syndrome).8
The leaders of most blood banks, however, be-
lieved that the risk of transmitting AIDS by
transfusion was too low to warrant surrogate
interventions.9 After the human immunodefi-
ciency virus (HIV) was isolated and identified
as the causative agent of AIDS, a donor screen-
ing test for antibody to this agent was rapidly
developed and implemented in 1985.
Once the HIV antibody test became avail-
able and cases of HIV were recognized in both
prior donors and transfusion recipients, it be-
came clear that the risk of transmitting HIV via
blood transfusion had been greatly underesti-
mated.10 The HIV experience highlighted the
fact that an infectious agent associated with a
lengthy asymptomatic carrier state could be
present in the blood supply for years without
being recognized.
In the wake of this realization, the ap-
proach to donor screening was extended be-
yond known agents. Current donor history
evaluations include screening for, and exclu-
sion of, donors with, an increased risk of expo-
sure to blood-borne or sexually transmitted
diseases. The intention is to reduce the likeli-
hood that the blood supply will contain other,
as-yet-unidentified agents that are potentially
transmissible by blood.
Rare transmissions of HIV persisted even
after implementation of donor testing due to a
delay of weeks or months between the time a
person is infected with HIV and the time the
screening test for HIV antibody shows positive
results.11 Blood donated during this “window
period” can contain infectious HIV but is not
detected by the donor screening tests.
The most straightforward means of pro-
tecting the blood supply from window-period
donations is to exclude potential donors with
an increased likelihood of exposure to HIV.
The FDA initially recommended that blood
banks provide informational materials to do-
nors listing HIV risk activities and requesting
that individuals not donate if they had en-
gaged in these activities. Later, in 1990, the
FDA recommended asking each donor directly
about each risk activity. In 1992, the FDA is-
sued comprehensive guidance describing this
questioning process.
In the years since the discovery of HIV, the
risk of transfusion-transmitted disease has
been progressively reduced through a variety
of measures:
1. Use of donor education and questioning to
minimize window-period donations and to
screen for infections for which no tests are
available.
2. Shortening of the window period for spe-
cific agents by improving and/or adding
tests to detect earlier stages of infection.
3. Use of questions and tests that exclude
donors at increased risk of blood-borne or
sexually transmitted infections.
4. Surveillance for transfusion-transmissible
diseases and implementation of new donor
screening tests, when available.
The approach used to screen potential
donors for a specific agent depends on wheth-
er specific risk factors are identifiable and
whether donor screening tests are available.
Table 8-2 lists the types of screening approach-
es used for different infectious agents.
DONOR SCREENING TESTS
The donor infectious disease tests required by
the FDA are specified in Title 21, Section
610.40, of the Code of Federal Regulations
(CFR).1 The process of amending the CFR is
slow; therefore, the FDA initially communi-
 CHAPTER 8 Infectious Disease Screening 䡲 183

TABLE 8-2. Approaches to Donor Screening

Approach Context for Use Example(s)

Questioning only Infectious agents with defined risk Malaria, prions

Testing only
Questioning and testing
Use of blood components that test
negative for specific recipients
Testing of blood components
factors and no sensitive and/or
specific test
Donor test is available, but no ques- West Nile virus
tion to distinguish individuals at risk
of infection
Agents for which there are both Human immunodeficiency
identified risk factors and effective virus, hepatitis B and C viruses
tests
Agents with a high prevalence in Cytomegalovirus
donors but for which an identifiable
subset of recipients can benefit
from blood components that test
negative
Infectious agent not detectable in Bacteria
donor samples

cates changes in its recommendations by issu-
ing guidance publications. Although FDA
guidance documents do not constitute legal
requirements, they define the expected stan-
dard of practice in the United States. The AABB
also issues recommendations to the blood
banking community, and these are communi-
cated either by Association Bulletins or by in-
clusion in AABB Standards for Blood Banks
and Transfusion Services. AABB recommenda-
tions and standards do not have the force of
law, except that one US state (California) has
incorporated some AABB standards into state
law.
AABB standards are often considered
throughout the United States as defining a
standard of practice in the blood banking
community and therefore are widely imple-
mented. Since 1985, the FDA and AABB have
issued a series of recommendations and/or
standards for additional screening tests in ad-
dition to the long-standing donor screens for
syphilis and HBsAg. Table 8-1 summarizes the
chronology of changes in donor infectious dis-
ease testing, and Table 8-3 lists the donor
screening tests that are currently performed by
US blood banks.
Logistics of Testing
All infectious disease testing for donor qualifi-
cation purposes is performed on samples col-
lected at the time of donation and sent to the
donor testing laboratory. In addition, platelet
components are tested for bacterial contami-
nation typically by the component manufac-
turing facility.
Laboratories that perform FDA-mandat-
ed donor testing must be registered with the
FDA as biologics manufacturers because this
“qualification of raw materials” is considered
part of the blood component manufacturing
process. The infectious disease tests and test-
ing equipment used to screen donors must be
approved (licensed or cleared) for this purpose
by the FDA Center for Biologics Evaluation and
Research. The tests must be performed exact-
ly as specified in the manufacturers’ package
inserts. Tests and test platforms that are ap-
proved only for diagnostic use may not be used
for screening whole blood donors.
184 䡲 AABB TECHNICAL MANUAL

TABLE 8-3. Blood Donor Screening Tests Performed in the United States

Agent Marker Detected Screening Test Method Supplemental Assays*

HBV 䡲 Hepatitis B surface ChLIA or EIA 䡲 Positive HBV DNA (FDA)†

antigen 䡲 Neutralization (FDA)

䡲 IgM and IgG antibody to ChLIA or EIA

hepatitis B core antigen

䡲 HBV DNA‡ TMA or PCR

HCV 䡲 IgG antibody to HCV ChLIA or EIA 䡲 Positive HCV RNA (FDA)†
peptides 䡲 RIBA (FDA)§

䡲 HCV RNA‡ TMA or PCR

HIV-1/2 䡲 IgM and IgG antibody to ChLIA or EIA 䡲 Positive HIV RNA (FDA)†
HIV-1/2 䡲 HIV-1: IFA or Western
blot (FDA)
䡲 HIV-2: EIA (FDA)

䡲 HIV-1 RNA‡ TMA or PCR

HTLV-I/II 䡲 IgG antibody to HTLV-I/II ChLIA or EIA IFA, Western blot, RIPA, and
line immunoblot

Syphilis 䡲 IgG or IgG + IgM antibody Microhemagglutination or T. pallidum antigen-specific

to Treponema pallidum EIA immunofluorescence or
antigens agglutination assays

or

䡲 Nontreponemal serologic Solid-phase red cell adher- T. pallidum antigen-specific

test for syphilis (eg, rapid ence or particle agglutination immunofluorescence or
plasma reagin) agglutination assays

WNV 䡲 WNV RNA‡ TMA or PCR

Trypanosoma 䡲 IgG antibody to T. cruzi ChLIA or EIA ESA (FDA)

cruzi (one time)|| RIPA

*Supplemental assays with “(FDA)” are FDA-approved supplemental assays. Other supplemental assays listed are not
required but may be useful for donor counseling.
†
Positive results on some nucleic acid tests are approved by FDA as providing confirmation for reactive HBsAg, HIV anti-
body, and HCV antibody serology tests. If a nucleic acid test result is negative, serologic supplemental test(s) must be
performed.
‡
Screening for DNA or RNA in the United States is usually performed on minipools of 6 to 16 donor samples.
§
As of 2013, RIBA is not available. FDA variance may be obtained to use a second licensed screening test as an alternative.
||
T. cruzi antibody testing may be limited to one-time testing of each donor.
HBV = hepatitis B virus; ChLIA = chemiluminescent immunoassay; EIA = enzyme immunoassay; DNA = deoxyribonucleic
acid, FDA = Food and Drug Administration; Ig = immune globulin; TMA = transcription-mediated amplification; PCR = poly-
merase chain reaction; HCV = hepatitis C virus; RNA = ribonucleic acid; RIBA = recombinant immunoblot assay; HIV-1/2 =
human immunodeficiency virus types 1 and 2; IFA = immunofluorescence assay; HTLV-I/II = human T-cell lymphotropic
virus, types I and II; RIPA = radioimmunoprecipitation assay, WNV = West Nile virus; ESA = enzyme strip assay.
 CHAPTER 8
Serologic Testing Process
Most of the serologic screening tests (assays
for the detection of antibody or antigen) are
enzyme immunosorbent assays or chemilumi-
nescent immunoassays. Typically, the process
involves performing each required screening
test once on each donor sample. If the screen-
ing test is nonreactive, the test result is consid-
ered negative (ie, there is no evidence of infec-
tion). If a test is reactive during the first round
of testing (“initially reactive”), the package in-
sert for the test typically requires that the test
be repeated in duplicate. If both of the repeat
results are nonreactive, the final interpretation
is nonreactive or negative, and the unit may be
used. If one or both of the repeat results are re-
active, the donor sample is characterized as
“repeatedly reactive,” and the blood unit is not
permitted to be used for allogeneic transfu-
sion. (In the case of cellular therapy products,
there are some circumstances in which repeat-
edly reactive donations may be used. See
“Considerations in Testing Donors of Human
Cells, Tissues, and Cellular and Tissue-Based
Products” later in this chapter.)
The infectious disease tests that are ap-
proved for donor screening have performance
characteristics chosen to make them highly
sensitive. They are designed to detect almost
all infected individuals and minimize false-
negative results. However, to achieve this sen-
sitivity, the assays also react with samples from
some individuals who are not infected (false-
positive results). Because the blood donor
population is preselected by questioning to be
at low risk of infection, the vast majority of re-
peatedly reactive results in donors do not rep-
resent true infections. To determine whether a
repeatedly reactive screening result represents
a true infection rather than a false-positive re-
sult, additional, more specific testing may be
performed on the donor sample.
FDA requires that repeatedly reactive do-
nor specimens be further evaluated by FDA-
approved supplemental/confirmatory assays
when such assays are available.1 FDA has ap-
proved confirmatory assays for HBsAg, HIV
type 1 (HIV-1) antibody, hepatitis C virus
(HCV) antibody, and antibody to Trypanoso-
Infectious Disease Screening 䡲 185
ma cruzi. The HCV antibody confirmatory test
(the recombinant immunoblot assay) recently
became unavailable, but blood centers can ob-
tain FDA approval to use an alternative HCV
supplemental testing pathway.12 If no licensed
confirmatory assay is available, unlicensed
supplemental tests or retests of the donor
sample using a second licensed screening test
may be helpful for donor counseling. Table 8-3
displays the available confirmatory and sup-
plemental assays.
A donation that is repeatedly reactive on a
screening test is not permitted to be used for
allogeneic transfusion, regardless of the results
of confirmatory or supplemental testing.
Syphilis is the only agent for which negative
results on supplemental tests can, in some cir-
cumstances, enable use of a screening test-
reactive unit. (This applies only to donations
screened using a nontreponemal assay.) This
situation is discussed more fully in the “Syphi-
lis” section.
Nucleic Acid Testing
Nucleic acid testing (NAT) was implemented
to reduce the window periods described
above. The process of screening donor sam-
ples for viral nucleic acid [ribonucleic acid
(RNA) or deoxyribonucleic acid (DNA)] is
somewhat different from the serologic screen-
ing process. NAT requires the extraction of nu-
cleic acid from donor plasma followed by use
of a nucleic acid amplification test to amplify
and detect viral genetic sequences.
The test systems that were initially devel-
oped in 1999 to screen donors for HIV and
HCV RNA were semiautomated and had insuf-
ficient throughput to allow individual testing
of each donor sample. Testing of seroconver-
sion panels showed little loss of sensitivity if
donor plasma samples were tested in small
pools [minipools (MPs)] because levels of HIV
and HCV RNA are typically high in the blood of
infected individuals and NAT assays are exqui-
sitely sensitive.
Thus, in the initially approved NAT donor
screening systems, MPs of 16 to 24 donor sam-
ples were prepared and tested together. If a
pool tested negative, all donations contribut-
186 䡲 AABB TECHNICAL MANUAL
ing to that pool were considered negative for
HIV and HCV RNA. If a pool showed reactivity
on the nucleic acid test, further testing of
smaller pools and, ultimately, individual sam-
ples was performed to determine which dona-
tion was responsible for the reactive test result.
Donations that were nonreactive on this addi-
tional testing could be released for transfu-
sion. Donations that were reactive at the indi-
vidual sample level were considered positive
for viral nucleic acid and could not be released
for transfusion.
In recent years, fully automated NAT sys-
tems have been developed. The two automat-
ed test platforms that are approved by the FDA
for donor screening use multiplex assays that
detect HIV RNA, HCV RNA, and HBV DNA in
one reaction chamber. Reactive donations are
subjected to discriminatory testing to identify
which virus is present. These systems are ap-
proved for testing of individual donations and
pools of 6 to 16 donor samples, depending on
the platform. The availability of fully automat-
ed NAT platforms raises the possibility of mov-
ing to routine screening of individual donor
samples [individual donation (ID) screening],
rather than testing of pools (MP-NAT).
However, it has been estimated that ID-
NAT screening would minimally increase de-
tection of infected donors, whereas the associ-
ated testing cost would be significantly higher
than with MP-NAT.13 Furthermore, it is not
clear that ID-NAT screening of the entire US
blood supply would be logistically feasible us-
ing the available platforms. An additional con-
cern is that donors might be deferred for false-
positive results more frequently with ID-NAT
screening than pooled screening.
In contrast to serologic testing policies,
repeat testing is not permitted by the FDA for
an individually reactive NAT sample to deter-
mine whether the initially reactive result rep-
resents a true-positive result. If an individual
(unpooled) specimen is reactive on a NAT
screen for HIV, HCV, or HBV, the FDA requires
that the corresponding blood component be
discarded and the donor be deferred indefi-
nitely.
ID-NAT screening rather than MP-NAT
screening is recommended when West Nile vi-
rus (WNV) activity is high in a specific geo-
graphic area. Circulating levels of viral RNA are
often low during WNV infection. When donor
samples are combined into MPs, the RNA from
a WNV-infected sample may become diluted
to below the detectable level. It has been esti-
mated that MP-NAT screening for WNV RNA
may fail to detect 50% or more of infected do-
nations.14-16 Therefore, both the FDA and AABB
have recommended ID-NAT screening for
WNV RNA at times of high WNV activity in a
region.16,17
Implications of Reactive Test Results
A repeatedly reactive result on a screening test
(or individually reactive NAT result) typically
results in mandatory discarding of the reactive
donation. Most blood bank computer systems
prevent labeling and/or release of products
from donations with reactive test results. A re-
active test result may also indicate that the do-
nor should be prohibited from making future
donations, because many infections are per-
sistent. Furthermore, past donations may also
be considered suspect because the exact date
of onset of a donor’s infection cannot be deter-
mined.
Both the FDA and AABB have issued rec-
ommendations regarding whether reactive
test results affect a donor’s eligibility for future
donations, components from prior donations
should be retrieved (and if so, how far back in
time), and patients who previously received
components from that donor should be noti-
fied. These recommendations are often guided
by the results of supplemental or confirmatory
testing performed on the donor sample. Many
of these recommendations have evolved over
time.
Because these recommendations are
complex, blood bank laboratories typically
create checklists that list each of the actions to
be performed after a specific reactive test re-
sult is obtained. Staff use these checklists to
document completion of each action as they
perform it.
Table 8-416-37 lists federal regulations, FDA
guidance documents, AABB standards, and
AABB Association Bulletins with recommen-
TABLE 8-4. FDA, CMS, and AABB Recommendations for Blood Donor Testing and Actions Following Reactive Results*

Topics

Donor Donor Product Recipient Donor

Document Agent/Test Testing Management Retrieval Notification Reentry

Title 21, CFR Part 610.40 HIV-1/2, HBV, HCV, HTLV-I/II, and X
Syphilis
Title 21, CFR Part 610.41 HIV-1/2, HBV, HCV, HTLV-I/II, and X
Syphilis
Title 21, CFR Parts 610.46, 610.47, and HIV and HCV X X
610.48
Title 42, CFR Part 482.2724 HIV and HCV X X
26
CHAPTER 8

FDA guidance, October 2012 HBV X X X

25
FDA guidance, November 2011 HBV (vaccine) X
FDA guidance, December 201027 HCV X X
29
FDA guidance, December 2010 Trypanosoma cruzi X X X X
28
FDA guidance, May 2010 HIV and HCV X X X X X
30
FDA guidance, May 2010 Anti-HBc X
FDA guidance, November 200917 WNV X X X
Infectious Disease Screening

31
FDA guidance, August 2009 HIV-1 group O X X X
32 †
FDA guidance, July 2009 Parvovirus B19
33
FDA guidance, June 2005 WNV X X X
䡲

FDA guidance, October 200434 NAT for HIV-1 and HCV X

(Continued)
187
TABLE 8-4. FDA, CMS, and AABB Recommendations for Blood Donor Testing and Actions Following Reactive Results* (Continued)
188

Topics
䡲

Donor Donor Product Recipient Donor

Document Agent/Test Testing Management Retrieval Notification Reentry

FDA guidance, August 199735 HTLV X X X

36 ‡
FDA memorandum, July 1996 HBsAg and anti-HBc X
37
FDA memorandum, December 1991 Syphilis X X X§ X§ X
FDA memorandum, December 198723 HBsAg X X X
AABB Standard 5.8.518(p31) Required donor tests for HIV, HCV, HBV, X
HTLV, WNV, and syphilis
AABB Standards 5.1.5.1, 5.1.5.1.1, 5.1.5.2, Bacteria X X||
and 7.1.318(pp11,34,86)
Association Bulletin #10-0520 Bacteria X X||
Association Bulletin #13-0216 WNV X
AABB TECHNICAL MANUAL

Association Bulletin #05-0219 Bacteria X X

21
Association Bulletin #04-07 Bacteria X X X
22
Association Bulletin #99-9 HTLV X X
*Recommendations in effect as of March 2014. Blood centers may be bound by additional requirements, such as specifications in recovered plasma contracts.
†
Plasma for further manufacture only.
‡
Memorandum also includes recommendations regarding HCV and HTLV, but these recommendations have been superseded by subsequent documents.
§
Not recommended.
||
Co-components of current donation.
FDA = Food and Drug Administration; CMS = Centers for Medicare and Medicaid Services; CFR = Code of Federal Regulations; HIV-1/2 = human immunodeficiency virus, types 1 and 2;
HBV = hepatitis B virus; HCV = hepatitis C virus; HTLV-I/II = human T-cell lymphotropic virus, types I and II; anti-HBc = antibody to hepatitis B core antigen; WNV = West Nile virus;
NAT = nucleic acid testing; HBsAg = hepatitis B surface antigen.
 CHAPTER 8
dations regarding management of blood do-
nors with reactive test results, retrieval of other
components, and notification of prior recipi-
ents. These regulations and recommenda-
tions are described briefly below.
Donor Eligibility
FDA regulation Title 21, CFR Part 610.41, ad-
dresses donors with reactive screening test re-
sults. FDA guidance documents and AABB As-
sociation Bulletins contain more detailed
recommendations regarding additional test-
ing, donor eligibility, and donor counseling for
these and other tests. Donors should be noti-
fied of any test results that affect their eligibili-
ty or that could have important implications
for their health. Blood banks should have sys-
tems that prevent future collections from ineli-
gible donors and the release of any compo-
nents inadvertently collected from such
individuals.
For donors deferred for reactive screening
tests, Title 21, CFR Part 610.41, provides for re-
instatement by means of FDA-defined requali-
fication algorithms. The FDA has issued guid-
ance documents (listed in Table 8-4) that
define reentry pathways for donors deferred
for reactivity on HIV, HCV, HBsAg, anti-HBc,
serologic syphilis, and HIV/HCV/HBV NAT
tests. Most of the pathways require that the do-
nor have negative results on specified tests af-
ter a defined waiting period. Blood banks that
desire to reenter donors must follow the FDA-
defined algorithms explicitly.
Retrieval of Prior Donations and
Notification of Prior Recipients
(“Look-Back”)
The FDA and AABB offer guidance with regard
to the appropriate management of previously
collected blood components from donors
whose current donation is repeatedly reactive
(or, in the case of NAT, individually reactive) on
an infectious disease screening test. These rec-
ommendations address the concern that at the
time of the previous donation, the donor could
have been in the window period of an early in-
Infectious Disease Screening 䡲 189
fection even though the screening test results
were negative.
For HIV and HCV tests, the algorithms for
managing prior donations and recipients of
prior donations are spelled out in Title 21, CFR
Parts 610.46 and 610.47. These requirements
are replicated in the Centers for Medicare and
Medicaid Services regulations (Title 42, CFR
Part 482.27) to ensure hospital transfusion ser-
vice compliance with recipient notification
requirements. For other agents, recommenda-
tions for the management of previously donat-
ed components may be found in the FDA
guidance documents or AABB Association Bul-
letins (or both) listed in Table 8-4.
In most cases, the FDA and AABB recom-
mend retrieval and quarantine of any remain-
ing components from prior donations of that
donor. It is essential that the retrieval of in-
date components be initiated immediately af-
ter the repeatedly reactive result is obtained.
This approach prevents transfusion of these
components while confirmatory testing is per-
formed. The FDA requires initiation of retriev-
al within 3 calendar days of a reactive HIV or
HCV test and within 1 week of reactive HBsAg,
anti-HBc, or anti-HTLV screening tests. If con-
firmatory test results on the current donation
are negative, the FDA, in some circumstances,
permits rerelease of the prior donations. In
many cases, some or all of the components
from prior donations will have been trans-
fused. For some infectious agents, the FDA
and AABB recommend that the recipients of
prior donations from confirmed positive do-
nors be notified of their possible exposure to
the infectious agent.
Recommendations for notification of re-
cipients of prior donations (“look-back”) are
usually issued by the AABB, FDA, or both at the
time a new test is implemented, but these rec-
ommendations may evolve as confirmatory
tests become available or medical treatments
are developed for the infection in question.
Look-back is required by law only for HIV and
HCV tests (Title 21, CFR Parts 610.46 and
610.47). For an HIV look-back investigation in-
volving a deceased prior recipient, the next of
kin must be notified. The CFR spells out spe-
cific timelines for component retrieval and
190 䡲 AABB TECHNICAL MANUAL
recipient notification. It also specifies how far
back in time (ie, to which donations) the re-
trieval and notification should extend. For oth-
er agents, such as WNV and T. cruzi, recom-
mendations regarding retrieval and recipient
notification are included in FDA guidance
documents and AABB Association Bulletins.
Table 8-4 indicates which of these documents
address product retrieval or recipient notifica-
tion.
In the absence of published guidance, it is
not always obvious whether or when it is ap-
propriate to notify prior recipients of their
possible exposure to infection. If there is no
confirmatory assay available, it may be diffi-
cult to determine whether a repeatedly reac-
tive screening test result for a donor represents
a true infection. Furthermore, if there is no ef-
fective treatment for that infection, there may
be no obvious benefit to the recipient of being
told that he or she might have been exposed.
There could, however, be a public health bene-
fit from such a notification. Specifically, a re-
cipient who is alerted of a potential exposure
can be tested and, if the results are positive,
take precautions to avoid further spread of the
infection.
Cytomegalovirus Testing of Products
for Immunocompromised Recipients
Some common infections cause relatively in-
nocuous illnesses in immunocompetent indi-
viduals but can cause severe disease in immu-
nocompromised patients. Such is the case
with cytomegalovirus (CMV).
CMV is a lipid-enveloped DNA virus in
the Herpesviridae family. Like other herpesvi-
ruses, CMV causes lifelong infection, typically
in a latent state, with the potential for reactiva-
tion. Primary CMV infection in immunologi-
cally competent individuals is mild, with
symptoms ranging from none to an infectious
mononucleosis-type syndrome. In immuno-
compromised patients, however, both primary
infection and reactivation disease can be over-
whelming and even fatal. CMV can be trans-
mitted by blood transfusion, primarily
through intact white cells contained in cellular
blood components. Frozen/thawed plasma
components do not appear to transmit CMV
infection. Immunocompromised patients who
are at increased risk of transfusion-transmit-
ted disease include fetuses, low-birthweight
premature infants who are born to CMV-sero-
negative mothers, and CMV-seronegative re-
cipients of solid-organ or allogeneic hemato-
poietic cell transplants from seronegative
donors.38
The majority of blood donors have had
prior exposure to CMV, indicated by the fact
that they have CMV antibodies. Therefore, it
would not be possible to produce an adequate
supply of blood if all CMV-antibody-positive
donations were discarded.
It is possible, however, to minimize CMV
transmission to patients at risk of severe CMV
disease, such as those described above. These
patients should be supported with cellular
blood components that have a reduced risk of
transmitting CMV. These reduced-risk options
include using blood components from donors
who are CMV antibody negative or compo-
nents that have been effectively leukocyte re-
duced. The literature suggests that these two
methods have similar but not identical effica-
cy, with an estimated transmission risk by
seronegative components of 1% to 2% vs a risk
of 2% to 3% with leukocyte-reduced compo-
nents.38-40 A recent study, however, found no
CMV transmissions among 100 carefully mon-
itored allogeneic hematopoietic cell transplant
recipients who received CMV-untested, leuko-
cyte-reduced components.41 Because many at-
risk patients receive leukocyte-reduced com-
ponents and are monitored closely for CMV
infection, treated early with anti-CMV drugs,
or both, it is difficult to measure a benefit from
also providing CMV-seronegative components
to these patients.
Autologous Donations
The FDA requires infectious disease testing of
autologous donations that are shipped from
one facility to another. If the receiving facility
does not permit autologous donations to be
crossed over to the general inventory, the FDA
requires testing of only the first donation in
each 30-day period [Title 21, CFR Part
 CHAPTER 8 Infectious Disease Screening 䡲 191

610.40(d)]. The labeling of the unit must be
consistent with its testing status. Units from
donors with repeatedly reactive tests must be
labeled with biohazard labels. Some hospitals
have policies that prohibit acceptance of au-
tologous units with positive results on some
tests because there is a potential for an infec-
tious unit to be transfused to the wrong pa-
tient. The AABB has warned that refusal of
test-positive units could be interpreted as vio-
lating the Americans with Disabilities Act.42
Considerations in Testing Donors of
Human Cells, Tissues, and Cellular
and Tissue-Based Products
Both the questions and tests required by FDA
to screen donors of human cells, tissues, and
cellular and tissue-based product (HCT/Ps)
differ from those for blood donors, and screen-
ing requirements vary by type of tissue. These
requirements are spelled out in Title 21, CFR
Part 1271 and an August 2007 FDA guidance
document and are summarized in Table 8-
5.43,44 The time frames for testing HCT/P do-
nors are also specified in these documents.
In most cases, the samples for infectious
disease testing must be obtained 7 days before
or after the tissue donation. However, samples
of peripheral blood hematopoietic progenitor
cells or marrow may be tested up to 30 days
before donation. Autologous tissues and re-
productive tissues from sexually intimate part-
ners may be exempt from some testing re-
quirements.
Blood bank laboratories that test samples
from HCT/P donors must take care to check
package inserts for HCT/P testing methods; a
package insert may require a different testing
method for HCT/P donors than for blood

TABLE 8-5. FDA Testing Requirements for HCT/Ps (as of March 2014)

Tissue Type Agent Tests

HIV Antibody to HIV-1 and HIV-2*

HIV-1 RNA*

HBV Hepatitis B surface antigen*

All tissues Antibody to hepatitis B core antigen*

HCV Antibody to hepatitis C*

HCV RNA*

Treponema pallidum FDA-cleared screening or diagnostic test

For donors of viable leukocyte-rich HTLV-I/II Antibody to HTLV-I/II*

HCT/Ps (eg, hematopoietic progenitor
CMV FDA-cleared screening test for anti-CMV
cells or semen) also test for the follow-
(total IgG and IgM)
ing in addition to the above:

For donors of reproductive tissues, test Chlamydia trachomatis FDA-licensed, approved, or cleared diag-
for the following in nostic test
addition to the above:
Neisseria gonorrhea FDA-licensed, approved, or cleared diag-
nostic test

*These tests must be FDA licensed for donor screening.

HCT/Ps = human cells, tissues, and cellular and tissue-based products; HIV = human immunodeficiency virus; RNA = ribo-
nucleic acid; HBV = hepatitis B virus; HCV = hepatitis C virus; FDA = Food and Drug Administration; HTLV = human T-cell
lymphotropic virus; CMV = cytomegalovirus; Ig = immune globulin.
192 䡲 AABB TECHNICAL MANUAL
donors. For example, NAT for most types of
HCT/P donors must be performed on individ-
ual donor samples, and MP-NAT is not permit-
ted for most HCT/P donor categories.
In some cases, FDA regulations permit
the use of HCT/P donations that are reactive
on infectious disease screening tests. These
exceptions are listed in Title 21, CFR Part
1271.65. FDA has issued specific labeling, stor-
age, and notification requirements for these
tissues.
Testing of HCT/P donors for WNV RNA
and antibody to T. cruzi is not required by FDA
as of March 2014. However, FDA has indicated
that it considers WNV to be a “relevant com-
municable disease,” and draft guidance docu-
ments indicate that FDA is considering a re-
quirement to test at least some HCT/P donors
for these agents.44
International Variations in Donor
Testing
Although this chapter focuses on infectious
disease screening in the United States, the
general approach to donor screening is similar
in other countries. However, the specific donor
questions and tests vary from country to coun-
try based on the regional epidemiology of in-
fections and tests available. For example, most
countries where WNV is not endemic do not
test for this agent, although they may question
donors about travel to WNV-affected coun-
tries. Countries where HBV is hyperendemic
cannot exclude donations from individuals
who test positive for anti-HBc without ad-
versely affecting the adequacy of their blood
supply. The AABB Standards Program Unit in
conjunction with the AABB Subcommittee for
the Evaluation of International Variances con-
siders variance applications from facilities in
countries where national practices and avail-
able tests differ from those in the United States.
R E SI D UA L I NF E C T I O U S R IS K S
OF TRANSFUSION
Despite donor screening, blood components
may still transmit infections. The residual risk
of transmission varies according to the inci-
dence of the infection in the donor population
and the nature of the donor screening process-
es in place.
Agents for Which Blood Is Tested
Transfusion transmission of HIV, HCV, and
HBV is now so rare that the rate of transmis-
sion cannot be measured by prospective clini-
cal studies. The risk can only be estimated by
theoretical modeling.
One theoretical source of risk is a virus
strain that the current test kits do not detect.
The Centers for Disease Control and Preven-
tion and the test manufacturers conduct sur-
veillance for such emerging strains. Over time,
the FDA has required that test manufacturers
expand their detection capabilities to include
new strains. A second potential cause of trans-
mission is a quarantine failure (ie, a blood
bank’s failure to quarantine a unit that tests
positive). Quarantine errors are thought to be
rare in blood banks that use electronic systems
to control blood component labeling and re-
lease because these systems are designed to
prevent the release of any unit with incom-
plete testing or a reactive test result. Erroneous
releases appear to occur more frequently in
blood banks that rely on manual records and
quarantine processes.45
The primary cause of residual transmis-
sions is thought to be donations from individ-
uals in the window period of early infection,
before test results are positive. Figure 8-1 dis-
plays the sequence in which different types of
donor screening tests demonstrate reactivity.
Over time, the window periods have been
shortened by implementation of donor
screening tests that detect earlier infections.
However, because no test gives a positive re-
sult immediately after an individual acquires
an infection, a window period remains. With
NAT, the average duration of the window peri-
od is estimated to be 9.0-9.1 days for HIV and
7.4 days for HCV.13,46 The window period for
HBV is longer, as discussed in the “HBV” sec-
tion below.
The likelihood that a blood donation has
been obtained from a donor in the window pe-
 CHAPTER 8
FIGURE 8-1. Time sequence of the appearance of various markers of infection.
riod can be estimated mathematically using
the incidence-window period model46:
Probability a donation was made during
the window period = length of window
period × incidence of infections in the
donor population
The incidence of infections in donors can
be calculated from the observed number of
donors with a negative test result for one do-
nation but a positive result for a subsequent
donation (ie, seroconverting donors). This
method measures incidence rates only in re-
peat donors and does not permit assessment
of the likelihood that first-time donors might
be in the window period.
Other methods permit measurement of
new infection rates in both first-time and re-
peat donors using tests that differentiate new
from established infections. Such tests include
nucleic acid tests (donor blood that contains
HIV or HCV RNA but not antibody most
likely represents a very early infection) and
“sensitive/less sensitive” antibody testing.13,46-49
When these alternative methods have been
used, new HIV and HCV infections were two to
four times more common among first-time
donors than among repeat donors.46-48 Howev-
Infectious Disease Screening 䡲 193
er, both of these donor populations have sig-
nificantly lower infection rates than the gener-
al population. The continued importance of
using donor questioning to select donors with
a low incidence of infection is explored in
more detail in the “HIV” section below.
The current estimated risks of HIV, HCV,
and HBV transmission in donors, based on
window-period and incidence calculations,
are shown in Table 8-6.
Agents for Which No Donor Screening
Tests Are Available
Essentially any infectious agent that can circu-
late in the blood of an apparently healthy per-
son could be transmitted by transfusion. It is
impossible to estimate the risk of transmission
for each of the infectious agents for which do-
nors are not tested. However, transfusion-
transmitted infections are rarely identified.
The infections that are most likely to be recog-
nized as transmitted by transfusion are those
that have a distinctive clinical presentation
and are otherwise rare in the United States.
The likelihood that an infection will be recog-
nized as transmitted by transfusion is en-
hanced if the infection is usually associated
with a behavioral risk that the transfusion
194 䡲 AABB TECHNICAL MANUAL

TABLE 8-6. Estimated Risks of Transfusion-Transmitted Infection in the United States Based on
Window-Period and Incidence Estimates*

Incidence per 105 Infectious Window Residual Risk per

Study Period Agent Person/Years Period (days) Donated Unit

2007-200846* HIV 3.1 9.1 1:1,467,000

46
2007-2008 * HCV 5.1 7.4 1:1,149,000
2009-201150† HBV 1.6 26.5-18.5 1:843,000 to
1:1,208,000
*HIV and HCV risk estimates are based on minipool nucleic acid testing in pools of 16.
†
HBV risk estimates are based on minipool nucleic acid testing in pools of 16 using the Novartis Ultrio Plus
assay. The range indicated for the HBV window period reflects uncertainty regarding the minimum infectious
dose of HBV (1 copy in 20 mL plasma vs 10 copies in 20 mL).
HIV = human immunodeficiency virus; HCV = hepatitis C virus; HBV = hepatitis B virus.

recipient lacks (eg, when malaria develops in a
transfusion recipient who has not traveled
outside the United States).
If a life-threatening agent is recognized as
a potential threat to the blood supply, both the
AABB and FDA typically consider whether a
donor screening question could be used to ex-
clude potentially exposed donors in the ab-
sence of a donor screening test. Donor ques-
tioning regarding travel to and residence in
endemic areas is currently the only means of
protecting the US blood supply from malaria
and variant Creutzfeldt-Jakob disease (vCJD).
Most infectious agents, however, do not have
such clear geographic risk areas. In general, it
is difficult to design donor questions that are
both sensitive (ie, detect most infected indi-
viduals) and specific (ie, exclude only infected
individuals).
An alternative method of protecting the
blood supply from infectious agents is
pathogen inactivation or reduction. Heat
inactivation, solvent/detergent treatment,
nanofiltration, chromatography, cold ethanol
fractionation, and other approaches have
been used with remarkable success to inacti-
vate or remove residual pathogens in plasma
derivatives. Pathogen reduction systems for
cellular blood components are not available in
the United States as of March 2014, although
systems for platelet components are in use in
some other countries. Pathogen reduction sys-
tems are discussed in the “Pathogen Reduc-
tion Technology” section later in this chapter.
The AABB Transfusion-Transmitted Dis-
eases Committee published an extensive re-
view of infectious agents that are possible
threats to the blood supply.51 Potential mitiga-
tion strategies were discussed for each agent,
including the documented or theoretical effi-
cacy of pathogen reduction processes. AABB
keeps these materials up to date and adds ma-
terials for new potential threats as they are
identified.52 The agents deemed to pose the
highest threat from either a scientific or public
perspective are briefly discussed in this chap-
ter. (See the 2009 supplement to TRANSFU-
SION and updates on the AABB website for a
more thorough review of these potential infec-
tious risks.51,52)
SCREENING FOR SPECIFIC
AGENTS
HIV
HIV-1, a lipid-enveloped, single-stranded RNA
virus, was identified in 1984 as the causative
agent of AIDS, and blood donor screening for
antibodies to this virus was implemented in
the United States in 1985. In 1992, donor
screening tests were modified to include de-
 CHAPTER 8
tection of antibodies to HIV-2, a closely related
virus identified initially in West Africa.
HIV can be transmitted sexually, paren-
terally, and from infected mothers to their in-
fants. Although heterosexual and vertical
spread of HIV predominate in some parts of
the world, new HIV cases in the United States
continue to be concentrated in men who have
sex with men (MSM) and individuals with
high-risk heterosexual contact (defined as
contact with an individual who is HIV positive
or in an identified risk group for HIV, such as
MSM or injection drug users).53
Current donor screening for HIV includes
NAT testing for HIV-1 RNA and serologic test-
ing for antibodies to HIV. The antibody tests
approved for donor screening detect both im-
mune globulin M (IgM) and IgG antibody to
both HIV-1 and HIV-2. Some of the approved
tests also detect antibody to the HIV-1 group
O, a strain of HIV-1 found primarily in Central
and West Africa. If the antibody test used by a
donor center does not have an HIV-1 group O
detection claim, the donor center must use
donor questioning to exclude individuals who
have resided in, received medical treatment in,
or had sex partners from HIV-1 group O en-
demic areas.31
The average lag time from HIV-1 exposure
to test detection (window period) is currently
estimated to be 9-9.1 days for MP-NAT.13,46
Based on window-period and incidence-rate
calculations, the current risk of acquiring HIV
from transfusion is estimated to be approxi-
mately 1 in 1.5 million units (Table 8-6).
In the United States, blood donor screen-
ing questions exclude very broadly defined
populations at increased risk of HIV. Given the
short delay of only days between exposure and
detection of infection by NAT, experts have
questioned whether donor interviews and ex-
clusion of donors with increased risk remain
medically necessary.54 The continued impor-
tance of a low-risk donor population becomes
evident if different HIV incidence figures are
entered into the blood safety calculation. For
example, HIV incidence rates as high as 1% to
8% have been observed in some high-risk pop-
ulations, such as young urban MSM.55,56 If an
individual from a population with a 1% inci-
Infectious Disease Screening 䡲 195
dence of HIV donates blood, the likelihood
that this individual is in the window period
and that the component will transmit HIV can
be calculated as follows:
Risk that the donation is in window period =
length of window period × incidence of infec-
tion in donor population = (9.0 days/365 days/
year) × (1/100 person-years) = 1/4100.
This is the likelihood that a unit from this
high-risk donor would harbor HIV but be
missed by the current donor screening. This
risk is clearly much higher than the estimated
HIV transmission risk of 1 in 1.5 million for a
unit of blood obtained from the current donor
population. Thus, despite the short window
period with current testing, inclusion of do-
nors with a high risk of HIV would have a pro-
foundly adverse impact on blood safety. Ac-
cordingly, questioning of donors for risk and
temporarily excluding those at increased risk
to minimize window-period donations contin-
ue to be critical for preserving blood safety.
Despite the efficacy of current donor
questioning approaches, there has been great
interest in developing a more specific donor-
screening algorithm for MSM that would
exclude only individuals who are truly at in-
creased risk of HIV. Although more specific do-
nor screening criteria for MSM are desirable,
the FDA states that no algorithm has yet been
developed that reliably identifies MSM whose
HIV risk is as low as that of current blood
donors.57
FDA’s rationale for permanently excluding
MSM from donation has been less clear. Most
other high-risk populations are excluded only
temporarily (for 1 year after the risk activity).
The FDA’s rationale for excluding MSM be-
yond 1 year appears to be based on two con-
cerns. One concern is that there are other po-
tentially transfusion-transmissible infections
that are more prevalent in MSM populations
(such as human herpesvirus 8, the causative
agent of Kaposi sarcoma), and donors are not
tested for all of these infections.54 The other
concern is “quarantine release errors,” or the
risk that a blood center might inadvertently
release a unit with a positive test result.
196 䡲 AABB TECHNICAL MANUAL
However, based on current electronic controls
used by most US blood centers, release errors
appear to be extremely rare.
Mathematical modeling suggests that the
deferral period for MSM could be shortened
without a substantial increase in risk to recipi-
ents.45 A US Department of Health and Human
Services (DHHS) advisory committee conclud-
ed in 2010 that available scientific data were
inadequate to support any specific alternative
policy.58 DHHS subsequently issued requests
for proposals to evaluate alternative ap-
proaches to donor screening that could main-
tain the current level of blood safety.
HBV
HBV is a lipid-enveloped, double-stranded
DNA virus. Like HIV, HBV is transmitted par-
enterally, sexually, and perinatally. Jaundice is
noted in only 25% to 40% of adult cases and in
a smaller proportion of childhood cases. A
large percentage of perinatally acquired cases
result in chronic infection, but most HBV in-
fections acquired in adulthood are cleared.
HBV is highly prevalent in certain parts of the
world, such as Eastern Asia and Africa, where
perinatal transmission and resultant chronic
infection have amplified infection rates in the
population. In the United States, the incidence
of acute HBV infection has decreased dramati-
cally with the implementation of routine vac-
cination programs. Perinatal screening and
newborn prophylaxis have also been effective
in reducing perinatal transmission.
During HBV infection, DNA and viral en-
velope material (HBsAg) are typically detect-
able in circulating blood. Antibody to the core
antigen is produced soon after the appear-
ance of HBsAg, initially in the form of IgM an-
tibody, followed by IgG. As infected individuals
produce antibody to the surface antigen (anti-
HBsAg), the HBsAg is cleared.
The FDA requires donor screening for
HBsAg, HBV DNA, and total anti-HBc (IgM
and IgG antibody). Measurements of HBV inci-
dence in donors have been complicated by the
transience of HBsAg and false-positive results
on the HBsAg test.48,50 Published estimates of
the infectious window have varied because of
differences in the sensitivity of various HBV as-
says and lack of certainty regarding the level of
virus in a blood component that is required for
infectivity.50,59 Recent publications provide
window-period estimates for different poten-
tial infectious doses of virus (eg, 10 copies/20
mL of plasma vs 1 copy/20 mL of plasma). The
infectious window prior to a positive result on
the Abbott PRISM (Abbott Laboratories, Ab-
bott Park, IL) HBsAg test has been estimated to
be 30 to 38 days.59 With the addition of HBV
DNA testing in MPs of 16, the window period is
estimated to have been reduced to 18.5 to 26.5
days.50 Using these MP testing estimates, US
HBV transfusion-transmission risk has been
estimated to be between 1 in 843,000 dona-
tions to 1 in 1.2 million donations (Table 8-6).50
Donor screening for HBV DNA can be of
value at a variety of points in infection. HBV
DNA may be detected during the infectious
window period before HBsAg detection; how-
ever, DNA levels may be low and could be be-
low the limits of detection of MP-NAT as-
says.50,59 Later in infection, following the
clearance of HBsAg, HBV NAT may detect per-
sistent (ie, “occult” HBV) infection.60 Such in-
fections are interdicted in the United States by
the donor screening test for anti-HBc. HBV
NAT testing can also detect acute HBV infec-
tions in individuals who have previously been
vaccinated.61,62 Such individuals may never de-
velop detectable HBsAg, but they may have
detectable DNA. The infectivity of such dona-
tions is not known because these units contain
vaccine-induced antibodies to HBsAg in addi-
tion to the virus. Routine HBV DNA screening
of US blood donations detects at least some of
these infections.
HCV
HCV is a lipid-enveloped, single-stranded RNA
virus. HCV was shown to be the cause of up to
90% of cases previously called NANB transfu-
sion-related hepatitis.63 The majority of HCV
infections are asymptomatic. However, HCV
infection is associated with a high risk of chro-
nicity, which can result in liver cirrhosis and
hepatocellular carcinoma.
 CHAPTER 8
HCV is thought to be transmitted primari-
ly through blood exposure. In the United
States, about 55% of HCV infections are associ-
ated with injection drug use or receipt of
transfusion before 1992, but the risk factors for
the remainder of the infections are not clear.64
Sexual and vertical transmissions are uncom-
mon, although coinfection with HIV may in-
crease transmission rates by these routes.
Current donor screening for HCV in-
cludes NAT testing for HCV RNA and serologic
testing for antibodies to HCV. The average
window period between exposure and detec-
tion of infection by MP-NAT is estimated to be
7.4 days.13 The serologic test detects only IgG
antibody, a relatively late marker of infection.
Therefore, there may be a significant lag (1.5 to
2 months) between detection of RNA and de-
tection of antibody.65 Donor questioning has
limited potential to exclude individuals who
may be harboring HCV infection because a
large proportion of infected individuals are
asymptomatic and have no identifiable risk
factors. Despite this limitation, the current es-
timated US risk of HCV transmission by trans-
fusion is extremely low—approximately 1 in
1.1 million (Table 8-6).46
Human T-Cell Lymphotropic Virus,
Types I and II
Human T-cell lymphotropic virus type I
(HTLV-I) is a lipid-enveloped RNA virus. It was
the first human retrovirus identified, isolated
in 1978 from a patient with cutaneous T-cell
lymphoma. A closely related virus, HTLV-II,
was later isolated from a patient with hairy cell
leukemia. Both viruses are highly cell associat-
ed, infect lymphocytes, and cause lifelong in-
fections, but most of these infections are
asymptomatic. Approximately 2% to 5% of
HTLV-I-infected individuals develop adult T-
cell leukemia/lymphoma after a lag of 20 to 30
years. A smaller percentage develop a neuro-
logic disease called HTLV-associated myelopa-
thy or tropical spastic paraparesis. HTLV-II
disease associations remain unclear. Both in-
Infectious Disease Screening 䡲 197
fections are thought to be spread through
blood, sexual contact, and breast feeding.
HTLV-I infection is endemic in certain
parts of the world, including regions of Japan,
South America, the Caribbean, and Africa. In
the United States, infections are found in im-
migrants from endemic areas, injection drug
users, and the sexual partners of these individ-
uals. Approximately one-half of the HTLV
infections in US blood donors are from HTLV-
II.66,67
The only FDA-approved donor tests for
HTLV infection are screening assays for IgG
antibody to HTLV-I and HTLV-II. Units that are
reactive on the screening assay may not be re-
leased for transfusion. Because there is no
FDA-approved confirmatory assay and the
majority of reactive donor samples are
thought to represent false-positive results, the
FDA does not require permanent deferral of
donors after one reactive donation. Donors are
permanently excluded only if their sample re-
acts again on a subsequent donation (Title 21,
CFR Part 610.41). Unlicensed supplemental
tests, such as immunoblots or immunofluo-
rescence assays, may be very helpful for coun-
seling donors about the likelihood that the
screening test reactivity represents a true in-
fection. Some of these supplemental assays
can also differentiate between HTLV-I and
HTLV-II infection.66,67 Rather than use unli-
censed supplemental tests, some blood cen-
ters retest reactive samples on a different FDA-
licensed donor screening assay; samples with
nonreactive results on the alternative screen-
ing assay are most likely false-positive. Do-
nors whose samples are reactive on the second
screen, however, must be counseled and de-
ferred.35
Risk estimates for transfusion-transmit-
ted HTLV are somewhat uncertain, given the
absence of well-defined window periods for
the current HTLV antibody tests and the ab-
sence of confirmatory assays to definitively
measure true case rates in donors.48 Like CMV,
HTLV is thought to be transmitted only by
white-cell-containing blood components and
not by frozen/thawed plasma components.66,67
198 䡲 AABB TECHNICAL MANUAL
Syphilis
Syphilis is caused by the spirochete bacterium
Treponema pallidum. Donor screening for
syphilis has been performed for more than 60
years. Donors were initially screened by non-
treponemal serologic tests that detect anti-
body to cardiolipin (eg, rapid plasma reagin).
In recent years, however, most blood banks
have begun using tests that detect specific an-
tibodies to T. pallidum because these tests can
be performed with automated testing instru-
ments.
The vast majority of reactive donor test
results do not represent active cases of syphi-
lis. Most reflect either biologic false-positive
results or persistent antibody in previously
treated individuals (the latter are detected by
treponemal-specific antibody screening tests).
The FDA has permitted use of additional,
treponemal-specific, confirmatory assays (eg,
fluorescent treponemal antibody absorption
test) to guide the management of both donors
and components. Specifically, the FDA has
permitted release of units from donors who
have reactive nontreponemal screening test
results and negative treponemal confirmatory
results if the units are labeled with both test
results.1,37 If, however, the result of the trepo-
nemal-specific confirmatory test is positive or
if no additional testing is performed, the com-
ponent may not be used and the donor must
be deferred for at least 12 months.
The current value of donor screening for
syphilis is controversial.68-70 Although numer-
ous cases of transfusion-transmitted syphilis
were reported before World War II, no cases
have been reported in the United States for
more than 40 years. The low transmission risk
is probably related to a declining incidence of
syphilis in donors as well as the limited surviv-
al of the T. pallidum spirochete during blood
storage.
One issue that has been considered is
whether the syphilis screen improves blood
safety by serving as a surrogate marker of high-
risk sexual activity. However, studies have
demonstrated that donor screening for syphi-
lis does not provide incremental value in de-
tecting other blood-borne and sexually trans-
mitted infections, such as HIV, HBV, HCV, or
HTLV.70
Other Bacteria
Bacterial contamination of blood components
(mainly platelets) continues to cause some
transfusion-related fatalities.71 Bacteria are re-
portedly present in approximately 1 in 3000
cellular blood components.72 The source of the
bacteria can be either the donor’s skin or
asymptomatic bacteremia in the donor.
The level of bacteria in components just
after collection is generally too low to detect or
to cause symptoms in the recipient. However,
bacteria can multiply during component stor-
age, particularly in platelet components,
which are kept at room temperature. Bacteria
proliferate to a lesser extent in refrigerated Red
Blood Cells, and septic reactions occur much
less commonly with these components. To re-
duce the risk of septic transfusion reactions
associated with platelets, the AABB imple-
mented a requirement for processes to limit
and detect bacterial contamination in all
platelet components in 2004. (Since 2009 the
requirement has been to “limit and detect or
inactivate” bacterial contamination in platelet
components.18(p11))
To limit blood component contamina-
tion by bacteria from donor skin, two elements
of the blood collection process are critical. Be-
fore venipuncture, the donor skin must be
carefully disinfected using a method with
demonstrated efficacy. Most of these methods
involve iodophors, chlorhexidine, or alcohol.73
Second, diversion of the first 10 mL to 40 mL of
donor blood, which can contain skin and ap-
pendages, away from the collection container
(eg, into a sample pouch) further reduces the
likelihood that skin contaminants will enter
the component.73,74 Since 2008, the AABB has
required that collection sets with diversion
pouches be used for all platelet collections, in-
cluding whole-blood collections from which
platelets are made.18(p22)
A variety of technologies are available for
detection of bacteria in platelet components.
AABB Standards requires blood centers to use
a bacteria detection method that is approved
 CHAPTER 8
by the FDA or validated to provide sensitivity
equivalent to FDA-approved methods. None of
these methods is sensitive enough to detect
bacteria immediately after collection. All
methods require a waiting time for bacteria
contaminants to multiply before the compo-
nent is sampled.
The process most commonly used in the
United States to screen apheresis platelets is a
culture-based system that requires the plate-
let component to be stored for 24 hours before
sampling. After that time, a sample is with-
drawn and inoculated into one or more cul-
ture bottles. The bottles are then incubated in
the culture system. Some blood centers con-
tinue to hold the component during the first
12 to 24 hours of culture and release it for use
only if the culture is negative at the end of that
time. In all cases, the culture is continued for
the shelf life of the unit. If the culture becomes
positive after the component is released, the
blood center attempts to retrieve it. If the com-
ponent has not been transfused, resampling of
the product for culture is very informative be-
cause approximately two-thirds of the initially
positive signals are determined to be caused
by either contamination of the bottle (and not
the component) or false signals from the cul-
ture system.73,74All positive cultures should be
tested to determine the identity of the organ-
ism. If a true-positive result is related to an or-
ganism that is not a skin contaminant, the do-
nor should be notified and advised to seek
medical consultation.19
Other methods approved in the United
States for platelet quality control testing early
in the storage period include a culture-based
system with a one-time-point readout and an
optical scanning system. All of the methods
are approved for testing leukocyte-reduced
apheresis platelets, and some are approved for
testing pools of leukocyte-reduced, whole-
blood-derived platelets. These methods are
not generally used for routine screening of
individual (unpooled) whole-blood-derived
platelet concentrates. Low-technology meth-
ods for screening platelets just before issue—
such as visually inspecting the platelets for
swirling or testing them for low glucose or
pH—lack both sensitivity and specificity and
Infectious Disease Screening 䡲 199
do not fulfill the AABB standard for bacteria
detection.18(p11),20,73 However, two FDA-cleared
point-of-issue assays can be used for bacteria
detection testing of platelet concentrates that
are pooled immediately before issue.
Since the implementation of routine bac-
terial screening of apheresis platelets, the fre-
quency of FDA-reported fatalities from con-
taminated apheresis platelets has declined.71
However, some contaminated apheresis plate-
lets escape detection by this early testing, pre-
sumably because bacterial concentrations are
below limits of detection at the time of sam-
pling; thus, septic, and even fatal, reactions do
still occur. AABB has recommended consider-
ation of policies to further reduce the risk
of bacterially contaminated platelets.75 The
point-of-issue assays mentioned above are
cleared by FDA as adjunct (time-of-issue) tests
for apheresis platelets that have been screened
by another method. In a large clinical trial, one
of these assays detected nine bacterially con-
taminated components among 27,620 aphere-
sis platelets (1 in 3069 components) previously
screened by an early-storage culture-based as-
say.76 There were also 142 false-positive results.
As of March 2014, point-of-issue retesting of
apheresis platelets had not been widely imple-
mented in the United States.
All of the above methods provide incom-
plete assurance of bacteria detection, and
none is practical for screening the red cell in-
ventory. Pathogen-reduction methods, which
impair proliferation of bacteria in the blood
component, could theoretically be used to re-
duce the risk of septic reactions from bacteria
in blood components. Indeed, in some regions
outside the United States, pathogen reduction
has replaced bacteria detection testing for
platelets, but these technologies are not
approved for use in the United States as of
March 2014.
Infections Transmitted by Insect
Vectors
Until recently, malaria was the only vector-
transmitted disease that was widely recog-
nized as having the potential for secondary
transmission by transfusion in the United
200 䡲 AABB TECHNICAL MANUAL
States. Malaria is rare in the United States, and
the blood supply has been effectively protect-
ed by questions that exclude donors who have
recently traveled to or resided in malaria-
endemic regions. In the past decade, however,
other vector-transmitted infections have been
recognized as threats to the US blood supply,
and these infections are targeted by the newest
blood donor screening assays.
WNV
WNV is a lipid-enveloped RNA virus in the Fla-
viviridae family. First detected in the United
States in 1999, it subsequently spread through-
out North America, appearing in annual epi-
demics every summer and autumn. Birds are
thought to be the primary reservoir for WNV,
with infection spreading to humans through
mosquito bites. Approximately 80% of human
cases are asymptomatic; 20% are associated
with a self-limited febrile illness; and less than
1% are associated with severe neuroinvasive
disease, such as meningoencephalitis or acute
flaccid paralysis.
Transfusion transmission of WNV was
first recognized in 2002 and traced to blood
donations containing viral RNA in the ab-
sence of antibody. Thus NAT, rather than sero-
logic testing, was required to protect the blood
supply. Donor screening was widely imple-
mented in 2003 using investigational NAT as-
says. Donor tests for WNV RNA are now ap-
proved by FDA and required by both the FDA
and AABB.17,18(pp31,32) The predominant method
of testing is in MPs.
However, as discussed in the “NAT” sec-
tion above, circulating levels of viral RNA are
frequently low during WNV infection, and a
donor sample containing a low concentration
of RNA may escape detection if it is diluted in a
minipool. Therefore, both the AABB and FDA
recommend testing of individual donor sam-
ples, not minipools, when WNV activity is high
in a particular collection region.16,17 Regional
WNV activity is monitored through active
communications between neighboring blood
collection agencies about viremic donors de-
tected as well as by public health reports of
clinical WNV cases and animal and mosquito
surveillance in the area.
Two documented cases of WNV transmis-
sion by transfusion were traced to donations
screened by MP-NAT during periods when
neighboring blood collection facilities were
screening donors by ID-NAT.77 The most re-
cent reported transmission was traced to a do-
nation containing WNV antibody and a low
level of virus that was not reproducibly detect-
able even by ID-NAT.78 It is possible that some
transmissions have escaped recognition be-
cause most WNV infections lack distinctive
symptoms.
T. cruzi
T. cruzi, the protozoan parasite that causes
Chagas disease, is endemic in parts of Mexico,
Central America, and South America. It is
transmitted to humans by an insect vector, the
reduviid bug. Acute infection is usually self-
limited but may be severe in immunocompro-
mised patients. Most infections become
chronic but asymptomatic. Decades after the
initial infection, 10% to 40% of infected indi-
viduals develop late-stage manifestations,
including intestinal dysfunction or cardiac
disease, which can be fatal. Transfusion trans-
mission of T. cruzi from the blood of chronical-
ly infected, asymptomatic donors has been re-
ported in endemic areas.
A blood donor screening enzyme immu-
noassay for antibodies to T. cruzi was ap-
proved by the FDA for US use in December
2006. Although not initially required by the
FDA, the test was widely implemented by US
blood centers during 2007. Supplemental test-
ing of reactive specimens using an FDA-
licensed enzyme strip assay or unlicensed
radioimmunoprecipitation assay (RIPA) is very
helpful for guiding donor counseling. Based
on the results of the latter assay, about 25% of
reactive US donors appear to be truly infect-
ed.79,80 All donors whose samples are reactive
on the screening assay, however, must be per-
manently deferred, regardless of the results of
the supplemental testing, pending FDA ap-
proval of a reentry algorithm.29
 CHAPTER 8
The vast majority of US donations with
reactive T. cruzi screening test results and pos-
itive supplemental results are from donors
born in T. cruzi-endemic areas. Other con-
firmed-positive donors appear to have con-
genitally acquired infections (ie, the donor’s
mother is from a T. cruzi-endemic area), and
only a small number of donor infections ap-
pear to have been acquired from vector expo-
sure within the United States (autochthonous
cases). In the first 2 years of US donor screen-
ing, no donor seroconversions were identi-
fied.79,80 In December 2010, the FDA issued
guidance recommending one-time screening
of every US donor for T. cruzi.29
Before implementation of donor screen-
ing, seven cases of transfusion-transmitted
T. cruzi had been identified in the United
States and Canada; all of the cases with avail-
able data were linked to platelet transfusions.
Since implementation of donor screening,
RIPA-positive donors have been identified,
and recipients of their prior donations have
been notified and tested. Thus far, only two
prior recipients (of platelets from one donor)
appear to have positive test results. Thus, de-
spite reported transmission rates of 10% to
20% from components from infected donors
in endemic areas, no T. cruzi transmissions by
red cells in the United States have been docu-
mented to date. The lower infectivity of US red
cell components compared to those in endem-
ic areas may be at least partly attributable to
more frequent use of fresh whole blood in en-
demic areas.
Babesia
Babesia are intraerythrocytic parasites. Cases
of transfusion-transmitted babesiosis are be-
ing identified with increasing frequency, and
some have been fatal.71,81,82 There is no FDA-
approved donor screening test for this infec-
tion.
Human Babesia infections are zoonotic,
usually acquired through the bite of an infect-
ed tick. In the northeastern and Midwestern
United States, the most common Babesia spe-
cies is B. microti. The vector is Ixodes scapular-
is, the same tick that transmits Lyme disease.
Infectious Disease Screening 䡲 201
Reported human infections with B. microti are
becoming more frequent. In the western Unit-
ed States, Babesia infections appear to be less
common, and a different species, B. duncani,
predominates. The vector for B. duncani has
not been clearly defined.
Babesia infection is usually asymptomat-
ic, even though parasites can circulate for
months to years. In some individuals, however,
Babesia infection presents as a severe malaria-
like illness that can be fatal. Immunocompro-
mised, elderly, and asplenic patients are at in-
creased risk of severe disease.
Babesia infection is diagnosed when the
intraerythrocytic parasites are seen on a blood
smear. If a patient is suspected of having ac-
quired the infection by transfusion, donors of
the patient’s components can be recalled and
tested for antibodies to Babesia using an im-
munofluorescence assay; the presence of
high-titer antibody in the donor is suggestive
of recent infection. Most of the donors impli-
cated in transfusion-transmitted cases have
been residents of endemic areas, although
some were residents of nonendemic areas who
were apparently exposed to Babesia during
travel to endemic areas.82
Studies in Connecticut have found B. mi-
croti antibodies in approximately 1% of blood
donors, suggesting that B. microti infections
are highly prevalent in the state’s population
and grossly underrecognized.83
In the absence of FDA-approved tests for
blood donor screening, there have been public
discussions of potential interventions to re-
duce transfusion risk in the most highly en-
demic regions.81,84 Clinical trials of some inves-
tigational serologic and nucleic acid tests are
currently under way.85
Malaria
Malaria is caused by an intraerythrocytic para-
site of the genus Plasmodium. Infection is
transmitted to humans through a mosquito
bite. Five species account for most human in-
fections: P. falciparum, P. vivax, P. malariae,
P. ovale, and P. knowlesi.
No FDA-approved test is available to screen
US blood donations for malaria infection.
202 䡲 AABB TECHNICAL MANUAL

Screening is accomplished solely by donor recent attention because donations contain-

questioning. Donors are excluded temporarily ing viral nucleic acid have been documented
from donating blood after traveling to malaria- during epidemics outside the continental
endemic areas, residing in malaria-endemic United States. Dengue activity has also been
countries, or after recovery from clinical ma- documented within the southern United States
laria. Donor questioning has been remarkably and Hawaii.93 The degree to which these agents
effective at preventing transfusion-transmit- threaten the US blood supply is unclear.94
ted malaria in the United States, with only six
cases reported between 1999 and 2012. All six Prions
cases were linked to donors with a history of
Prions are proteinaceous infectious particles
residence in Africa; on re-interview, five of the
that induce disease by triggering conforma-
six donors were verified to have met accep-
tional changes in naturally occurring protein
tance criteria, but one had emigrated less than
counterparts. These agents cause fatal infec-
3 years before donation.86,90
tions of the nervous system called “transmissi-
This level of transfusion safety has been
ble spongiform encephalopathies” (TSEs).
achieved at substantial cost in terms of donor
Classical CJD is a TSE with both a sporad-
loss; malaria-related questions have excluded
ic form and familial forms and an incidence of
hundreds of thousands of otherwise accept-
about 1 per million. CJD has been transmitted
able US donors annually. Although travel to
by infusion or implantation of products from
Mexico has accounted for the largest propor-
infected central nervous system tissues. Blood
tion of deferred donors, the risk of acquiring
components do not appear to transmit classi-
malaria during tourist travel to Mexico is ex-
cal CJD. Nevertheless, blood donations are not
ceedingly low.91 The FDA recently issued guid-
accepted from donors who are at increased
ance redefining malaria-endemic areas as only
risk of this disease.95
those for which chemoprophylaxis is recom-
Another TSE, vCJD, does appear to be
mended.92 With this new definition, travel to
transmissible by blood transfusion. This TSE is
many popular tourist locations will no longer
caused by the same prion that causes bovine
be considered a malarial risk. However, the
spongiform encephalopathy (BSE), also known
new guidance adds a complex algorithm for
as “mad cow disease.” As of March 2014, four
evaluating travel by donors who have lived for
cases of vCJD transmission by transfusion had
more than 5 years in malaria-endemic coun-
been reported in the United Kingdom, the area
tries because of a concern of partial immunity
in which BSE was most endemic. In addition,
in such donors.
one latent vCJD infection was identified in a
Some other countries that exclude donors
patient with hemophilia in the United King-
after travel to malaria-endemic areas permit
dom who died of other causes. This patient
reinstatement of these donors if they test neg-
had received UK-plasma-derived Factor VIII,
ative for malaria antibodies 4 to 6 months after
including material from a donor who later
completion of travel. In the absence of an ap-
developed vCJD, suggesting that vCJD might
proved assay, such a “test-in” reinstatement
have been transmitted by clotting factor con-
strategy has not been accepted by the FDA.
centrates.51,52 vCJD infection is extremely rare
in the United States. The few reported cases
Other Vector-Transmitted Infections
have been in individuals who most likely
There are many other vector-transmitted in- acquired their infections elsewhere, and no US
fections that could be secondarily transmitted transfusion-transmitted cases have been
by transfusion. These agents and potential reported.
intervention strategies are reviewed in the There are no FDA-approved donor
publicly available AABB emerging infectious screening tests for prion infections. Blood do-
disease resources.51,52 Two of these agents, nors in the United States are screened solely by
dengue and chikungunya viruses, have received questioning and are excluded if they have an
 CHAPTER 8
increased risk of either CJD or vCJD. CJD ex-
clusions are based on family history of the dis-
ease, receipt of human growth hormone de-
rived from pituitary glands, or receipt of a dura
mater tissue graft. vCJD exclusions are for resi-
dence in the United Kingdom or Europe dur-
ing specified times when BSE was endemic, re-
ceipt of a transfusion in the United Kingdom
or France, or receipt of UK bovine insulin.95 It is
thought that plasma derivative manufacturing
processes remove substantial amounts of TSE
infectivity.95
In-Process Screening for Plasma
Derivatives
Commercial plasma derivatives are prepared
from large pools of plasma derived from thou-
sands of donors. Before the incorporation of
specific pathogen-reduction processes, con-
tamination of these large pools with viral
agents was common. Today, plasma derivative
manufacturing processes incorporate meth-
ods—such as prolonged heat or solvent/deter-
gent (SD) treatment—that remove or inacti-
vate most known pathogens. SD treatment
inactivates lipid-enveloped agents, such as
HIV, HCV, and HBV. Pathogen infectivity may
also be reduced by nanofiltration, chromatog-
raphy, or cold ethanol fractionation, which are
used in the production of certain products.
Not all infectious agents, however, are re-
moved or inactivated by these processes.
One agent that can persist in plasma-
derivative products is human parvovirus B19.
This small, nonenveloped DNA virus is ex-
tremely resistant to physical inactivation.
Acute infection is typically mild and self-
limited; clinical manifestations include “fifth
disease” (erythema infectiosum) and polyar-
thropathy. Acute infection is associated with
transient red cell aplasia that may be clinically
significant in immunodeficient individuals
and those with underlying hemolytic process-
es. The aplasia in immunodeficient individu-
als can be prolonged. Intrauterine infection is
associated with severe fetal anemia and hy-
drops fetalis.
Parvovirus B19 infection is very common;
most adults have antibodies to this agent, indi-
Infectious Disease Screening 䡲 203
cating previous exposure. Levels of viral DNA
during acute infection may exceed 1012 IU/mL,
decreasing over weeks to months in associa-
tion with antibody production.
Viral DNA, mostly at low concentrations,
has been detected in approximately 1% of
blood donations and in essentially all lots of
pooled plasma derivatives. Transmission of
parvovirus B19 by transfusion has been
linked only to blood components or plasma
products that contain high concentrations of
viral DNA; only one transmission has been
documented with a product containing less
than 104 IU/mL.52
Currently, there is no FDA-approved test
to screen fresh blood donations for parvovirus
B19 infection. However, plasma-derivative
manufacturers require screening of incoming
plasma units for the presence of high-titer par-
vovirus B19. This is accomplished by perform-
ing NAT on pools of samples from plasma
units, with sensitivity adjusted to detect only
units with a high concentration of virus. By ex-
cluding high-titer units from the plasma pools,
the final titer in the plasma pool is kept below
104 IU/mL.
Other Agents
The AABB maintains a publicly accessible
electronic resource containing expert analyses
of emerging infectious disease (EID) agents
that have received attention as potential
threats to the US or global blood supply.52 This
digital resource contains up-to-date fact
sheets on a variety of agents. Each fact sheet
includes information about clinical manifesta-
tions and epidemiology of infection, evidence
of transfusion transmissibility, and analyses
of the potential effectiveness of various
mitigation strategies (eg, donor questioning,
serologic testing or NAT, or pathogen reduc-
tion). Readers are encouraged to use this rich
resource.
One agent that has received recent atten-
tion is hepatitis E virus (HEV), a small, nonen-
veloped, single-stranded RNA virus. HEV is
thought to be primarily transmitted through
food and water sources. However, recent stud-
ies have demonstrated asymptomatic viremia
204 䡲 AABB TECHNICAL MANUAL
in blood and plasma donors, and transfusion
transmission has been documented.52,96 As a
nonenveloped agent, HEV is not susceptible to
SD treatment. NAT screening of donors and/or
heat inactivation of plasma pools may be miti-
gation strategies if this agent is deemed to
pose a clinically important threat.
PAT H OG E N RED UCT IO N
TE CH NOLO G Y
Donor screening reduces, but cannot eliminate,
the infectious risks of blood transfusion. The ef-
ficacy of blood donor testing is limited by a
number of factors, including the following:
1. It is not logistically feasible to test donors
for every infection that is conceivably
transmissible by transfusion.
2. For every test, there is a lag time (window
period) between when a person becomes
infected and when the test detects infec-
tion.
3. Every test has limited sensitivity (concen-
tration of the target marker that can be
detected by the test).
4. Developing a donor test is a long, multi-
phase process that includes identification
of the infectious agent, selection of the type
of test that would be effective in interdict-
ing infectious donations (eg, serology vs
NAT), development of a test suitable for
donor screening, performance of clinical
trials of the test, and regulatory approval.
During this development process, infec-
tions can be transmitted.
Pathogen reduction technology (PRT)
provides an attractive alternative to relying on
donor testing to interdict all infectious dona-
tions. PRT processes reduce the infectivity of
residual pathogens in blood components. This
approach could reduce the transmission of in-
fectious agents for which there are no donor
screening tests and further reduce the residual
transmission risks of known agents. PRT could
theoretically enable discontinuation of some
testing that is currently performed (eg, CMV
testing and bacterial testing of platelets), and
some PRT methods could obviate the need for
irradiation, potentially offsetting some of the
cost of PRT.
As discussed above, PRT is now an essen-
tial component of the plasma-derivative man-
ufacturing process. SD treatment can also be
applied to pools of plasma for transfusion. An
SD-treated pooled plasma product has been
approved for use in the United States.
No PRT processes are approved in the
United States for treatment of individual units
of plasma or for cellular blood components,
although some technologies are in use outside
the United States. Processes that are available
or in development have been recently re-
viewed in detail and are summarized in Table
8-7.51,52,97,98
The benefit to be gained from PRT in the
United States is primarily the mitigation of
emerging pathogens and platelet-associated
bacterial sepsis. Currently, the quantifiable in-
fectious risks of transfusion in the United
States are low. Therefore, it is critically impor-
tant to demonstrate that PRT treatments do
not introduce new hazards to patients. Rigor-
ous preclinical and clinical studies are re-
quired for US regulatory approval of PRT. Toxi-
cology studies are critical because most of
these agents interact with nucleic acid, raising
the theoretical potential of carcinogenicity
and mutagenicity. Treated products should be
assessed for neoantigen formation and the im-
pact of the PRT on the final product’s clinical
efficacy. The evaluation process for PRT in
North America was the subject of a recent con-
sensus conference.97,99
Documented transmission of new infec-
tious agents for which there are no donor
screening tests could influence the risk/bene-
fit assessment of PRTs. It is important, though,
to keep in mind that there are limits to the viral
loads that are inactivated by these processes,
and not all agents are inactivated by PRTs.
Pooled SD plasma, for example, would have a
reduced risk of transmitting most infectious
agents but a potentially increased risk of trans-
mitting an agent that lacks a lipid envelope.
 CHAPTER 8 Infectious Disease Screening 䡲 205

TABLE 8-7. Pathogen Reduction Technologies for Transfusable Blood Components

Component Technology Manufacturer

Plasma: commercially prepared pools Solvent/detergent treatment Octapharma

Plasma: individual units 䡲 Amotosalen (psoralen) + UV light Cerus
䡲 Riboflavin (vitamin B2) + UV light Terumo BCT
䡲 Methylene blue + light Macopharma
Platelets 䡲 Amotosalen (psoralen) + UV light Cerus
䡲 Riboflavin (vitamin B2) + UV light Terumo BCT
䡲 UV light Macopharma
Red Blood Cells 䡲 Frangible nucleic acid crosslinker Cerus
䡲 Riboflavin (vitamin B2) + UV light Terumo BCT
UV = ultraviolet.

SUM MARY Current quantifiable risks of infectious

The current level of safety of blood compo-
nents is based on two critical elements of do-
nor screening: donor questioning, which is the
sole method of screening for certain agents,
such as malaria and prions, and donor testing.
Testing must be performed carefully and in ac-
cordance with manufacturers’ instructions,
and facilities must have robust systems for
quarantining components of donations that
test positive and for retrieving prior donations
from donors whose samples have tested posi-
tive.
disease transmission are very low; the estimat-
ed risk of HIV transmission by transfusion in
the United States is approximately 1 in 1.5 mil-
lion units, the risk of HCV transmission is ap-
proximately 1 in 1.1 million units, and the risk
of HBV transmission is approximately 1 in
800,000 to 1 in 1.2 million units.46,50 However, it
is critical to remain vigilant for evidence of
new infectious agents and to implement miti-
gation measures as quickly as feasible. PRT
may, in the future, provide some protection
against emerging infectious agents for which
no screening is in place.

KEY POINTS

1. Infectious disease screening of donors is accomplished by 1) questioning potential donors

and excluding those with an increased risk of infection, and 2) testing donated blood.
2. There is a delay between the time when an individual is exposed to an infection and the
time when the donor screening test for the infection yields a positive result. Blood donated
during this window period can transmit infections.
3. The estimated window period with MP-NAT of donor samples is less than 10 days for HIV
and HCV and less than 28 days for HBV.
4. The residual risk of transfusion-transmitted infection is a function of the length of the win-
dow period and the incidence of infection in the donor population. Maintaining a donor
population that has a low incidence of infection continues to play a key role in preserving
blood safety.
206 䡲 AABB TECHNICAL MANUAL

5. Based on window-period and incidence calculations, the current risk of HIV transmission
by transfusion in the United States is approximately 1 in 1.5 million units, the risk of HCV
transmission is approximately 1 in 1.1 million units, and the risk of HBV transmission is ap-
proximately 1 in 800,000 to 1 in 1.2 million units.
6. There are no donor screening tests approved by the FDA for malaria or vCJD. Donor ques-
tioning about potential exposure is the sole means of protecting the US blood supply from
these diseases.
7. AABB requires blood banks to have processes that limit and detect or inactivate bacteria in
platelet components. Pathogen reduction methods have replaced bacteria testing in some
regions outside the United States.
8. Infections transmitted to humans by vectors are increasingly recognized as a potential
source of transfusion-transmitted infection. These include WNV, T. cruzi, Babesia species,
and dengue virus.
9. PRTs may reduce the transmission of infectious agents for which there are no donor screen-
ing tests, and PRTs may further reduce the residual transmission risks of known agents. PRT-
treated plasma derivatives and pooled plasma are available in the United States. Some PRT
systems are available outside the United States for treatment of individual platelet and plas-
ma components, but as of early 2014, these were not approved for US use.
10. Blood banks must have processes in place to ensure that donations with positive test results
are not released for transfusion. In some circumstances, 1) prior donations from those do-
nors must also be retrieved and quarantined, and 2) recipients of prior donations must be
notified of their possible exposure to infection.

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48. Dodd RY, Notari EP, Stramer SL. Current preva-
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49. Glynn SA, Kleinman SH, Wright DJ, Busch MP.
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56. Truong HM, Kellogg T, Klausner JD, et al. In-
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57. Food and Drug Administration. Blood dona-
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58. US Department of Health and Human Servic-
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60. Stramer SL. Pooled hepatitis B virus DNA test-
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63. Alter HJ. Descartes before the horse: I clone,
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74. Eder AF, Kennedy JM, Dy BA, et al. Limiting
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 CHAPTER 8
culture-negative apheresis platelets on day of
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comparative evaluation of serologic assays for
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81. Gubernot DM, Nakhasi HL, Mied PA, et al.
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82. Herwaldt BL, Linden JV, Bosserman E, et al.
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83. Johnson ST, Cable RG, Tonnetti L, et al. Sero-
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89. Gonzalez C, Layon A, Arguin P, et al. Transfu-
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90. Mali S, Tan KR, Arguin PM. Malaria surveil-
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212 䡲 AABB TECHNICAL MANUAL

97. Webert KE, Cserti CM, Hannon J, et al. Pro- 99. Klein HG, Anderson D, Bernardi MJ, et al.
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98. AuBuchon J, Prowse C, eds. Pathogen inactiva-
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da, MD: AABB Press, 2010.
 C h a p t e r 9

Hospital Storage, Monitoring,

Pretransfusion Processing,
Distribution, and Inventory
Management of Blood Components

Nancy M. Dunbar, MD

BLOOD COMPONENT MANUFAC- BLOOD AND BLOOD COMPONENT

TURING is highly regulated and must S TO R AG E A ND M O N I TO R I NG
comply with Food and Drug Administration
(FDA) current good manufacturing practice General Considerations
(cGMP) regulations from the time of collection Transport and storage requirements must be
to product issue.1,2 The intent of cGMP is to followed when blood components are trans-
maintain the safety, purity, potency, quality, ferred from the collection site to the process-
and identity of blood components. Collection ing facility, from the supplier to the blood
and transfusion facilities must develop poli- bank, or from the blood bank to the patient.
cies, processes, and procedures and retain rec-
Storage requirements and expiration dates
ords to demonstrate compliance with regula-
vary by product type and are based on factors
tions for storage, monitoring, pretransfusion
such as in-vitro red cell metabolism for Red
processing, and distribution of blood compo-
Blood Cell (RBC) components in various stor-
nents. These policies, processes, and proce-
age solutions or coagulation protein stabiliza-
dures must also comply with additional regu-
tion for plasma products (Table 9-1). Failure to
lations (Clinical Laboratory Improvement
adhere to these storage and expiration require-
Amendments and state laboratory statutes)
ments can result in decreased product potency
and policies and standards applicable to blood
and/or safety.
banking from voluntary laboratory accrediting
Temperature requirements during trans-
organizations (eg, College of American Pathol-
port of blood components differ from those
ogists, AABB,3 The Joint Commission, and
American Osteopathic Association). during storage.4 Shipping from the supplier to

Nancy M. Dunbar, MD, Assistant Professor, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire
The author has disclosed no conflicts of interest.

213
214 䡲 AABB TECHNICAL MANUAL
the hospital blood bank is considered trans-
port, and applicable temperature require-
ments must be met. When blood components
are issued from the blood bank to the patient
care area, maintenance of appropriate tem-
perature requirements allows for the possibili-
ty of returning the component to inventory if it
is not transfused.
Refrigerators, freezers, and platelet incu-
bators for blood and blood component storage
can be equipped with continuous-tempera-
ture-monitoring devices to allow detection of
temperature deviations before products are af-
fected. Automated electronic monitoring de-
vices that are available include: 1) weekly pen
and chart recorders, 2) sets of hard-wired or
radio-frequency temperature-recording devic-
es, and 3) centralized temperature-monitor-
ing systems. Thermometers or thermocouples
should be strategically placed in the equip-
ment for optimal temperature monitoring. If
an automated temperature-recording device is
not used, temperatures of the blood storage
environment must be recorded manually ev-
ery 4 hours. This requirement includes ambi-
ent room temperature monitoring of platelets
that are not stored in a platelet chamber or in-
cubator.
Recorded temperatures should be checked
daily to ensure proper operation of the equip-
ment and recorder. Deviations from acceptable
temperature ranges should be documented and
explained (including any actions taken), dated,
and initialed by the person noting the devia-
tion.
Most product-storage devices are equipped
with audible alarms to alert personnel that tem-
perature ranges are approaching unacceptable
levels. Central alarm monitoring allows facili-
ties that do not have personnel in the vicinity
of the equipment to alert designated staff at
another location when an alarm is activated.
Because platelets must be gently agitated dur-
ing storage, typically using horizontal flatbed
or elliptical rotators, alarm systems should
also emit alerts when the platelet agitator has
malfunctioned.
Transfusion services may locate blood
storage refrigerators in other areas of the hos-
pital to allow immediate access to blood in
emergency situations. Such a practice re-
quires that the same blood component storage
monitoring standards be met in these other ar-
eas.
If an equipment failure occurs and pre-
vents acceptable temperature ranges from be-
ing maintained, the facility should have poli-
cies, processes, and procedures in place to
relocate blood and blood components. The
secondary storage location may be another
on- or off-site refrigerator or freezer, qualified
storage boxes, or coolers used with a validated
process that has been shown to maintain re-
quired storage temperatures during storage.
Because the safety, purity, potency, and quality
of the blood components could be affected by
delays in relocation to a secondary storage lo-
cation, it is recommended that the relocation
occur before upper or lower acceptable storage
temperatures are exceeded. This can be ac-
complished by setting the alarm points of the
storage devices so that an alarm sounds before
the unacceptable tempature limit is reached.
Some facilities may use temperature-
monitoring indicators for each blood compo-
nent container. Such indicators monitor the
liquid temperature of the immediate inner
bag, not the liquid core temperature in the
unit, which may be cooler. Policies, processes,
and procedures should specify how the facility
will determine the disposition of blood com-
ponents when using temperature-monitoring
indicators.
Specific Considerations
Holding blood components that have been
dispensed from the blood bank to other hospi-
tal areas before transfusion is considered to be
“storage.” If the blood components are not
kept in a monitored device, they must be
stored in containers (eg, boxes or coolers) vali-
dated to maintain the correct temperature
during storage.
Red Blood Cells
RBC components are stored in plastic bags of
different types with a variety of added antico-
agulants and additive solutions that modify
TABLE 9-1. Requirements for Storage, Transportation, and Expiration3(pp51-59)
Item
No. Component
Whole Blood Components
1 Whole Blood
2 Whole Blood Irradiated
Red Blood Cell Components
3 Red Blood Cells (RBCs)
4 Deglycerolized RBCs
5 Frozen RBCs
40% Glycerol
6 RBCs Irradiated
Storage Transport Expiration1 Additional Criteria
1-6 C Cooling toward 1-10 C. ACD/CPD/CP2D: 21 days
If intended for room tempera- If intended for room tempera- CPDA-1: 35 days
ture components, then store ture components, cooling
CHAPTER 9
at 1-6 C within 8 hours after toward 20-24 C
collection
1-6 C 1-10 C Original expiration or 28 days
from date of irradiation, which-
ever is sooner
1-6 C 1-10 C ACD/CPD/CP2D: 21 days
CPDA-1: 35 days
Additive solution: 42 days
Open system: 24 hours
1-6 C 1-10 C Open system: 24 hours
Closed system: 14 days or as
FDA approved
–65 C if 40% glycerol or as Maintain frozen state 10 years Frozen within 6 days of col-
FDA approved (A policy shall be developed if lection unless rejuvenated
Storage, Processing, Distribution, and Inventory
rare frozen units are to be Frozen before Red Blood Cell
retained beyond this time) expiration if rare unit
1-6 C 1-10 C Original expiration or 28 days
䡲
from date of irradiation, which-
ever is sooner
(Continued)
215
TABLE 9-1. Requirements for Storage, Transportation, and Expiration3(pp51-59) (Continued)
216

Item
No. Component Storage Transport Expiration1 Additional Criteria
䡲

7 RBCs Leukocytes Reduced 1-6 C 1-10 C ACD/CPD/CP2D: 21 days

CPDA-1: 35 days
Additive solution: 42 days
Open system: 24 hours
8 Rejuvenated RBCs 1-6 C 1-10 C CPD, CPDA-1: 24 hours AS-1: freeze after rejuvena-
tion
9 Deglycerolized Rejuvenated 1-6 C 1-10 C 24 hours or as approved by
RBCs FDA
10 Frozen Rejuvenated RBCs –65 C Maintain frozen state CPD, CPDA-1: 10 years
AS-1: 3 years
(A policy shall be developed if
rare frozen units are to be
retained beyond this time)
11 Washed RBCs 1-6 C 1-10 C 24 hours
AABB TECHNICAL MANUAL

12 Apheresis RBCs 1-6 C 1-10 C CPDA-1: 35 days

Additive solution: 42 days
Open system: 24 hours
13 Apheresis RBCs Leukocytes 1-6 C 1-10 C CPDA-1: 35 days
Reduced Additive solution: 42 days
Open system: 24 hours
14 Platelets 20-24 C with continuous As close as possible to 24 hours to 5 days, depending Maximum time without
gentle agitation 20-24 C2 on collection system agitation: 24 hours
Platelet Components
15 Platelets—Irradiated 20-24 C with continuous As close as possible to No change from original expi- Maximum time without
gentle agitation 20-24 C2 ration date agitation: 24 hours
16 Platelets—Leukocytes 20-24 C with continuous As close as possible to Open system: 4 hours Maximum time without
Reduced gentle agitation 20-24 C2 Closed system: No change in agitation: 24 hours
expiration
17 Pooled Platelets Leukocytes 20-24 C with continuous As close as possible to 4 hours after pooling or 5 days Maximum time without
Reduced gentle agitation 20-24 C2 following collection of the old- agitation: 24 hours
est unit in the pool3
18 Pooled Platelets 20-24 C with continuous As close as possible to Open system: 4 hours
(in open system) gentle agitation 20-24 C2
CHAPTER 9

19 Apheresis Platelets
20 Apheresis Platelets
Irradiated
21 Apheresis Platelets Leuko-
cytes Reduced
22 Apheresis Platelets Platelet
Additive Solution Added
Leukocytes Reduced
Granulocyte Components
23 Apheresis Granulocytes
24 Apheresis Granulocytes
20-24 C with continuous As close as possible to 24 hours or 5 days, depending Maximum time without
gentle agitation 20-24 C2 on collection system agitation: 24 hours
20-24 C with continuous As close as possible to No change from original expi- Maximum time without
gentle agitation 20-24 C2 ration date agitation: 24 hours
20-24 C with continuous As close as possible to Open system: within 4 hours Maximum time without
gentle agitation 20-24 C2 of opening the system agitation: 24 hours
Closed system: 5 days
20-24 C with continuous As close as possible to 5 days Maximum time without
gentle agitation 20-24 C2 agitation: 24 hours
20-24 C As close as possible to 24 hours Transfuse as soon as possi-
20-24 C ble; Standard 5.28.10
applies3(45)
20-24 C As close as possible to No change from original Transfuse as soon as possi-
Storage, Processing, Distribution, and Inventory

Irradiated 20-24 C expiration date ble; Standard 5.28.10

applies3(45)
䡲

(Continued)
217
TABLE 9-1. Requirements for Storage, Transportation, and Expiration3(pp51-59) (Continued)
218

Item
No. Component Storage Transport Expiration1 Additional Criteria
䡲

25 Cryoprecipitated AHF
Plasma Components
26 Cryoprecipitated AHF (after
thawing)
27 Pooled Cryoprecipitated AHF
(pooled before freezing)
28 Pooled Cryoprecipitated AHF
–18 C Maintain frozen state 12 months from original Thaw the FFP at 1-6 C
collection Place cryoprecipitate in the
freezer within 1 hour after
removal from refrigerated
centrifuge
20-24 C As close as possible to Single unit: 6 hours Thaw at 30-37 C
20-24 C
–18 C Maintain frozen state 12 months from earliest Thaw the FFP at 1-6 C
date of collection of product Place cryoprecipitate in the
in pool freezer within 1 hour after
removal from refrigerated
centrifuge
20-24 C As close as possible to Pooled in an open system: Thaw at 30-37 C
AABB TECHNICAL MANUAL

(after thawing) 20-24 C 4 hours

If pooled using a sterile
connection device: 6 hours
29 Fresh Frozen Plasma (FFP)5 –18 C or –65 C Maintain frozen state –18 C: 12 months from
collection
–65 C: 7 years from
collection
Place in freezer within
8 hours of collection or as
stated in FDA-cleared opera-
tor’s manuals/package
inserts
Storage at –65 C requires
FDA approval if product is
stored for longer than
12 months
30 FFP (after thawing)5 1-6 C 1-10 C If issued as FFP: 24 hours Thaw at 30-37 C or using an
FDA-cleared device
31 Plasma Frozen Within 24 –18 C Maintain frozen state 12 months from collection
Hours After Phlebotomy
(PF24)5
32 Plasma Frozen Within 24 1-6 C 1-10 C If issued as PF24: 24 hours Thaw at 30-37 C or using an
Hours After Phlebotomy FDA-cleared device
(after thawing)5
CHAPTER 9

33 Plasma Frozen Within 24 –18 C or colder Maintain frozen state 12 months from collection
Hours After Phlebotomy Held
At Room Temperature Up To
24 Hours After Phlebotomy
(PF24RT24)
34 Plasma Frozen Within 24 1-6 C 1-10 C If issued as PF24RT24: Thaw at 30-37 C or using an
Hours After Phlebotomy Held 24 hours FDA-cleared device
At Room Temperature Up To
24 Hours After Phlebotomy
(after thawing)
35 Thawed Plasma5 1-6 C 1-10 C 5 days from date product was Shall have been collected
thawed or original expiration, and processed in a closed
whichever is sooner system
36 Plasma Cryoprecipitate –18 C Maintain frozen state 12 months from collection
Reduced
Storage, Processing, Distribution, and Inventory

37 Plasma Cryoprecipitate 1-6 C 1-10 C If issued as Plasma Cryopre- Thaw at 30-37 C

Reduced (after thawing) cipitate Reduced: 24 hours
䡲

(Continued)
219
 220

TABLE 9-1. Requirements for Storage, Transportation, and Expiration3(pp51-59) (Continued)

䡲

Item
No. Component Storage Transport Expiration1 Additional Criteria
38 Thawed Plasma Cryoprecipi- 1-6 C 1-10 C If issued as Thawed Plasma Shall have been collected
tate Reduced Cryoprecipitate Reduced: 5 and processed in a closed
days from date product was system
thawed or original expiration,
whichever is sooner
39 Liquid Plasma 1-6 C 1-10 C 5 days after expiration of 21 CFR 610.53(c) applies
Whole Blood
40 Recovered Plasma, liquid or Refer to short supply agree- Refer to short supply agree- Refer to short supply agree- Requires a short supply
frozen ment ment ment agreement4
Tissue and Derivatives
41 Tissue Conform to source facility’s Conform to source facility’s Conform to source facility’s 21 CFR 1271.3(b),
AABB TECHNICAL MANUAL

written instructions written instructions written instructions 1271.3(bb), and

21 CFR 1271.15(d) apply
42 Derivatives Conform to manufacturer’s Conform to manufacturer’s Conform to manufacturer’s
written instructions written instructions written instructions
1
If the seal is broken during processing, components stored at 1 to 6 C shall have an expiration time of 24 hours, and components stored at 20 to 24 C shall have an expiration time of 4 hours,
unless otherwise indicated. This expiration shall not exceed the original expiration date or time.
2
21 CFR 600.15(a).
3
Storage beyond 4 hours requires an FDA-cleared system.
4
21 CFR 601.22.
5
These lines could apply to apheresis plasma or whole-blood-derived plasma.
 CHAPTER 9 Storage, Processing, Distribution, and Inventory
the cellular and protein environment. The
storage temperature must be maintained at
1 to 6 C throughout the duration of stor-
age.3(pp51-53) Changes that may occur during
storage directly affect how long RBC units may
be stored. Product approval by the FDA re-
quires in-vivo labeling studies demonstrating
that at least 75% of the transfused red cells are
present in circulation 24 hours after transfu-
sion with less than 1% hemolysis.
During ex-vivo storage of RBC units, bio-
chemical and morphologic changes to the red
cells occur that have been termed the “storage
lesion.” These changes include cell membrane
shape change and microvesiculation; de-
creased pH, adenosine triphosphate, and 2,3-
diphosphoglycerate; and increased lysophos-
pholipids, potassium, and free hemoglobin.5
Although these ex-vivo observations are well
described, there is currently insufficient evi-
dence to conclude that storage duration corre-
lates with clinical outcomes.6,7 One situation
where evidence supports the use of fresher
RBCs is large-volume transfusions (>25 mL/
kg) in neonates.8
Platelets
Platelet storage conditions and shelf life are af-
fected by morphologic and functional changes
during storage. Metabolic changes include the
glycolytic production of lactic acid and the oxi-
dative metabolism of free fatty acids, which re-
sults in the production of carbon dioxide.
Platelet pH is maintained above 6.2 via buffer-
ing of lactic acid by bicarbonate and promo-
tion of oxidative metabolism facilitated by the
diffusion of oxygen and carbon dioxide across
a gas-permeable storage bag during gentle agi-
tation.9 The maximum time for platelets to be
without agitation is 24 hours.3(pp53-55)
Platelet shelf life also is limited because of
an increased risk of bacterial growth due to
room-temperature storage. Blood banks and
transfusion services are required to have
methods to detect or inactivate bacteria in all
platelet components.3(p11)
䡲 221
Granulocytes
Granulocytes are fragile, deteriorate rapidly in
vitro, and should be transfused as soon as pos-
sible after receipt from the supplier. Granulo-
cytes are stored at 20 to 24 C, should not be ag-
itated, and must never be leukocyte reduced.
PRETRANSF USION
PROC ESSING
Thawing Plasma and Cryoprecipitate
Fresh Frozen Plasma (FFP), Plasma Frozen
within 24 Hours After Phlebotomy (PF24), and
Plasma Frozen within 24 Hours After Phlebot-
omy Held at Room Temperature up to 24
Hours After Phlebotomy (PF24RT24) must be
thawed at 30 to 37 C using a waterbath or other
FDA-approved device. Thawing in a waterbath
requires the frozen component to be in a plas-
tic overwrap before insertion into the water to
prevent contamination of the container entry
ports. Thawed plasma products (FFP, PF24,
and PF24RT24) are stored at 1 to 6 C and expire
24 hours after thawing.
These products must be relabeled as
“Thawed Plasma” if they are stored for longer
than 24 hours. Although not licensed by the
FDA, Thawed Plasma is included in the AABB
Standards for Blood Banks and Transfusion
Services3(p28) and the Circular of Information for
the Use of Human Blood and Blood Compo-
nents.10 Thawed Plasma is stored at 1 to 6 C
and expires 5 days after it was originally
thawed. Facilities may label such products as
“Thawed Plasma” at the initial time of thaw-
ing. By maintaining a Thawed Plasma invento-
ry, transfusion services may decrease wastage
of thawed plasma products.11
Levels of labile coagulation factors (Fac-
tor V and Factor VIII) and stable factors are
well above 50% of immediate post-thaw levels
in Thawed Plasma that has been stored for up
to 5 days.12 Thawed Plasma does, however,
contain reduced concentrations of Factor V,
Factor VII, and Factor VIII. For this reason,
Thawed Plasma is not suitable for single-factor
replacement when antihemophilic factor de-
rivatives are unavailable.
222 䡲 AABB TECHNICAL MANUAL
Cryoprecipitate is thawed at 30 to 37 C, is
gently resuspended, and can be pooled for
ease of transfusion using small quantities of
0.9% sodium chloride, injection (USP) to rinse
the contents of the bag into the final container
(Method 6-11). Thawed cryoprecipitate is
stored at 20 to 24 C and expires within 4 hours
of pooling if it is pooled in an open system or
within 6 hours for single units or units pooled
using an FDA-cleared sterile connecting de-
vice.10
Thawing and Deglycerolizing RBCs
RBC units may be frozen and stored for up to
10 years following the addition of glycerol as a
cryopreservation agent (Methods 6-6 and
6-7).13,14 Frozen units can be thawed using a
37 C dry heater or 37 C waterbath. After being
thawed, the glycerol must be removed before
the component is transfused. Commercial in-
struments for batch or continuous-flow wash-
ing are available for deglycerolization. The
manufacturer’s instructions should be fol-
lowed to ensure maximal red cell recovery and
minimal hemolysis. Measurement of free he-
moglobin in the final wash can be used to con-
firm adequate free hemoglobin removal and as
a surrogate marker for adequate deglyceroliza-
tion (Method 6-8).
Integrally attached tubing must be filled
with the deglycerolized red cells and sealed
appropriately so that a segment may be de-
tached and available for crossmatch testing.
The shelf life of Deglycerolized RBCs de-
pends on the type of system used. Closed-
system devices allow storage for up to 14 days,
but components prepared using open systems
expire within 24 hours of deglycerolization.
Platelet Gel Production
Platelet gel is produced when thrombin and
calcium are added to platelet-rich plasma to
produce a glue-like substance for surgical ap-
plication.15 This product is typically prepared
at the bedside immediately before use. Facili-
ties involved in the production of this compo-
nent should refer to the current edition of the
AABB Standards for Perioperative Autologous
Blood Collection and Administration for guid-
ance and quality oversight of this manufactur-
ing process.
Irradiation
Irradiation of cellular components is intended
to prevent transfusion-associated graft-vs-
host disease (TA-GVHD), which is caused by
proliferation of donor T lymphocytes. People
at increased risk of TA-GVHD include pro-
foundly immunocompromised patients, re-
cipients of intrauterine transfusion, patients
undergoing marrow or peripheral blood stem
cell transplantation, and recipients of cellular
components from blood relatives or donors
selected for HLA compatibility.
Sources of radiation include gamma rays
(cesium-137 or cobalt-60 radioisotopes) and
x-rays. The required gamma radiation dose
needed to prevent proliferation of donor T
lymphocytes in the recipient is a minimum of
25 Gy (2500 cGy/rad) to the central point of the
blood container and 15 Gy (1500 cGy/rad) to
any other part of the container. Confirmation
that the blood container has received an ade-
quate radiation dose can be achieved with the
use of commercially available radiographic
film labels.
Irradiation is associated with damage to
the red cell membrane, which may result in in-
creases in extracellular free hemoglobin and
potassium during product storage. For this
reason, the expiration date of irradiated RBCs
is 28 days after irradiation or the original expi-
ration date, whichever is earlier.
Hospital transfusion services may pur-
chase irradiated blood components from their
supplier or perform irradiation within the
blood bank using approved and monitored ra-
diation devices. Hospitals that perform their
own irradiation may irradiate their inventory
on demand or in batches. Maintenance of a
dual inventory (irradiated and nonirradiated)
requires policies and procedures to ensure
that transfusion recipients receive the appro-
priate component for their clinical situation.
Poststorage Leukocyte Reduction
Poststorage leukocyte reduction can be per-
formed by the blood bank before issuing a
 CHAPTER 9 Storage, Processing, Distribution, and Inventory
component using a leukocyte reduction filter
attached by a sterile connection. It can also be
performed at the bedside during transfusion
using a blood-administration filter designed
for this purpose. Leukocyte reduction filters
are designed to remove >99.9% of white cells
(3-log reduction) and meet the AABB standard
of less than 5 × 106 leukocytes in 95% of sam-
pled units for RBCs and Apheresis Plate-
lets.3(p24) The manufacturer’s instructions must
be followed for the filtration device used to
achieve acceptable leukocyte reduction.
Prestorage leukocyte reduction is general-
ly the preferred method for leukocyte reduc-
tion because it prevents accumulation of cyto-
kines during product storage. Quality control
of bedside filtration is challenging, and the
process has been associated with hypotensive
transfusion reactions.16
Volume Reduction
Volume reduction results when plasma and
additive solutions are partially removed from
RBC or platelet components following centrif-
ugation. This process may be used to aggres-
sively manage volume in patients at risk of
transfusion-associated circulatory overload,
reduce exposure to plasma proteins or addi-
tives, or achieve a target hematocrit level.
Volume reduction of platelets is described
in Method 6-13. The speed of centrifugation
may affect the degree of platelet loss. Higher g
forces are associated with better platelet reten-
tion but raise the theoretical concern of plate-
let damage and activation as platelets are
forced against the container wall. When plate-
let volumes are reduced, platelets should rest
at room temperature for 20 to 60 minutes fol-
lowing centrifugation and before resuspension
in remaining plasma or added saline. The
manufacturer’s instructions must be followed
regarding the minimum volume necessary to
maintain proper air exchange across the gas-
permeable platelet-storage bag. The shelf life
of volume-reduced platelets is 24 hours for
components stored at 1 to 6 C and 4 hours for
those stored at 20 to 24 C.
䡲 223
Washing
Cellular components are typically washed to
remove plasma proteins. Washing can also be
performed to remove glycerol from frozen RBC
units after thawing. Indications for washing
RBC or platelet components include a patient
history of severe allergic reactions to compo-
nents containing plasma, the presence of anti-
bodies against immune globulin A (IgA) in an
IgA-deficient recipient when IgA-deficient cel-
lular components are not available, the pres-
ence of maternal anti-HPA-1a (eg, when using
maternal blood for a neonatal transfusion),
and the need for complement removal.
Washing is accomplished with the use of 1
to 2 L of sterile normal saline (preferably using
automated equipment). As with volume re-
duction, washed platelets should rest at room
temperature without agitation between cen-
trifugation. Up to 20% of the red cell yield or
33% of the platelet yield may be lost during
washing. Because washing creates an “open
system” and removes anticoagulant-preserva-
tive solutions, washed RBC units expire 24
hours after washing and washed platelet units
expire 4 hours after washing. It is recommend-
ed that hospitals performing washing comply
with the manufacturer’s recommendations re-
garding minimum volumes needed for com-
ponent storage bags to maintain optimal stor-
age conditions.
Pooling
Certain blood components (whole-blood-
derived platelets, cryoprecipitate, or RBCs and
plasma to produce reconstituted whole blood)
may need to be pooled to provide clinically ef-
fective transfusion therapy without the need to
transfuse multiple single components.
Pooled whole-blood-derived platelets
may contain a significant number of red cells,
and, therefore, ABO compatibility and risk for
RhD alloimmunization are recipient factors
that must be considered. If whole-blood-
derived platelets are pooled using an open sys-
tem, the expiration time is 4 hours from the
start of pooling. A commercially available, FDA-
approved, prestorage, whole-blood-derived,
224 䡲 AABB TECHNICAL MANUAL
platelet pooling system allows storage for up
to 5 days and the ability to perform culture-
based bacterial testing.17 When this system is
used, the pool maintains the expiration date of
the earliest collected component in the pool.
Single cryoprecipitate units are pooled af-
ter thawing in a manner similar to that used
for platelets. The expiration time of cryopre-
cipitate pools depends on the method used for
pooling. Cryoprecipitate pooled in an open
system expires within 4 hours of pooling.
Thawed single concentrates and pooled con-
centrates using sterile connecting devices ex-
pire 6 hours after thawing. Thawed cryopre-
cipitate is stored at 20 to 24 C. As an
alternative, the blood center may pool single
concentrates before freezing.
Reconstituted whole blood consists of
RBCs combined with ABO-compatible FFP.
This product can be used for neonatal ex-
change transfusion. The conventional ap-
proach is to combine group O RBCs (Rh com-
patible with the neonate) and group AB FFP to
achieve a 50% ± 5% hematocrit of the final
product. The volumes of the two components
before pooling can be adjusted to achieve a
desired hematocrit level. Following recombi-
nation, the product can be stored at 1 to 6 C for
up to 24 hours.
Current FDA uniform guidelines should
be followed when pooled products are la-
beled.18 A unique pool number should be af-
fixed to the final container, and all units in the
pool must be documented in electronic or
manual records.
Aliquoting
Patients requiring low-volume transfusions
may receive aliquots of smaller volumes de-
rived from the original unit via an FDA-cleared
sterile connecting device or integrated transfer
bags. Available products designed for use with
sterile connecting devices include transfer
packs, smaller-volume bags, and tubing with
integrally attached syringes.
The expiration date of the aliquot and
minimum residual volumes that must be
maintained depend on the storage container
used. Hospital transfusion services must de-
velop policies and procedures for aliquot
preparation and storage that comply with
manufacturer specifications. The use of ali-
quots for neonatal transfusion has been shown
to result in a decreased number of donor expo-
sures.19 The process of preparing aliquots for
small-volume transfusion in neonates and
children is discussed in greater detail in Chap-
ter 23. Lower-volume components (split units)
may also be prepared for adult patients who
require slow rates of transfusion due to con-
cerns about fluid overload. Split units are rec-
ommended when the component volume can-
not be transfused at a rate that ensures
completion of the transfusion within 4 hours.
DISTR IBUTION
Inspection
Inspection is a critical control point in blood
component manufacturing and must occur
before shipping, upon receipt, and before is-
sue for transfusion. Proper documentation of
this process includes 1) date of inspection, 2)
donor identification number, 3) description of
any visual abnormalities, 4) action(s) taken,
and 5) identity of the staff member performing
the inspection. Visible abnormalities may in-
clude discoloration of the segments, compo-
nent, or supernatant fluid or the presence of
visible clots, particulate matter, or other for-
eign bodies. Detection of any such abnormali-
ties should result in product quarantine for
further investigation that may include return-
ing the component to the supplier.
If a component is determined to be bacte-
rially contaminated, the component manufac-
turer must be notified so that an immediate
investigation can take place. Other compo-
nents prepared from that collection should be
quarantined until the investigation is com-
plete. If the component (or co-component)
has been transfused, the recipient’s attending
physician should be notified, and consultation
with the medical director is recommended.
 CHAPTER 9 Storage, Processing, Distribution, and Inventory
Shipping
Blood components may be transported be-
tween blood centers, hospitals, and blood cen-
ters. All containers used to transport blood
components must be qualified before use to
ensure that the proper component transport
temperature is maintained. The shipping tran-
sit time, mode of transport, and climatic con-
ditions must also be validated. All components
should be inspected upon receipt to confirm
appropriate transport conditions, component
appearance, and expiration date. Any devia-
tion from routine shipping or component con-
ditions should be reported to the shipping fa-
cility and documented according to each
location’s policies, processes, and procedures.
Whole Blood, RBCs, and Thawed
Plasma Products
Whole blood, RBCs, and thawed plasma prod-
ucts must be transported at a temperature of 1
to 10 C. A variety of options exist for maintain-
ing transport temperature, including bagged
wet ice, commercial cooling packs, and spe-
cially designed containers. All transport cool-
ers must be qualified to maintain the transport
temperature when packed using a validated
process.
Blood components transported at 1 to 10
C and stored at 1 to 6 C may need to be tempo-
rarily removed from those temperatures for
entry into inventory, irradiation, or other pro-
cessing. The maximum number of units that
can be manipulated before the component
reaches an unacceptable temperature should
be determined and not exceeded. Validation of
this process may be accomplished using man-
ual temperature monitoring indicators affixed
to the blood components or electronic devices
that can measure the temperature of the blood
components without opening the bag.
Platelets, Thawed Cryoprecipitate, and
Granulocytes
Platelets, thawed cryoprecipitate, and granu-
locytes must be transported at a temperature
of 20 to 24 C. All transport coolers must be
qualified to maintain this transport tempera-
䡲 225
ture when packed using a validated process.
For platelets, the maximum time without agi-
tation is 24 hours.
Frozen Components
Frozen components should be packaged to
minimize breakage and maintain a frozen
state. Dry ice in a suitable container is routine-
ly used for shipping these components. All
transport coolers must be qualified to main-
tain the transport temperature when packed
using a validated process.
Receiving
The receiving facility should notify the ship-
ping facility and document any deviation from
usual shipping container packing or appear-
ance of the shipped blood components. Any
blood component not complying with the fa-
cility’s policies, processes, and procedures
should be quarantined. Only after investiga-
tion of the deviation and determination that
the component meets acceptance criteria may
the component be removed from quarantine
and released into the general inventory.
Blood components should be fully trace-
able from collection to final disposition. Elec-
tronic or manual records indicating compli-
ance with policies, processes, and procedures
should be generated and maintained for the
applicable record-retention time. Any devia-
tion must be recorded, and blood components
not meeting requirements should be quaran-
tined. Deviations must be investigated to de-
termine appropriate product disposition and
possible corrective action. The results of any
corrective action should be reported to the
blood supplier as needed. Inventory manage-
ment should consist of routine determination
that all blood components are accounted for
and transfused or appropriately discarded.
Product Testing
Before transfusion, the ABO group of all units
and Rh type of any units labeled “Rh negative”
must be confirmed by serologic testing for all
red-cell-containing components (RBCs, whole
blood, and granulocytes). Any typing discrep-
226 䡲 AABB TECHNICAL MANUAL
ancies identified must be reported immediate-
ly to the supplier and resolved before the prod-
uct is dispensed for transfusion.
Issuance of Components
Ensuring that the correct blood component is
transfused to the correct patient is paramount
for transfusion safety. All requests for blood
components must contain two independent
identifiers so that the intended recipient can
be uniquely identified. Recipient compatibility-
testing records must also be reviewed. Current
testing results must be compared with histori-
cal records, if available, and any discrepancies
must be resolved before product selection.
Personnel must visually inspect and doc-
ument that the selected product is acceptable
for use. This inspection must include confir-
mation that the product does not have an ab-
normal color or appearance and that the con-
tainer is intact. Once selected for transfusion,
the blood component must have an attached
label or tie tag that contains the intended re-
cipient’s two independent identifiers, dona-
tion identification number, and compatibility
test result interpretation, if performed.
At the time of issue, there must be a final
check of each unit that includes the following:
1. The intended recipient’s two independent
identifiers, ABO group, and Rh type.
2. The donation identification number, donor
ABO group, and, if required, Rh type.
3. The interpretation of the crossmatch test
results, if performed.
4. Special transfusion requirements (eg, cyto-
megalovirus-reduced-risk, irradiated, or
antigen-negative components), if applica-
ble.
5. The expiration date and, if applicable, time.
6. The date and time of issue.
The transfusion service must confirm that
the recipient identifying information, transfu-
sion request, testing records, and blood com-
ponent labeling and compatibility are accu-
rate and in agreement. Any discrepancies
identified must be resolved before issue.
Final identification of the transfusion re-
cipient and blood component rests with the
transfusionist. This individual identifies the
patient and donor unit and certifies that the
identifying information on forms, tags, and la-
bels is in agreement.
Documentation
The patient’s medical record must include
proper documentation of all transfusions. For
each transfusion, this documentation must
contain the transfusion order, consent for
transfusion, component name, donation iden-
tification number, date and time of transfu-
sion, pre- and posttransfusion vital signs,
volume transfused, identification of the trans-
fusionist, and, if applicable, any transfusion-
related adverse events.
Return of Blood Components and
Reissue
The transfusion service may receive back into
inventory units that meet acceptance specifica-
tions. These conditions include the following:
1. The primary container has not been opened.
2. The component has been maintained at the
appropriate temperature.
3. At least one sealed segment remains inte-
grally attached to the container of RBCs.
4. Documentation indicates that the compo-
nent has been inspected and is acceptable
for reissue.
Individual unit temperature indicators or
temperature-reading devices can be used to
determine the acceptability of products for re-
turn to inventory. Blood and blood compo-
nents may also be transported or stored in
qualified containers using a validated process
that has been shown to maintain acceptable
temperatures for a defined interval. If time
frames are used to determine the acceptability
of a product’s return to inventory, the time
frame must be validated by the individual fa-
cility. The validation should demonstrate that
for the defined period, the appropriate tem-
perature of the product has been maintained.
 CHAPTER 9 Storage, Processing, Distribution, and Inventory
Components meeting the acceptance cri-
teria may be returned to the general blood
inventory and reissued. Components not
meeting the acceptance criteria must be quar-
antined for further investigation or discarded
in a biohazard container to prevent inadver-
tent return to inventory.
I N V E N TO RY M A N AG E M E N T
General Considerations
A sufficient number of ABO- and Rh-compati-
ble units should be available to meet routine
hospital needs, allow for unanticipated in-
creases in utilization due to emergency situa-
tions, and minimize component outdating.
Factors that influence determination of blood
bank component inventory levels include his-
toric usage patterns, outdate rates, and dis-
tance from suppliers. Inventory levels should
be periodically evaluated in response to insti-
tutional changes that may affect component
usage, including expansion of inpatient beds
or operating rooms; implementation of new
surgical procedures; or changes in hospital
guidelines or medical practice that may influ-
ence transfusion behavior.
The blood bank should also maintain a
reserve of universally compatible RBCs for
emergency use and have a reliable emergency
delivery system to ensure adequate availability
of blood components in unexpected situations
when demand exceeds supply. A disaster plan
should be developed and tested periodically
(see Chapter 4).
Inventory levels should be monitored dai-
ly to facilitate timely ordering from blood sup-
pliers and maintain adequate inventory levels.
This can be particularly challenging for plate-
lets due to their 5-day shelf life. Inventory-
management plans should also take into con-
sideration desirable inventory levels of special
products, such as cytomegalovirus-seronega-
tive, leukocyte-reduced, and irradiated com-
ponents. Antigen-negative RBC units and
HLA-matched or HLA-selected platelets are
typically ordered on an as-needed basis from
suppliers.
䡲 227
Surgical Blood Ordering Practices
Component outdate rates are influenced by
surgical ordering practices. For example, when
RBC units are crossmatched for surgical pa-
tients, the shelf life of the unit is shortened if
the component is unused. When crossmatch-
to-transfusion (C:T) ratios are monitored, a
C:T ratio of >2.0 may indicate excessive order-
ing of crossmatched blood.
One approach to reducing excessive C:T
ratios is to identify procedures that do not typ-
ically require blood, and use this information
to develop guidelines for the use of type and
screen units instead of crossmatched units.
Maximum surgical blood order schedules for
common elective procedures can also be de-
veloped based on local transfusion utilization
patterns.20 This practice is particularly useful
in hospital transfusion services that lack the
ability to perform electronic crossmatching.
These schedules can also be applied to pa-
tients undergoing elective surgery who are
known to have clinically significant alloanti-
bodies and require crossmatch-compatible,
antigen-negative blood. Once a surgical
blood-ordering schedule has been estab-
lished, the transfusion service routinely cross-
matches the predicted number of units for
each patient undergoing the designated pro-
cedures. Routine orders may need to be modi-
fied for patients with anemia, bleeding disor-
ders, or other conditions in which increased
blood use is anticipated. As with other circum-
stances that require rapid availability of blood
components, the transfusion service staff
should be prepared to provide additional
blood components if the need arises.
Emergent Transfusion
If transfusion is deemed medically necessary
and blood is released before pretransfusion
testing is complete, the records must contain a
signed statement from the requesting physi-
cian indicating that the clinical situation was
sufficiently urgent to require the release of
blood components before completion of
compatibility testing. If there has been time to
confirm the recipient’s ABO group, the
transfusion service should issue ABO- and
228 䡲 AABB TECHNICAL MANUAL

Rh-compatible blood. If the patient’s ABO
group is unknown, group O RBCs should be is-
sued.
A more detailed discussion of emergent
transfusion that covers clinical concerns rath-
er than inventory management can be found
in Chapter 15.
Massive Transfusion
Massive transfusion can be defined as the ad-
ministration of 8 to 10 RBC units to an adult
patient in less than 24 hours, acute adminis-
tration of 4 to 5 RBC units in 1 hour, or ex-
change transfusion of an infant.
To ensure the ability to accurately inter-
pret ABO group testing results, the patient
specimen should be obtained for testing as
early as possible during massive transfusion. If
the patient ABO type cannot be determined,
continued support with group O RBCs is rec-
ommended. Unexpected and significant usage
of group O RBCs in the setting of massive
transfusion should be considered when deter-
mining component inventory levels. In mas-
sive transfusion situations where large
amounts of blood may be required, policies
may be developed to provide D-positive RBCs
to select patients, such as all adult males and
postmenopausal females.
Many hospitals have developed massive-
transfusion protocols to standardize the re-
sponse to hemorrhage.21, 22 These protocols are
designed to rapidly provide blood compo-
nents in a balanced ratio of plasma and plate-
lets to RBCs, particularly when laboratory test-
ing is not rapid enough to guide transfusion
support. Additional studies are needed to clar-
ify whether the use of these protocols is associ-
ated with improved patient outcomes.

KEY POINTS

1. Ensuring that the correct blood component is transfused to the correct patient is para-
mount for transfusion safety. It must be confirmed that the recipient’s identifying informa-
tion, transfusion request, testing records, and blood component labeling and compatibility
are accurate and in agreement. Any discrepancies identified must be resolved before com-
ponents are issued or transfused.
2. Visual inspection of the blood component is a critical control point in the manufacturing
process and must occur before shipping, upon receipt, and before issue for transfusion.
3. Refrigerators, freezers, and platelet incubators for blood component storage must be moni-
tored to ensure that proper storage conditions are maintained. Because the safety, purity,
potency, and quality of the blood components may be affected by improper storage, alarm
settings should be configured to notify necessary personnel before the upper or lower ac-
ceptable storage temperatures are exceeded.
4. Temperature requirements during transport of blood components differ from those during
storage. Blood components held outside the blood bank before transfusion are considered
to be in storage. Validated processes must ensure that acceptable storage temperatures are
maintained.
5. Acceptable time frames for returning blood components to inventory after issue should be
validated by individual facilities. Individual unit-temperature indicators or temperature-
reading devices may be used to determine component acceptability for return to inventory.
6. Thawed FFP, PF24, and PF24RT24 expire within 24 hours of thawing. These products may be
labeled as “Thawed Plasma” to allow for a 5-day shelf life if they were originally collected in
a closed system.
 CHAPTER 9 Storage, Processing, Distribution, and Inventory
REFER ENCES
1. Food and Drug Administration. Drugs; current
good manufacturing practice in manufacture,
processing, packing, or holding. (June 19,
1963) Fed Regist 1963;133:6385-7.
2. Code of federal regulations. Title 21, CFR Parts
210 and 211. Washington, DC: US Government
Printing Office, 2014 (revised annually).
3. Levitt J, ed. Standards for blood banks and
transfusion services. 29th ed. Bethesda, MD:
AABB, 2014.
4. Nunes E. Transport versus storage: What is the
difference? AABB News 2013;15(2):4-5.
5. Klein HG, Spahn DR, Carson JL. Red blood cell
transfusion in clinical practice. Lancet 2007;
370:415-26.
6. van de Watering L. Red cell storage and prog-
nosis. Vox Sang 2011;100:36-45.
7. Zimrin AB, Hess JR. Current issues to the
transfusion of RBCs. Vox Sang 2009;96:93-103.
8. Strauss RG. Data-driven blood banking prac-
tices for neonatal RBC transfusions. Transfu-
sion 2000;40:1528-40.
9. Shrivastava M. The platelet storage lesion.
Transfus Apher Sci 2009;41:105-13.
10. AABB, American Red Cross, America’s Blood
Centers, Armed Services Blood Program. Cir-
cular of information for the use of human
blood and blood components. Bethesda, MD:
AABB, 2013.
11. Werhli G, Taylor NE, Haines, AL, et al. Institut-
ing a thawed plasma procedure: It just makes
sense and saves cents. Transfusion 2009;49:
2625-30.
12. Tholpady A, Monson J, Radovancevic R, et al.
Analysis of prolonged storage on coagulation
Factor (F)V, FVII, and FVIII in thawed plasma:
Is it time to extend the expiration date beyond
5 days? Transfusion 2013;53:645-50.
13. Meryman HT, Hornblower M. A method for
freezing and washing RBCs using a high glyc-
erol concentration. Transfusion 1972;12:
14556.
14. Valeri CR, Ragno G, Pivacek LE, et al. A multi-
center study of in vitro and in vivo values in
䡲 229
human RBCs frozen with 40-percent (wt/vol)
glycerol and stored after deglycerolization for
15 days at 4 degrees C in AS-3: Assessment of
RBC processing in the ACP 215. Transfusion
2001;41:933-9.
15. Borzini P, Mazzucco L. Platelet gels and releas-
ates. Curr Opin Hematol 2005;12:473-9.
16. Cyr, M, Hume H, Sweeney JD, et al. Anomaly of
the des-Arg9-bradykinin metabolism associat-
ed with severe hypotensive reaticon during
blood transfusions: A preliminary report.
Transfusion 1999;39:1084-8.
17. Benjamin RJ, Kline L, Dy BA, et al. Bacterial
contamination of whole-blood-derived plate-
lets: The introduction of sample diversion and
prestorage pooling with culture testing in the
American Red Cross. Transfusion 2008;48:
2348-55.
18. Food and Drug Administration. Guidance: In-
dustry consensus standard for the uniform la-
beling of blood and blood components using
ISBT 128 version 2.0.0, November 2005. (Sep-
tember 22, 2006) Silver Spring, MD: CBER Of-
fice of Communication, Outreach, and Devel-
opment, 2006.
19. Liu EA, Mannino FL, Lane TA. Prospective,
randomized trial of the safety and efficacy of a
limited donor exposure transfusion program
for premature neonates. J Pediatr 1994;125:92-
6.
20. Boral LI, Dannemiller FJ, Standard W, et al. A
guideline for anticipated blood usage during
elective surgical procedures. Am J Clin Pathol
1979;71:680-4.
21. Young PP, Cotton BA, Goodnough LT. Massive
transfusion protocols for patients with sub-
stantial hemorrhage. Transfus Med Rev 2011;
25:293-303.
22. Hendrickson JE, Shaz BH, Pereira G, et al. Im-
plementation of a pediatric trauma massive
transfusion protocol: One institution’s experi-
ence. Transfusion 2012;52:1228-36.
 C h a p t e r 1 0

Molecular Biology and

Immunology in Transfusion
Medicine

James C. Zimring, MD, PhD, and Steven L. Spitalnik, MD

I N T H I S C H A P T E R , the fundamental Moreover, this chapter predominantly focuses

principles for analyzing deoxyribonucle-
ic acid (DNA), ribonucleic acid (RNA), and
protein are described. In transfusion medi-
cine, these techniques are used to 1) detect in-
fectious pathogens; 2) predict the phenotype
of red cell, platelet, and neutrophil antigens; 3)
detect and identify red cell and platelet anti-
bodies; 4) determine HLA type; and 5) perform
relationship testing. In addition to describing
the principles behind these analyses, this
chapter describes potential problems with the
assay systems. This chapter also addresses the
basic mechanisms by which the immune sys-
tem initiates a response to foreign antigens.
Entire textbooks have been written to de-
scribe these processes, and it is outside the
scope of this chapter to provide detailed and
comprehensive explanations. Instead, this
chapter focuses on topics immediately rele-
vant to the practice of transfusion testing.
on humoral immunity because the role of cel-
lular immunity in transfusion medicine re-
mains unclear and is rarely, if ever, of conse-
quence in the pathophysiology of hemolytic
transfusion reactions. This chapter does not
provide specific assay protocols; rather, it
seeks to provide an understanding of the sci-
entific principles underlying the molecular bi-
ological and immunological assays used in
transfusion medicine.
NUCLEIC ACID ANALYSIS
The clinical utility of nucleic acid analysis in
transfusion medicine lies in detecting infec-
tious pathogens and genotyping blood donors
and recipients. The human genome is encod-
ed in polymers of DNA, and, likewise, many
pathogens utilize DNA to encode their ge-
nome. Alternatively, many viral pathogens

James C. Zimring, MD, PhD, Director, Transfusion Medicine Research, Puget Sound Blood Center Research
Institute, Seattle, Washington, and Steven L. Spitalnik, MD, Professor of Pathology and Cell Biology, and
Director of Clinical Laboratories, Columbia University, New York, New York
J. Zimring has disclosed financial relationships with Immucor Inc and Haemonetics. S. Spitalnik has disclosed
no conflicts of interest.

231
232 䡲 AABB TECHNICAL MANUAL
encode their genome as RNA. In addition, be-
cause normal flora may have a substantial ef-
fect on human biology, DNA and RNA analysis
may become important to monitor normal
nonpathological and commensal microorgan-
isms. With the possible exception of prion-
associated disorders, either DNA or RNA en-
codes all known genetic material relevant to
transfusion medicine.
Basic Chemistry and Structure of
Nucleic Acids
DNA is a nucleic acid polymer consisting of
long chains of nucleotides linked together.1
Nucleotides consist of a pentose (a carbohy-
drate with five carbon atoms), a phosphate
group attached to the fifth carbon atom (C5) of
the pentose, and a base group attached to C1
[Fig 10-1(A)]. Variations in the chemistry of the
base group give rise to the four different nucle-
FIGURE 10-1. Chemical structure of nucleic acids and DNA.
otides comprising DNA: the purines (adenine
and guanine) and pyrimidines (cytosine and
thymine). In addition, C3 of the pentose is
modified by a hydroxyl group [Fig 10-1(A)].
The individual nucleotides form DNA poly-
mers when the phosphate group on C5 of one
nucleotide forms a covalent bond with the free
hydroxyl group on C3 of another nucleotide
[Fig 10-1(B)]. DNA molecules vary from each
other based on the sequence of nucleotides
that are incorporated into the polymer. Each
DNA strand has a terminal 5' end that contains
a free phosphate (attached to C5) and a termi-
nal 3' end that has a free hydroxyl group (at-
tached to C3).
The human genome consists of double-
stranded DNA. The bases contained within a
single strand of DNA form hydrogen bonds to
complementary bases on another strand of
DNA. In particular, thymidine (T) binds to ad-
CHAPTER 10 Molecular Biology and Immunology in Transfusion Medicine
enine (A), and guanine (G) binds to cytosine
(C). When two strands have complementary
sequences, they can hybridize to form a
double-stranded molecule [Fig 10-1(C)]. The
two complementary strands hybridize such
that the 5' and 3' ends have opposite orienta-
tions and form a double helix in which the
phosphodiester backbone is on the outside of
the helix and the hydrogen-bond-paired bases
are on the inside.
When genes are expressed, the DNA en-
coding a given gene is transcribed into RNA
[Fig 10-1(C)]. The structure of RNA is similar to
that of DNA with the following exceptions: 1)
ribonucleotides have an additional hydroxyl
group on C2 of the pentose sugar (thus form-
ing ribose), 2) uracil (U) replaces thymine (T),
and 3) RNA coding for gene products is
typically single-stranded (although double-
stranded RNA has important regulatory roles).
Several classes of RNA exist in human
cells; the type described in this paragraph,
which is subsequently translated into protein,
is messenger RNA (mRNA). When a gene is ex-
pressed, the transcription machinery unwinds
the DNA double helix, synthesizes a mRNA
strand of complementary sequence, and rehy-
bridizes the DNA in its wake [Fig 10-1(C)]. In
this way, the RNA is essentially a copy of the
sequence found in the DNA. RNA is synthe-
sized in the 5' to 3' direction, and, thus, only a
single strand of the DNA is transcribed into
RNA by any given run of a polymerase. After
synthesis in the nucleus, RNA is processed and
exported to the cytoplasm, where ribosomes
translate it into protein.
Isolation of Nucleic Acids
The first step in most DNA and RNA analyses is
the isolation of nucleic acids. All nucleated
cells of an individual contain identical genom-
ic DNA, with some notable exceptions (eg, re-
arranged genes in mature T and B cells). Ge-
nomic DNA can be isolated from readily
obtainable cellular sources, such as peripheral
blood leukocytes and buccal swabs. In con-
trast, mRNAs are distinct in different cell pop-
ulations because their expression patterns are
䡲 233
important in defining the phenotype of these
cells.
For mRNA analysis, then, the choice of
starting cell types is critical. Multiple manu-
facturers offer reagents that simplify and expe-
dite isolation of cellular DNA and/or mRNA as
well as for purifying viral nucleic acids from
plasma. Depending on the type of testing to be
performed, the quantity and quality of the nu-
cleic acids isolated may be important, as de-
termined by the relative purity of DNA or RNA
and the absence of contaminating protein.
Hybridization-Based Methods of
Nucleic Acid Detection
Before the advent of techniques that allowed
the amplification of a specific nucleic acid se-
quence (see Polymerase Chain Reaction be-
low), detection of nucleic acids depended on
hybridization-based methods. Probes with a
particular sequence of nucleotides were syn-
thesized and labeled with one of a variety of
detectable markers. The probe could then hy-
bridize to complementary sequences of DNA
or RNA, allowing the detection and quantifica-
tion of the corresponding complementary tar-
get. This could be done in the solid phase (ie,
Southern and Northern blots), the fluid phase
(ie, RNAse protection assay or S1 protection
assay) or as a result of enzymatic extension (ie,
primer extension assay).2-5 However, compared
to more recent amplification-based techniques,
hybridization-based methods have limited
sensitivity and are less amenable to automa-
tion. Although hybridization-based methods
are useful in basic research and still play a mi-
nor role in some diagnostic laboratories, most
analyses of nucleic acids are performed using
amplification-based methods.
The Polymerase Chain Reaction
Nucleic acid detection and analysis were revo-
lutionized by the invention of the polymerase
chain reaction (PCR). PCR was the first ampli-
fication-based technique for generating nucle-
ic acid fragments for direct analysis.6 The
concept of amplification-based systems has
234 䡲 AABB TECHNICAL MANUAL
grown to include many other techniques and
applications.
A PCR reaction requires 1) a DNA sample
to be analyzed; 2) gene-specific primers; 3) a
thermostable DNA polymerase; and 4) compo-
nents that allow the DNA polymerase to enzy-
matically synthesize DNA, including nucleo-
tides (A, T, C, and G) and the proper salts and
buffer. The PCR reaction involves repeat cycles
of heating/cooling (thermocycling), allowing
exponential DNA amplification of the frag-
ment of interest. This reaction is carried out in
a thermocycler that rapidly changes tempera-
ture with accuracy and precision. The thermo-
cycling reaction involved is 1) heat denatur-
ation of double-stranded DNA, 2) cooling to
FIGURE 10-2. Graphic overview of the polymerase chain reaction.
allow primer annealing, and 3) extension and
synthesis of DNA on the primer strand. This
procedure continues for multiple cycles, typi-
cally 20 to 40, depending on the abundance of
the template and the required sensitivity of the
assay.
An overview of the PCR process is pre-
sented in Fig 10-2. This example begins with a
single copy of a double-stranded DNA tem-
plate. The DNA is denatured by heating to near
boiling (typically at 94-95 C), which disrupts
the hydrogen bonds between complementary
bases, thereby separating the two strands. The
temperature is then lowered (annealing reac-
tion) to allow gene-specific primers to anneal
to their complementary target. Typically, an
CHAPTER 10 Molecular Biology and Immunology in Transfusion Medicine
annealing temperature of 5 C below the melt-
ing temperature of the target DNA-primer
complex increases specificity to the correct
target sequence. The exact melting tempera-
ture of a given target DNA primer complex de-
pends on the primer length, abundance of
guanines and cytosines in the sequence (ie, GC
content), and any mismatches with the target.
At this point, the temperature is raised to that
at which the DNA polymerase functions opti-
mally (typically 72 C), and the primers are ex-
tended along the length of the DNA by the in-
corporation of the correct complementary
nucleotides by DNA polymerase. Thus, at the
end of extension, there are two copies of the
DNA. This process repeats itself with denatur-
ing, annealing, and extension. With each sub-
sequent cycle, an exponential increase in DNA
copy number occurs. PCR results in a geomet-
ric expansion of a selected DNA “amplicon,”
defined as the sequence that is flanked by the
two chosen primers.
PCR Considerations
Although PCR is a robust and reliable method
of detecting nucleic acids, as with all methods,
technical problems can affect PCR and other
amplification-based techniques.
Specimen Processing and Template
Degradation
DNA is stable and can usually withstand some
variations in storage temperature and han-
dling before being processed for genomic
analysis. Exceptions include samples in which
the target DNA is present in low quantity, such
as fetal typing from maternal plasma and viral
testing. RNA is far less stable than DNA and is
susceptible to autocatalytic degradation by
RNAse enzymes found in many biological
specimens.
Inhibitors
PCR amplification depends on the enzymatic
activity of DNA polymerase and can be inhibit-
ed by any substance that negatively affects
synthesis by DNA polymerase. Heparin can in-
hibit PCR in some situations, and hemoglobin
䡲 235
or lactoferrin released from erythrocytes or
leukocytes also inhibit PCR.7,8 Most analytic
systems are optimized such that the risk of in-
terference by an inhibitor is minimized, but
care should be taken not to deviate from es-
tablished protocols because such deviations
may introduce unintended inhibitory sub-
stances. Amplifying ubiquitous target se-
quences present in all samples (ie, conserved
genomic DNA or housekeeping genes, such as
hemoglobin) and/or spiking the specimen
with an internal positive control can be used
to assess whether inhibitors are present.
Primer Design
Inferior performance of primers should not be
a concern in the context of commercially avail-
able tests. Nevertheless, a thorough under-
standing of primer design is important in trou-
bleshooting assays, and primer design is a key
component in developing PCR-based assays
for new targets. Although ideal primers hy-
bridize to a target that is found in only one lo-
cation in the entire genome, given the com-
plexity of genomic DNA, this is not always
possible. Primer annealing to unintended tar-
gets can occur, resulting in ongoing consump-
tion of primers with each cycle and the poten-
tial to amplify unintended targets.
In addition to cross annealing to unin-
tended genomic sites, primers can anneal to
each other and form a short amplicon. For ex-
ample, if the 3' ends of two primers bind to
each other, the polymerase fills in the 5' over-
hangs [Fig 10-3(A)]. A primer modified in this
way may still be able to anneal to its genomic
target sequence. However, the additional bas-
es added to the 3' end no longer anneal to the
genomic target and may prohibit proper ex-
tension. This “primer-dimer formation” can
take place between two different primers in a
reaction or two molecules of the same primer
[Fig 10-3(B)]. Primers can also form hairpin
loops and anneal to themselves [Fig 10-3(C)].
A self-complementary sequence in a primer
may allow this to occur, particularly in primers
over 20 bp in length, and this reaction is fa-
vored because of its intramolecular nature. If
self-annealing results in a 5' overhang, the
236 䡲 AABB TECHNICAL MANUAL

PCR Primers Normal Amplification

5’ 3’ 3’ 5’
5’ 3’
5’ 3’

A. B. C.
5’
5’ 3’ 5’ 3’
3’ 5’ 3’ 5’ 3’

5’
5’ 3’ 5’ 3’
3’ 5’ 3’ 5’ 3’

FIGURE 10-3. Potential problems in primer design that may inhibit polymerase chain reaction (PCR).

molecule can self-prime with the polymerase Using this approach, DNA extraction is sepa-
and extend the 3' end to be complementary to rate from testing, PCR reactions are assembled
the 5' end, increasing the tendency of the in one room and amplified in a second room,
primer to self-anneal. The presence of the ad- and downstream analysis (if required) is per-
ditional sequence prevents amplification of formed in a third room. There should be no
the authentic target amplicon. retrograde flow, and no materials or instru-
ments used in the amplification or analysis
Contamination between Specimens (post-PCR) rooms should make their way into
the PCR setup (pre-PCR) room. Pipette filter
One of the greatest strengths of PCR is its abili-
tips that minimize carry-over contamination
ty to amplify very small amounts of genetic
and sample aerosols are routinely used.
material. In theory, single-copy sensitivity can
Other ways to minimize potential con-
be achieved. In practice, detection of 10 copies
tamination include the addition of deoxyuri-
of DNA or fewer is not uncommon, depending
dine triphosphate (dUTP) to PCR reactions.
on the sensitivity of the assay readout. This
Polymerases incorporate dUTP in place of de-
level of sensitivity also makes the assay sus-
oxythymidine triphosphate (dTTP), and the
ceptible to false-positive results due to con-
added enzyme uracil-DNA glycosylase (UNG)
tamination from specimens being analyzed or
specifically cleaves DNA containing uracil but
amplicons generated in previous amplifica-
not normal DNA or RNA.9 Thus, adding UNG
tion reactions. Beginning with just 10 mole-
to PCR reactions destroys contaminating am-
cules of DNA, 30 rounds of amplification in a
plicons from previous amplifications but not
PCR reaction yields more than 1 × 1010 ampli-
native DNA in the specimen. The UNG is heat
cons. Thus, if only 0.0000001% of a previous
inactivated during the initial denaturation
reaction is inadvertently introduced onto a pi-
PCR step, allowing amplification of the new
pette used to set up a subsequent reaction, a
sample.
false-positive result may occur.
To minimize the possibility of contamina-
Reverse-Transcriptase PCR
tion from previously generated amplicons and
a false-positive signal, PCR laboratories rou- When amplification and analysis of mRNA is
tinely process samples in only one direction. required, a reverse-transcriptase (RT) enzyme
CHAPTER 10 Molecular Biology and Immunology in Transfusion Medicine 䡲 237

that synthesizes DNA from an RNA template is
used.10,11 Like DNA polymerase, RT synthesizes
in the 5' to 3' direction and requires the an-
nealing of a primer to initiate transcription.
When RNA is transcribed, the resulting DNA is
a single-stranded complementary copy of the
RNA and is referred to as “complementary
DNA” (cDNA). The cDNA is then a suitable
substrate for PCR, as described above.
Transcription-Mediated Amplification
and Nucleic Acid Sequence-Based
Amplification
Since the advent of PCR, several additional nu-
cleic acid amplification strategies have been
devised. Among the most useful are two relat-
ed techniques, transcription-mediated ampli-
fication (TMA) and nucleic acid sequence-
based amplification (NASBA).12,13 Although
TMA and NASBA have some differences (de-
scribed in this section), they are conceptually
similar and, thus, are described together (see
Fig 10-4).
TMA plays a large role in nucleic acid test-
ing for human immunodeficiency virus (HIV),
hepatitis C virus, and West Nile virus (ie, using
the Chiron/Gen-Probe system). Both tech-
niques use RNA as the amplification target.
The reaction contains two primers, RT, DNA
polymerase, RNAse H, and T7 polymerase. The
reaction begins when a specific downstream
primer (Primer 1) hybridizes to the 3' end of
the target RNA and RT synthesizes a cDNA
copy (Fig 10-4, Step 1). Primer 1 not only con-
tains a complementary sequence at its 3' end
that hybridizes to the target RNA, but the 5'
end also encodes a bacteriophage T7 promot-
er. The RNA template is degraded by RT in the
TMA assay or by RNAse H in an additional step
with NASBA (Fig 10-4, Step 2). A second 5' up-
stream primer (Primer 2) then binds to the
newly synthesized cDNA (Fig 10-4, Step 3) and
utilizes DNA polymerase to synthesize a
double-stranded DNA molecule (Fig 10-4, Step
4). This molecule has a T7 promoter at one
end, and T7 polymerase then drives transcrip-
tion of RNA (Fig 10-4, Step 5). Numerous RNA

FIGURE 10-4. Schematic overview of transcription-mediated amplification (TMA) and nucleic acid sequence-
based amplification.
RNA = ribonucleic acid; cDNA = complementary deoxyribonucleic acid; mRNA = messenger RNA; RNAse =
ribonuclease.
238 䡲 AABB TECHNICAL MANUAL
transcripts are synthesized from a single DNA
template. These new RNA molecules can reen-
ter the amplification cycle with Primer 2 initi-
ating reverse transcription, followed by RNA
degradation and subsequent synthesis of DNA
using Primer 1 and DNA polymerase. This
leads to additional amplification with ongoing
cycles of transcription and template synthesis.
One distinct advantage of NASBA compared to
PCR is that repeat nucleic acid denaturation is
not required. Therefore, NASBA amplifies RNA
sequences in an isothermal reaction that does
not require a thermocycler.
In addition to the amplification methods
described above, several other techniques use
nucleic acid polymerizing or ligating enzymes
to amplify DNA and/or RNA. These include
strand displacement amplification (SDA) and
the ligase chain reaction (LCR).14-16 There are
also probe/signal-amplification methods,
such as the Cleavase Invader, branched DNA,
and hybrid capture assays. Although these
techniques have been adapted to detect
pathogens, they are not widely used in transfu-
sion medicine and are not described further
here.17
Detection of Nucleic Acid
Amplification Products
PCR, RT-PCR, TMA, and NASBA amplify spe-
cific nucleic acid sequences. However, the am-
plified sequences must still be detected. Tradi-
tionally, this has been accomplished by
separating the amplification reactions by gel
electrophoresis in the presence of a fluores-
cent dye (eg, ethidium bromide), which makes
the nucleic acids visible in the gel under ultra-
violet light. This allows visualization of the
bands and, if appropriate standards are in-
cluded in the gels, provides a molecular weight
by which one can distinguish amplicons of the
correct size from cross-reactive products of
different sizes. To confirm that the amplified
nucleic acids have the correct sequence, the
amplification products can be digested with
restriction endonucleases and the sizes of the
resulting molecules determined. Because the
amplicon sequence is known, one can predict
the correct restriction sites [this is known as
“restriction fragment length polymorphism”
(RFLP) analysis]. Even greater specificity can
be achieved by hybridization analysis. After
electrophoresis, the amplified products are
transferred to nitrocellulose paper, which is
then hybridized with DNA probes specific for
the amplified sequences.
Each of these approaches requires gel
electrophoresis, which is time consuming, in-
compatible with the throughput needs of
donor-testing laboratories, and not easily
automated. Moreover, it requires opening
tubes containing amplification reactions and
manipulating the products, which increases
the risk of contamination of reagents and
false-positive results in subsequent samples.
More advanced techniques for detecting
amplification products rely on the chemistry
of fluorescent molecules. Probes that fluoresce
only when the correct amplicon is present are
included in the amplification reactions. By in-
cluding a fluorescence spectrophotometer in
the thermocycler, the generation of amplifica-
tion products can be measured at each cycle.
This approach, called “real-time PCR,” is high-
ly sensitive; much more quantitative than gel
electrophoresis-based methods; and, by using
multiple reporter dyes with distinct fluores-
cence spectra, makes detection of multiple
targets in a single reaction feasible (ie, multi-
plex nucleic acid amplification). In addition,
including fluorescent probes in the reaction
allows analysis without ever opening the tube
containing the amplification products; this de-
creases the risk of laboratory contamination
with amplicons and subsequent false-positive
results.
Two of these fluorescent methods rely on
the juxtaposition of a fluorescent molecule
with a quencher molecule that prevents fluo-
rescence. In the TaqMan system, a sequence-
specific probe has the fluorescent molecule at
one end and the quencher at the other. When
the probe hybridizes to its target, it essentially
blocks the DNA polymerase that is synthesiz-
ing the next round of DNA by extending from
the primer. When the DNA polymerase en-
counters the hybridized probe, it degrades the
probe, thus separating the fluorescent and
quencher molecules [Fig 10-5(A)]. Because
CHAPTER 10 Molecular Biology and Immunology in Transfusion Medicine 䡲 239

FIGURE 10-5. Methods of detection by sequence-specific probes during real-time polymerase chain reaction.
Pol = polymerase enzyme.
240 䡲 AABB TECHNICAL MANUAL
this occurs only when the probe hybridizes to
its target, fluorescence is generated as a func-
tion of amplicon generation.
A second approach uses molecular bea-
cons that also have a fluorescent molecule on
one end of a DNA probe and a quencher mole-
cule on the other end. In this case, the se-
quence-specific probe is flanked by two se-
quences that are complimentary to each other
and form a hairpin loop, thus juxtaposing the
fluorescent molecule with its quencher. How-
ever, when the probe hybridizes to its target,
the hairpin loop unfolds, separating the
quencher from the fluorescent molecule and
allowing fluorescence to occur [Fig 10-5(B)].
A third approach uses two molecules that
do not fluoresce unless they are in close prox-
imity to each other. These molecules are
linked to two separate DNA probes that hy-
bridize to adjacent sequences on the ampli-
con. If the amplicon is present, the probes an-
neal in such a way that the two molecules are
in close proximity, providing a fluorescent sig-
nal [Fig 10-5(C)].
A fourth method uses SYBR® green dye,
which fluoresces when bound to double-
stranded DNA. SYBR® green is not sequence
specific and, thus, detects all amplicons. How-
ever, authentic amplicons can be distin-
guished from cross-reactive products by melt-
ing curve analysis. Because the melting curve
is a function of amplicon size and GC content
and the size and sequence of the correct am-
plicon is known, the melting curve can be used
to confirm the identity of the amplicon.
As with PCR, these fluorescent-probe
techniques can be applied to other amplifica-
tion technologies, such as TMA.
Analysis of Single-Nucleotide
Polymorphisms
The vast majority of blood group antigens con-
sist of single-nucleotide polymorphisms
(SNPs). The gene product is present in most
people, but the difference that determines the
identity of the blood group antigen is a small
change in sequence, often a single nucleotide.
Some SNPs destroy or create recognition
sites for restriction endonucleases. In these
cases, PCR-amplified material can be digested
by restriction enzymes and then analyzed by
agarose electrophoresis to observe the result-
ing fragment sizes. RFLP analysis has low
throughput and depends on the presence of a
restriction enzyme recognition site associated
with the SNP in the gene of interest.
Several different methods for detecting
SNPs consist mostly of modifications of PCR or
array technologies. With these methods, prim-
ers or probes are engineered such that hybrid-
ization depends on the presence of the correct
SNP. Both multiplex PCR and DNA array sys-
tems can determine the genotype of blood
group antigens in individual specimens.17-19
These systems offer the advantage of higher
throughput and automated readout while
avoiding the need for complex interpretation.
Genotyping of red cell antigens may be
more efficient than traditional serologic typ-
ing. In addition, when a patient has received
multiple RBC units and it is not possible to dis-
tinguish the patient’s own red cells from trans-
fused red cells, genotyping the patient’s DNA
may be the only reliable way to predict the pa-
tient’s red cell phenotype. However, occasion-
ally the genotype may not correlate with the
phenotype. It is worth noting that genotyping
typically focuses on known polymorphisms
but does not provide the entire sequence of
the gene or its regulatory regions. Therefore,
genotyping might not predict phenotype when
1) new, hitherto unknown, polymorphisms in
the coding region alter protein structure; 2)
new polymorphisms in the coding, promoter,
or other regulatory regions prevent gene ex-
pression despite the presence of the correct
coding sequence; or 3) changes alter the epit-
ope (ie, modification by bacterial enzymes)
when epitopes depend on posttranslational
modification.
PROT E IN A NA LY SI S
Nucleic acid analysis, as described in the “Nu-
cleic Acid Analysis” section above, detects the
presence of DNA or RNA that encodes genom-
ic material and/or measures gene expression
at the RNA level. However, due to regulation of
protein translation, the presence of mRNA
CHAPTER 10 Molecular Biology and Immunology in Transfusion Medicine
does not always correlate with the presence of
the encoded protein. In addition, the detec-
tion of antibodies, which are proteins, cannot
be determined by detecting or measuring nu-
cleic acids. Accordingly, measuring protein-
protein and/or protein-carbohydrate interac-
tions provides relevant biological information
in transfusion medicine that may not be ob-
tainable by nucleic acid analysis.
Fluid-Phase Assays (Agglutination-
Based Methods)
Depending on antibody isotype, immunoglob-
ulins have 2 to 10 antigen-binding sites per
molecule. Each antibody can bind more than
one target molecule, allowing antibodies to
cross-link antigens present in multiple copies
on particles, such as red cells or beads. This
cross-linking can aggregate particles that have
the relevant antigen on their surface, a process
known as “agglutination.” Agglutination is an
old, but generally reliable, serologic method
for detecting antibody-antigen interactions
and is used extensively in transfusion medi-
cine.
Agglutination can be detected by several
methods. The antigen copy number and den-
sity varies depending on the blood group. Ag-
glutination is used for serological crossmatch-
ing (donor red cells incubated with recipient
plasma or serum), screening for unexpected
antibodies (reagent red cells of known blood
group antigen composition incubated with
patient plasma or serum), and blood group
antigen phenotyping of the donor or recipient
(test red cells incubated with monoclonal anti-
bodies or reagent-quality antisera of known
specificity).
Because red cells are easily visible due to
their red color, several systems were devised to
detect agglutination. These include 1) tube
testing in which agglutination is visually de-
tected by the adhesion of red cells to one an-
other in the postcentrifuge pellet, 2) passive
agglutination in microtiter plates where agglu-
tination is visualized by the spread pattern of
red cells in individual wells, and 3) gel testing
in which unagglutinated red cells pass through
a matrix but agglutinated complexes are re-
䡲 241
tained at the top or within the matrix due to
their greater size.20 In addition to blood bank
serology, red cells can be used to detect anti-
bodies against other antigens by linking these
particular antigens to the red cell surface. Ag-
glutination-based assays can also be engi-
neered using particles other than red cells,
such as latex beads. Each of these techniques
uses the same basic principles and has broad
applicability in clinical diagnostic testing.
Although agglutination reactions are very
sensitive and easy to perform, the formation of
agglutinates depends on the proper stoichio-
metric ratio of antibody to antigen. When the
ratio of antigen to antibody is in a range such
that agglutination readily occurs, it is referred
to as the “zone of equivalence.” In this situa-
tion, each arm of the antibody binds to a dif-
ferent particle, and a network (or lattice) of
linked particles results in agglutination [Fig
10-6(B)]. A false-negative result is generated if
the stoichiometric ratio is outside the zone of
equivalence at either extreme.
A prozone effect can occur when such a
high concentration of antibody is present that
the likelihood of an antibody being able to
bind to two separate particles or red cells is di-
minished [Fig 10-6(A)]. This is seen most com-
monly with non-red-cell-based agglutination
assays, such as the rapid plasma reagin screen-
ing test for syphilis serology. Although prozone
effects are unusual in classical red cell serolo-
gy, they have been observed when titers of red
cell antibodies are very high. In particular, dis-
crepant reverse ABO typing due to prozone ef-
fects has been reported.21,22 Diluting the se-
rum being tested and using EDTA containing
diluents decreases the likelihood of prozone
effects. One reason why prozone effects are
uncommon in red cell serology is that the use
of antihuman globulin (AHG) largely over-
comes these problems. However, a secondary
“prozone-like” effect occurs if there is inade-
quate washing before adding AHG.23 In this
case, residual immune globulin G (IgG) in so-
lution binds the AHG and competes for AHG
binding to IgG on the red cells. This may occur
with very high titers of blood group antibodies
or even in the presence of highly increased lev-
els of polyclonal antibodies, as in hypergam-
242 䡲 AABB TECHNICAL MANUAL
FIGURE 10-6. Effects of relative concentrations of antigen and antibody on the outcome of agglutination
reactions.
maglobulinemia. In this setting, additional
washing steps remove the problem.23
In theory, false-negative agglutination re-
actions can occur due to a postzone effect in
which the antigen is in excess [Fig 10-6(C)]. In
this situation, every antibody binds to multi-
ple epitopes on the same particle, thereby pre-
venting the cross-linking that leads to aggluti-
nation. This could occur if a large excess of red
cells were used. This problem is easily con-
trolled by careful attention to methods and is
not common in transfusion medicine.
Solid-Phase Assays
Various solid-phase assays exist that utilize the
same general principle. As opposed to fluid-
phase assays, where the reaction occurs in a
solution or suspension, the antigen or anti-
body being studied in solid-phase assays is im-
mobilized on a solid matrix. The analyte is
then incubated with the coated solid phase,
and adherence of the analyte to the solid
phase is measured. Several combinations of
adherence and detection approaches have
been described.
Solid-Phase Red Cell Adherence
Techniques
SOLID-PHASE ASSAYS FOR PHENOT YPING
RE D CELLS. Antibodies specific for known
blood group antigens are coated onto round-
bottom microtiter plates [Fig 10-7(A)]. Cells
being analyzed are added to the microtiter
plate wells and allowed to adhere. If no bind-
ing occurs (negative reaction), the red cells all
cluster together as a “button” at the bottom of
the well. In contrast, specific binding results in
diffusion of the red cells over the surface of the
CHAPTER 10 Molecular Biology and Immunology in Transfusion Medicine
FIGURE 10-7. Schematic representation of (A) phenotyping red cells and (B) detecting antibodies by solid-
phase assay.
well (positive reaction). A positive reaction in-
dicates the presence of the antigen on the red
cells being tested.
S O L I D - PH A S E A S S AY S F O R D E T E C T I N G
A N T I B O D I E S TO R E D C E L L A N T I G E N S . An-
tigen-coated particles, consisting of red cells
or red cell fragments, are coated onto microti-
ter plate wells [Fig 10-7(B)]. Patient serum is
then added to each well, followed by incuba-
tion and then washing. If the patient serum
has antibodies against the red cell antigens
coated on the well, then the antibodies bind to
the red cells or their fragments. Indicator red
cells coated with antihuman IgG are then add-
ed. A positive reaction is demonstrated by dif-
fuse adherence of the indicator red cell to the
well, whereas a negative reaction is demon-
strated by clustering of indicator cells in a but-
ton.
Solid-Phase Assays for Platelet Testing
Solid-phase red cell adherence (SPRCA) tech-
nology has also been adapted to detect anti-
gens on platelets, such as HPA-1a, as well as
䡲 243
antibodies against platelet antigens using the
approaches described above.24
Enzyme-Linked Immunosorbent Assay
Enzyme-linked immunosorbent assay (ELISA),
also referred to as “enzyme immunoassay,”
can detect antibodies and antigens. A signal is
generated via an enzyme linked to a secondary
antibody or antigen that converts a substrate
to a measurable product (eg, a color change or
a chemiluminescent reaction). For this reason,
ELISAs have considerable signal amplification
and are significantly more sensitive than fluid-
phase agglutination or SPRCAs. In most cases,
ELISAs use purified or recombinant antigens
or antibodies, depending on the analyte de-
tected. However, intact red cells can be used to
screen for red cell antibodies, referred to as the
“enzyme-linked antiglobulin test.”25
DETECTION OF ANTI BODIES BY ELISA (IN-
DIRECT ELISA). To detect antibodies against
a given antigen, the antigen is coated onto mi-
crotiter plate wells [Fig 10-8(A)]. The test sam-
ple is then added, incubated, and washed. If
244 䡲 AABB TECHNICAL MANUAL
FIGURE 10-8. Schematic representation of (A) indirect enzyme-linked immunosorbent assay (ELISA), (B)
sandwich ELISA, and (C) competitive ELISA.
antibodies against the antigen are present,
they bind to the antigen-coated well and the
bound antibodies are detected by incubating
with anti-Ig (eg, anti-IgG) linked to an enzyme
(eg, alkaline phosphatase or horseradish per-
oxidase). After further washing, enzyme sub-
strate is added and is converted to a detectable
color if enzyme is present. Quantification is
performed using a standard curve and a spec-
trophotometer to measure the absorbance at
the wavelength appropriate for that enzyme/
substrate product. In some cases, samples
may need to be diluted to ensure that they
yield absorbance values in the linear range of
the assay.
DETECTION OF ANTI GENS BY SANDWICH
ELISA. In sandwich ELISA, two separate anti-
bodies are used that bind different epitopes on
the same target antigen; the antibodies bind
the target without interfering with each other.
Typically, this is accomplished by monoclonal
antibodies. Microtiter plate wells are coated
with one antibody, the “capture antibody” [Fig
10-8(B)]. The specimen is then incubated in
the well; if the antigen is present, it binds to
the solid-phase antibody coated in the well.
The plate is then washed and incubated with
the second antibody, which is linked to a re-
porter enzyme (the “conjugate”). Because the
second antibody is specific for the target anti-
gen, it binds to the well only if antigen was
bound to the capture antibody. After addition-
al washing, enzyme substrate is added and is
converted to a detectable color if enzyme is
present.
DETECTION OF ANTIGENS BY COMPETI-
TIVE ELISA. Competitive ELISA is similar to
indirect ELISA in that the target antigen is
CHAPTER 10 Molecular Biology and Immunology in Transfusion Medicine
bound to the microtiter plate well. The test
sample is incubated with antibody specific for
the target antigen and this mixture is added to
the well [Fig 10-8(C)]. If no antigen is in the
specimen, then the reagent antibodies bind to
the solid-phase antigen. However, if antigen is
present in the specimen, it combines with the
reagent antibodies and prevents them from
binding to the solid-phase antigen. Therefore,
as the amount of soluble antigen in the speci-
men increases, the amount of reagent anti-
body that is free to bind to the solid-phase an-
tigen on the well decreases. Similarly, as the
signal weakens, the amount of soluble antigen
in the specimen rises.
Competitive ELISA is also used for anti-
body detection. In this case, the test sample
along with a labeled reagent antibody is added
to an antigen-coated well. Both patient anti-
body and labeled reagent antibody compete
for antigen-binding sites in the well. Again, a
higher signal is generated if the antibody level
in the sample is low or absent. Although more
difficult to optimize, competitive ELISAs have
an advantage over sandwich ELISAs in not re-
quiring two separate antibodies against differ-
ent epitopes on the target antigen.
TE CHNICAL PROBLEMS WITH ELISA S. Typ-
ically, ELISAs are straightforward and robust.
Although low signals are possible due to
enzymatic inhibitors in the sample and false-
positive signals can occur due to enzymatic
activity, proper controls and washing prevent
these problems. Problems also occur if the
amount of the antigen being measured ex-
ceeds the amount of antibody present. This
phenomenon, called the “hook effect,” leads
to underestimates of the concentration of the
antigen. Like the prozone effect (see “Fluid-
Phase Assays” section above), excess amounts
of the analyte can cause the signal to decrease
in some sandwich ELISAs in which the analyte
and detection antibody are added simultane-
ously. Hook effects can be overcome by dilut-
ing the analyte. Finally, some patients have hu-
man antimouse antibodies that cross-link the
capture antibody and the detection antibody
in sandwich ELISAs, resulting in very high sig-
nals for essentially all analytes.
䡲 245
Protein Microarrays
Microarray technology has dramatically in-
creased the number of substrates that can be
simultaneously assayed by solid-phase meth-
ods. By spotting numerous different protein
substances on a small chip, a single specimen
can be assayed for binding activity to multiple
(in some cases thousands) substances simul-
taneously. For example, patient serum can be
tested for antibodies to blood-group antigens
by making a microarray chip with different
blood-group antigens and then incubating it
with patient serum.
Although microarray approaches are very
promising, like ELISA, they require that the an-
tigens being tested be spotted on a chip in a
pure form that maintains the structural con-
formation required for recognition. In the cas-
es of carbohydrate antigens or linear protein
epitopes, this is readily achieved. However,
with multipass transmembrane proteins that
require membrane insertion for proper con-
formation (eg, the Rh antigens), it is consider-
ably more difficult. The application of protein
microarrays to blood-bank serology is still in
early stages of development, and it remains
unclear to what extent it will mature into a
useful diagnostic platform.
Western Blotting
The ELISA techniques described above can be
highly sensitive. However, because the anti-
gens used may not be pure (eg, lysates of tissue
culture grown viruses), false-positive results
are possible due to cross-reactivity with other
components in the antigen preparation. West-
ern blotting is conceptually similar to ELISA,
except that instead of coating a well with the
antigen, the antigen mixture (eg, viral lysate) is
first separated by high-resolution protein elec-
trophoresis (typically using polyacrylamide
gels). The separated proteins are then trans-
ferred to nitrocellulose paper (or another suit-
able medium), which serves as the solid phase
to be probed with an antibody-containing pa-
tient sample. In this case, one can determine
the molecular weight of the antigens recog-
nized by the antibodies.
246 䡲 AABB TECHNICAL MANUAL
Although not often used clinically, other
methods can be used to separate antigens
based on physical properties other than size.
For example, isoelectric focusing separates
proteins of different charges over a pH gradi-
ent. Because the likelihood of having a cross-
reactive antigen with the same physical char-
acteristics as the authentic antigen is small,
Western blotting provides more specificity
than ELISA. Thus, Western blots have been
used as confirmatory tests for serologic
screening assays to detect infectious agents,
such as HIV. Similar techniques were devel-
oped using recombinant, or otherwise puri-
fied, proteins for detecting other infectious
disease agents, such as hepatitis C virus.
Flow Cytometry
Flow cytometry revolutionized the analysis of
cell populations. The basic principle is that an-
tibodies against cell-surface molecules are la-
beled with fluorescent tags. Cells are incubat-
ed with the antibodies, and these “stained”
cells are then passed through a flow cytometer.
The individual cells are exposed to lasers that
excite the fluorochromes, causing fluorescent
emissions detected by sensors in the flow cy-
tometer. The amount of fluorescence is deter-
mined on a cell-by-cell basis, allowing both the
quantification of the number of cell-surface
molecules and the visualization of small popu-
lations of cells in a complex mixture.
Clinical flow cytometry has been applied
primarily to the diagnosis of neoplasia, partic-
ularly for hematologic malignancies. Howev-
er, flow cytometry can also be applied to red
cell serology. For example, red cells can be
phenotyped using labeled antibodies of
known specificity. Alternatively, antibody
screens or crossmatching can be performed by
mixing red cells of known phenotype with pa-
tient antisera and then staining the cells with a
secondary antihuman Ig (eg, anti-IgG) that is
coupled to a fluorescent molecule. However,
flow cytometry has yet to be widely applied to
human transfusion medicine and is currently
utilized mostly in experimental systems.
BASIC IMMUNOLO GY
The process by which the immune system
generates antibodies against foreign antigens
and yet maintains tolerance to self-antigens is
complicated and elegant, with multiple cellu-
lar players and intricate regulation. The mech-
anistic details of this process fall outside the
scope of this chapter but can be found else-
where. This section covers antibody structure,
function, and role in transfusion complica-
tions.
Antibody Structure
At its simplest, an antibody is a tetramer of two
identical heavy chains and two identical light
chains (Fig 10-9). Each heavy and light chain
contains a variable region, the part of the mol-
ecule that varies from antibody to antibody
and binds antigen, and a constant region. Two
light-chain families (kappa and lambda) are
found in humans, and any given antibody has
either two identical kappa or two identical
lambda light chains. Likewise, the two heavy
chains in an antibody molecule are identical
and differ depending on isotype.
Immunoglobulins treated with the en-
zyme papain can be digested into two func-
tional fragments. The Fab fragment consists of
the heavy- and light-chain variable regions,
the light-chain constant region, and one
heavy-chain constant region domain. The Fab
fragment binds antigen but does not activate
effector mechanisms. In contrast, the Fc frag-
ment, consisting only of heavy-chain constant
regions, activates effector mechanisms, allow-
ing destruction of the antibody target. Fc con-
stant regions differ based on antibody isotype
and subclass.
There are five different antibody isotypes—
IgM, IgG, IgE, IgA, and IgD—determined by
the constant region of the heavy chain. Anti-
body isotypes can differ in the number of anti-
gen-binding sites per molecule and the poten-
cy of their effector functions [Fig 10-10(A)].
The number of antigen-binding sites per anti-
body affects binding avidity for antigen. For
example, IgM is expressed early in an immune
response before the onset of affinity matura-
tion. However, IgM has high avidity because it
CHAPTER 10 Molecular Biology and Immunology in Transfusion Medicine 䡲 247

FIGURE 10-9. General structure of a monomeric immunoglobulin.

FIGURE 10-10. Immune globulin (Ig) isotypes (A), IgG subclasses, and their relative activation of complement
and binding to Fc-gamma receptors (FcRs) (B).
248 䡲 AABB TECHNICAL MANUAL

consists of five Ig molecules held together by IgE antibodies bind to Fc receptors on

an additional protein (the J chain) and exten- mast cells and induce histamine release when
sive disulfide binding, resulting in 10 antigen- they encounter antigen; thus, IgE antibodies
binding sites. The high avidity of IgM compen- are the predominant cause of allergic and ana-
sates for its typically low affinity for antigen. phylactic responses (ie, Type I hypersensitivi-
Treatment with dithiothreitol (DTT) can de- ty). IgD primarily remains membrane bound
stroy IgM binding because it reduces disulfide on the B-cell surface, with only minimal levels
bonds, and DTT treatment is used to distin- in serum, and its functions remain unclear.
guish IgM from IgG antibodies in the laborato-
ry. IgM potently activates complement by The Role of Fc Receptors in Target
changing its three-dimensional structure after Destruction
antigen binding. Although an IgM-specific Fc
The Fc regions of antigen-bound IgGs are rec-
receptor has long been suspected and was re-
ognized by the gamma family of Fc receptors
cently cloned, its function is not yet fully un-
(FcRs). At least four FcRs have been de-
derstood. In general, IgM (and some IgG) is
scribed to date, each with subtly different
known to cause hemolysis during transfusion
properties that can have opposite functions.
reactions and autoimmune hemolytic anemia.
For example, FcR2a and FcR3 promote
IgG antibodies are important in mature
phagocytosis of targets. Due to the relatively
humoral immune effector function and are di-
low affinity of these receptors, monomeric IgG
vided into four subclasses: IgG1, IgG2, IgG3,
does not engage FcR2a and FcR3, making
and IgG4. Each subclass has a different con-
them specific for targets bound by multiple
stant region and a different capacity to activate
antibodies. In contrast, FcR2b is an inhibitory
complement and/or interact with Fc recep-
receptor that prevents phagocytosis. FcR1 has
tors on phagocytes [Fig 10-10(B)]. IgG1 and
an unusually high affinity and binds mono-
IgG3 are generally the most potent in these re-
meric IgG. The result is that FcR1 binds IgG
gards, IgG2 only weakly activates complement,
whether or not it is bound to a target; the func-
and IgG4 largely lacks effector activity. Consis-
tion of this activity is currently unclear.
tent with these observations, patients with iso-
Thus, FcR biology is complicated by the
lated IgG4 subclass red cell autoantibodies
fact that any given IgG-bound cell or particle
typically do not exhibit hemolysis. In contrast,
may simultaneously activate multiple, poten-
IgG1, IgG2, and IgG3 red cell antibodies can
tially antagonistic, receptors. This is further
induce hemolysis.
complicated by the fact that each of the four
IgA is the primary antibody isotype se-
different IgG subclasses (IgG1, IgG2, IgG3, and
creted at mucosal surfaces; therefore, it is
IgG4) has a different affinity for the different
largely responsible for neutralizing pathogens
FcRs (see Fig 10-11). A mixture of IgG1-IgG4
encountered in the gastrointestinal, genitouri-
may bind a particle or cell bearing a foreign
nary, and respiratory tracts. Although IgA ex-
antigen, and the net effect on enhancing or in-
ists in either monomeric or dimeric forms (a
hibiting phagocytosis will depend on the rela-
dimer is shown in Fig 10-10), it is often mono-
tive binding of different IgG subclasses and in-
meric in serum. IgA is further divided into IgA1
teractions with different FcRs. Thus, direct
and IgA2 subclasses (not shown). In the IgA di-
binding of Fc domains to FcRs can promote
meric form, IgA monomers are connected by
red cell clearance in many cases but does not
the J chain, similar to IgM. In rare instances,
always do so.
IgA red cell antibodies cause hemolysis. Most
antiglobulin (ie, Coombs) reagents do not typi-
The Role of Complement in Target Cell
cally detect IgA; thus, the potential presence of
Destruction and Opsonization
IgA red cell antibodies must be considered
when analyzing a patient with hemolysis and In addition to serving as ligands for FcRs, the
negative direct antiglobulin test-results. Fc regions of IgG antibodies can activate com-
CHAPTER 10 Molecular Biology and Immunology in Transfusion Medicine 䡲 249

FIGURE 10-11. Mechanisms of red cell destruction by antibody binding. Upon binding (A), an immune globulin
G (IgG) represents a ligand for Fc-gamma receptors (FcRs) on phagocytes (B). If the red cell avoids FcR-
mediated phagocytosis, opsonization may be increased by activation of complement with deposition of C3b (C).
if the combined opsonization of FcR binding and C3b is not sufficient to mediate clearance, completion of the
complement cascade may lead to insertion of the membrane attack complex (MAC) into the red cell surface,
resulting in lysis (D). In reality, these processes likely occur simultaneously, with the ultimate outcome being
the aggregate effect of competing pathways.
CR = complement receptor.

plement. The complement system consists of
a cascade of proteases that, once activated,
amplifies the initial signal, leading to the pro-
duction of a large number of effector mole-
cules. Although there are several complement-
activation pathways, this discussion focuses
on the “classical pathway” initiated by Fc re-
gions.
IgM is highly efficient in activating com-
plement. However, to avoid indiscriminant ac-
tivation, IgM undergoes a conformational shift
after binding antigen, thereby exposing com-
plement-binding sites in the heavy-chain con-
stant region. This interaction is so potent that,
in theory, a single antigen-bound IgM is suffi-
cient to lyse a target. In contrast, IgG does not
require a conformational change to bind com-
plement, but complement activation requires
clustered binding of multiple IgG molecules to
the same target. This prevents indiscriminate
activation of complement by unbound circu-
lating IgG.
Once activated, the complement system
provides at least two distinct mechanisms of
target destruction. The first involves target op-
sonization by complement components. Dur-
ing early events in complement activation, C3
covalently attaches to the antigen surface by
thioester bonds, providing multiple copies of
C3b, which are recognized by the complement
receptor 1 (CR1) and CRIg on phagocytes.
When a phagocyte encounters a C3b-coated
molecule, it ingests and destroys it. C3b also
rapidly degrades sequentially into iC3b, C3c,
and C3dg. Because C3dg is recognized by CR2,
which is not on phagocytes, complement can
degrade past the point of promoting phagocy-
tosis. In the second mechanism, downstream
of C3 activation, the cascade assembles the
membrane attack complex (MAC). The MAC
consists of complement proteins C5b-C9 ar-
ranged into a structure resembling a hollow
tube inserted into the membrane of the target
cell. This nonselective channel between the
250 䡲 AABB TECHNICAL MANUAL
inside of a target cell and its external environ-
ment results in osmotic lysis of the target (if it
is sensitive to osmotic shock).
Specific Outcomes of MAC Assembly,
C3 Opsonization, and Fc Opsonization
of Antibody-Coated Red Cells
The effector mechanisms induced by antibody
binding have different effects on bacteria, vi-
ruses, particles, and various human tissues. In
general, once an IgG antibody binds to a red
cell, the target cell may undergo FcR-mediated
phagocytosis by phagocytes (Fig 10-11). If the
antibody initiates the complement cascade,
C3b deposition on the red cell surface contrib-
utes to opsonization, leading to phagocytosis
mediated by CR1 and CRIg. Finally, if comple-
ment activation is complete, MAC insertion
causes red cell lysis. The relative contributions
of each pathway vary based on the relative
amounts of antibody isotype and subclass and
on the nature of the antigen (eg, antigen densi-
ty or linkage to cytoskeleton). The sections be-
low describe what is known about these pro-
cesses with regard to red cell destruction and
the clinical manifestations of hemolysis.
Extravascular Hemolysis
Consumption of antibody- and/or C3b-bound
red cells by phagocytes in the reticuloendothe-
lial system (RES; predominantly in the spleen
and liver) is referred to as “extravascular” he-
molysis because the red cells are destroyed
outside of their normal compartment, the in-
travascular space. This process is also com-
monly referred to as a “delayed hemolytic
transfusion reaction” (DHTR) because it often
occurs days after transfusion in contrast to
“intravascular” hemolysis (see below), which
is recognized acutely during the transfusion
[eg, an acute hemolytic transfusion reaction
(AHTR)]. The delayed kinetics of DHTRs are
due to the milder clinical manifestations of ex-
travascular hemolysis and/or the need for the
implicated antibodies to develop or increase
their titer before the onset of significant red
cell destruction.
The term “hemolysis” in this context can
cause confusion for health-care providers not
accustomed to blood bank terminology be-
cause they typically think of hemolysis as the
rupture of red cells within the circulation (ie,
intravascular hemolysis). In contrast, in extra-
vascular hemolysis, the red cells hemolyze in
the digestive compartment (ie, lysosomes) of
phagocytes. This is a very important distinc-
tion because phagocytes in the RES consume a
substantial number of senescent, autologous
red cells each day in the normal process of red
cell turnover. Thus, red cell consumption in
this fashion uses a pathway that evolved spe-
cifically to break down and recycle red cell
contents (eg, hemoglobin and iron) in a man-
ner that avoids tissue damage.
This does not mean, however, that extra-
vascular removal of antibody-coated red cells
is biologically equivalent to clearance of nor-
mal, senescent red cells. On the contrary,
DHTRs can cause substantial morbidity and
occasional mortality. Indeed, in murine mod-
els of incompatible transfusion, rapid clear-
ance of antibody-coated red cells induces
systemic inflammation and cytokine storm.
Nonetheless, extravascular hemolysis is dis-
tinctly different from intravascular hemolysis
in which red cell contents are directly released
into the circulating blood.
It is not clear why some red cell antibod-
ies preferentially promote opsonization and
phagocytosis instead of osmotic lysis by the
MAC. Substantial evidence indicates that the
antibody type and/or the topographical ar-
rangement of the target antigen on the red
cells are important. In addition, although
complement may be activated, the aggregate
opsonization of red cells by C3b and antibody
may result in phagocytosis before MAC-
induced lysis occurs. Consistent with these ex-
planations is the observation that extravascu-
lar hemolysis is typically induced by IgG red
cell antibodies, whereas intravascular hemoly-
sis is typically induced by IgM red cell antibod-
ies. The latter is much more efficient at fixing
complement and promoting MAC formation.
CHAPTER 10 Molecular Biology and Immunology in Transfusion Medicine 䡲 251

Intravascular Hemolysis match-incompatible red cells produce hemo-

lysis. However, somewhat surprisingly, the vast
In some cases of incompatible transfusion, the
majority of red cell antibodies are not hemo-
MAC rapidly assembles and lyses the red cells
lytic. For some blood-group antigens, hemoly-
before C3b and/or IgG opsonization can
sis is never, or only very rarely, observed fol-
induce phagocytosis. Because these red cells
lowing incompatible transfusion (eg, JMH,
lyse while still circulating, this is termed “in-
Chido, and Rodgers antigens). Indeed, approx-
travascular hemolysis.” In addition, because
imately 1% of healthy blood donors have posi-
antibody-mediated intravascular hemolysis
tive direct antiglobulin test results, indicating
occurs at a brisker pace than extravascular he-
that IgG autoantibodies are bound to their
molysis (or, at least, is more quickly noticed
own red cells, yet there is no evidence of he-
due to dramatic signs and symptoms), this he-
molysis in these donors. Even for antigens
molytic transfusion reaction is called “acute.”
known to be involved in antibody-mediated
As discussed above, AHTRs are typically
hemolysis (eg, in the Rh, Kell, Kidd, Duffy, and
caused by IgM antibodies, which efficiently ac-
Ss systems), hemolysis is variable. Indeed, in
tivate complement, leading to rapid forma-
patients mistakenly transfused with ABO-
tion of the MAC. Although an IgM-specific Fc
incompatible Red Blood Cell (RBC) units, no
receptor has been described (ie, FcR), it is
clinically significant hemolysis occurs in 50%
predominantly expressed on nonphagocytic
of cases, even for this robustly hemolytic anti-
lymphocytes; thus, it is unlikely to promote
gen/antibody combination.
phagocytosis of IgM-coated red cells. None-
Several explanations may account for the
theless, complement activation by IgM red cell
lack of hemolysis during incompatible trans-
antibodies can produce opsonization by C3b,
fusions. For antigens that are essentially never
leading to CR1- and CRIg-mediated phagocy-
involved in hemolysis, the antigen density or
tosis. Taken together, then, it is not surprising
surface topography may prevent hemolysis.
that IgMs predominantly induce intravascular
For antigens that are variably involved in he-
hemolysis.
molysis, idiosyncrasies in a given recipient’s
Intravascular hemolysis, unlike extravas-
antibodies (ie, titer, affinity, isotype, or IgG
cular hemolysis, does not normally occur at
subclass) may play a role. Based on these
any appreciable level. The release of red cell
properties, different antibodies (of the same
contents directly into the circulation can be
antigenic specificity) may have different ca-
highly toxic, with free hemoglobin inducing
pacities for activating complement. This is the
perhaps the greatest insult. Although much
rationale for including the anti-C3 compo-
free hemoglobin is scavenged by haptoglobin,
nent in the antiglobulin (Coombs) reagent: it
this system is easily overwhelmed. AHTRs of-
provides information on whether an antibody
ten result in tea-colored urine (ie, hemoglo-
can fix complement. A number of different ge-
binuria) and can induce renal dysfunction.
netic polymorphisms and/or deficiencies may
Moreover, the signs and symptoms of AHTRs
also regulate hemolysis vs red cell survival on a
can be very dramatic, including disseminated
patient-by-patient basis, including: perturba-
intravascular coagulation, shock, and death.
tions in complement, complement-regulatory
This type of reaction most often occurs as a
proteins, and allelic polymorphisms in FcRs.
result of a clerical error that leads to an ABO-
Thus, in some cases, regulation of hemolysis
incompatible transfusion, and many current
may be independent of the nature of the anti-
practices have evolved to prevent ABO-incom-
body.
patible AHTRs.
From a practical standpoint, crossmatch-
incompatible RBCs may be issued for transfu-
Nonhemolytic Red Cell Antibodies
sion if the offending entity is a “clinically insig-
Given the redundant pathways leading to the nificant antibody,” especially if the antigen is
destruction of antibody-coated red cells, it is of very high frequency and antigen-negative
not surprising that transfusions of cross- blood is difficult or impossible to obtain. The
252 䡲 AABB TECHNICAL MANUAL

blood bank must be prepared to address ap- Summary of Efferent Immunity

propriate concerns from health-care provid-
ers managing these patients. Also from a prac-
tical standpoint, although clinically significant
antibodies may have variable hemolysis in dif-
ferent patients, there are only very limited
means of predicting whether hemolysis will
occur in a given recipient. Thus, transfusion of
crossmatch-incompatible RBC units should
not be issued for clinically significant antibod-
ies unless this lifesaving procedure is specifi-
cally requested by the managing physician. If
compatible RBC units are unavailable, it might
be determined that hemolysis occurring over
several days (eg, in the case of a DHTR) is less
dangerous than the consequences of the pa-
tient’s severe anemia. Transfusion with a unit
that is antigen matched to the patient for com-
mon, clinically significant blood-group anti-
gens should be considered. Close and frequent
communication between managing physi-
cians and the blood bank is required in these
cases.
In aggregate, when an antibody binds a red
cell, multiple pathways are activated that can
lead to cell destruction. Complement activa-
tion promotes phagocytosis through the opso-
nizing properties of C3b and direct red cell
lysis through assembly of the MAC. The pres-
ence of IgG Fc domains promotes red cell
phagocytosis by ligating FcRs on the phago-
cyte surface. The relative contributions of
these different pathways vary depending on
the nature of the target antigen and the prop-
erties of the cognate antibodies. Moreover, the
immune-activation events that occur and the
toxicity of the released RBC units can produce
a scenario in which the negative effects of im-
mune destruction of transfused red cells go far
beyond simply losing the efficacy of the trans-
fused red cells. Indeed, substantial toxicity can
occur, leading to morbidity and, in some cases,
mortality. Should the reader desire more fine
mechanistic details on immunobiology and
the immune response, additional sources are
available.26,27

KEY PO I NT S

1. Hybridization-based methods can be used to detect genes, gene products, and polymor-
phisms. However, compared to amplification-based methods, straight hybridization meth-
ods lack sensitivity.
2. Amplification-based methods (PCR, TMA, NASBA, SDA, and LCR) are highly sensitive but
are also susceptible to problems with contamination and/or inhibitors due to the nature of
geometric amplification involved.
3. The presence of nucleic acids predicts, but does not always equate with, expression of the
corresponding protein or antigen structure.
4. Analysis of protein expression detects the actual gene product(s). Therefore, it does not suf-
fer from the problems of nucleic acid testing, which relies on predicting protein expression.
5. Protein analysis is less sensitive than nucleic acid testing because no amplification is in-
volved, but protein analysis is also less susceptible to contamination and inhibition, which
can lead to false-positive and false-negative results.
6. Methods of detecting protein suffer from nonamplification-based artifacts (ie, heterophilic
antibodies, prozone effects, and hook effects) that can lead to erroneous results.
7. There can be substantial variability in detecting antigens and antibodies based on different
methods.
8. Immunoglobulins can cause destruction of red cells by several different mechanisms, based
largely on the antigen recognized and the antibody structure.
CHAPTER 10 Molecular Biology and Immunology in Transfusion Medicine 䡲 253

9. Most IgG antibodies that cause red cell destruction induce extravascular hemolysis by pro-
moting consumption of red cells by phagocytes (through Fc receptors and/or complement-
based opsonization). This typically presents as a delayed hemolytic transfusion reaction.
10. IgM antibodies (and in some rare cases IgG) that cause red cell destruction typically induce
intravascular hemolysis through complement activation to the membrane attack complex.
This typically presents as an acute hemolytic transfusion reaction.
11. Despite the serious nature of hemolysis due to incompatible transfusion, many antibodies
to red cell antigens are clinically insignificant and do not result in destruction of red cells.
Incompatibility is best avoided whenever possible, but when compatible blood is not avail-
able, incompatible RBC units can be used when the antigens are known to be clinically in-
significant. Such maneuvers must be carefully considered on a-case-by-case basis and with
extensive communication with the clinicians who are requesting the blood products.

REF EREN CE S

1. Alberts B, Bray D, Lewis J, et al. Molecular biol-
ogy of the cell. 3rd ed. New York: Garland Sci-
ence, 1994:98-105.
2. Southern EM. Detection of specific sequences
among DNA fragments separated by gel elec-
trophoresis. J Mol Biol 1975;98:503-17.
3. Alwine JC, Kemp DJ, Stark GR. Method for de-
tection of specific RNAs in agarose gels by
transfer to diazobenzyloxymethyl-paper and
hybridization with DNA probes. Proc Natl
Acad Sci U S A 1977;74:5350-4.
4. Melton DA, Krieg PA, Rebagliati MR, et al. Effi-
cient in vitro synthesis of biologically active
RNA and RNA hybridization probes from plas-
mids containing a bacteriophage SP6 promot-
er. Nucleic Acids Res 1984;12:7035-56.
5. Berk AJ, Sharp PA. Sizing and mapping of early
adenovirus mRNAs by gel electrophoresis of
S1 endonuclease-digested hybrids. Cell 1977;
12:721-32.
6. Mullis KB, Faloona FA. Specific synthesis of
DNA in vitro via a polymerase-catalyzed chain
reaction. Methods Enzymol 1987;155:335-50.
7. Masukawa A, Miyachi H, Ohshima T, et al.
[Monitoring of inhibitors of the polymerase
chain reaction for the detection of hepatitis C
virus using the positive internal control].
Rinsho Byori Jap 1997;45:673-8.
8. Al-Soud WA, Radstrom P. Purification and
characterization of PCR-inhibitory compo-
nents in blood cells. J Clin Microbiol 2001;39:
485-93.
9. Pang J, Modlin J, Yolken R. Use of modified nu-
cleotides and uracil-DNA glycosylase (UNG)
for the control of contamination in the PCR-
based amplification of RNA. Mol Cell Probes
1992;6:251-6.
10. Temin HM, Mizutani S. RNA-dependent DNA
polymerase in virions of Rous sarcoma virus.
Nature 1970;226:1211-13.
11. Baltimore D. RNA-dependent DNA poly-
merase in virions of RNA tumour viruses. Na-
ture 1970;226:1209-11.
12. Compton J. Nucleic acid sequence-based am-
plification. Nature 1991;350:91-2.
13. Kwoh DY, Davis GR, Whitfield KM, et al. Tran-
scription-based amplification system and de-
tection of amplified human immunodeficien-
cy virus type 1 with a bead-based sandwich
hybridization format. Proc Natl Acad Sci U S A
1989;86:1173-7.
14. Walker GT, Fraiser MS, Schram JL, et al. Strand
displacement amplification—an isothermal,
in vitro DNA amplification technique. Nucleic
Acids Res 1992;20:1691-6.
15. Walker GT, Little MC, Nadeau JG, Shank DD.
Isothermal in vitro amplification of DNA by a
restriction enzyme/DNA polymerase system.
Proc Natl Acad Sci U S A 1992;89:392-6.
16. Wu DY, Wallace RB. The ligation amplification
reaction (LAR)—amplification of specific DNA
sequences using sequential rounds of tem-
plate-dependent ligation. Genomics 1989;4:
560-9.
17. Denomme GA, Van Oene M. High-throughput
multiplex single-nucleotide polymorphism
analysis for red cell and platelet antigen geno-
types. Transfusion 2005;45:660-6.
18. Bugert P, McBride S, Smith G, et al. Microarray-
based genotyping for blood groups: Compari-
son of gene array and 5'-nuclease assay tech-
niques with human platelet antigen as a mod-
el. Transfusion 2005;45:654-9.
254 䡲 AABB TECHNICAL MANUAL
19. Hashmi G, Shariff T, Seul M, et al. A flexible ar-
ray format for large-scale, rapid blood group
DNA typing. Transfusion 2005;45:680-8.
20. Langston MM, Procter JL, Cipolone KM, Stron-
cek DF. Evaluation of the gel system for ABO
grouping and D typing. Transfusion 1999;39:
300-5.
21. Voak D. Observations on the rare phenome-
non of anti-A prozone and the non-specific
blocking of haemagglutination due to C1 com-
plement fixation by IgG anti-A antibodies. Vox
Sang 1972;22:408-19.
22. Judd WJ, Steiner EA, O’Donnell DB, Oberman
HA. Discrepancies in reverse ABO typing due
to prozone. How safe is the immediate-spin
crossmatch? Transfusion 1988;28:334-8.
23. Salama A, Mueller-Eckhardt C. Elimination of
the prozone effect in the antiglobulin reaction
by a simple modification. Vox Sang 1982;42:
157-9.
24. Procter JL, Vigue F, Alegre E, et al. Rapid
screening of platelet donors for PIA1 (HPA-1a)
alloantigen using a solid-phase microplate im-
munoassay. Immunohematology 1998;14:141-
5.
25. Leikola J, Perkins HA. Enzyme-linked antiglob-
ulin test: An accurate and simple method to
quantify red cell antibodies. Transfusion 1980;
20:138-44.
26. Kindt TJ, Osborne BA, Goldsby RA. Kuby im-
munology. 6th ed. New York: WH Freeman and
Company, 2007.
27. Murphy K, Travers P, Walport M. Janeway’s im-
munobiology. 7th ed. New York: Garland Sci-
ence, 2008.
 C h a p t e r
Blood Group Genetics
Christine Lomas-Francis, MSc, FIBMS
T H E S C I E N C E O F genetics is the
study of heredity—that is, the mecha-
nisms by which particular characteristics are
passed from parents to offspring. This chapter
describes the genetics of blood groups. The
term “blood group” can be applied to any de-
tectable, variable characteristic of a compo-
nent of the blood including platelet and white
cell groups, serum groups, red cell enzymes,
and hemoglobin variants. In this chapter, the
term “blood group” applies primarily to anti-
gens on the surface of the red cell membrane
that are defined serologically by an antibody.
Platelet and white cell blood groups are dis-
cussed in Chapter 18.
That blood groups are inherited charac-
teristics was first shown by von Dungern and
Hirszfeld in 1910, 10 years after Landsteiner’s
discovery of the ABO blood group. Blood
groups became an ideal tool for geneticists be-
cause they could be identified by specific anti-
bodies in simple hemagglutination tests and,
once identified, their inheritance could easily
be followed in family studies. Red cell antigens
were (and still are) valuable as markers (de-
tectable characteristics to recognize a gene’s
presence) in genetic and anthropologic stud-
ies as well as in relationship testing.
Christine Lomas-Francis, MSc, FIBMS, Technical Director, Laboratory of Immunohematology and Genomics,
New York Blood Center, New York, New York
The author has disclosed no conflicts of interest.
1 1
The detection of inherited differences on
the red cells from different people is the basis
of safe blood transfusion. Therefore, an under-
standing of the principles of human genetics
(including the patterns of inheritance and the
language or terminology in use) is an impor-
tant aspect of immunohematology and trans-
fusion medicine. This chapter outlines the
fundamental principles of genetics as they ap-
ply to blood group antigens and relates them
to examples relevant to transfusion medicine.
This requires the use of numerous genetic
terms; each term, when first used or when fully
described, will be in bold text and usually is
closely followed by a definition.
Technical advances in genetics and mo-
lecular biology have ushered in the age of mo-
lecular genetics, when genes are routinely se-
quenced and inheritance and disease are
studied at the nucleic acid level.1,2 These ad-
vances have provided an understanding of the
regulatory elements and genes that control the
expression of blood groups so that the pres-
ence or absence of blood groups can be pre-
dicted through DNA-based analysis3 with the
potential to revolutionize transfusion medi-
cine and the care of patients. Knowledge of the
fundamental principles of classical genetics
255
256 䡲 AABB TECHNICAL MANUAL
aids understanding of the molecular aspects of
individual blood groups.
BASIC PRINCIPLES OF
GENE T IC S
Gregor Mendel established the basic tech-
niques of genetic analysis when, in 1865, he
published his classic breeding experiments
with pea plants. Mendel’s observations led him
to conclude that there is a “factor” or unit of
inheritance, now known as a gene, that is
passed from one generation to another ac-
cording to two simple rules: the principles of
independent segregation and independent as-
sortment (see “Inheritance of Genetic Traits”
section below).
Cytology studies, toward the end of the
19th century showed that each living cell has a
characteristic set of chromosomes in the nu-
cleus. In the early part of the 20th century, it
was realized that chromosomes carry genes.
Biochemical studies showed that the chromo-
somal material is primarily made up of nucleic
acids and associated proteins.1,2
Many excellent texts offer a greater in-
sight into classical genetics.4 The fundamental
principles of genetics outlined in this chapter
are intended to serve as a review of inheritance
and expression of blood group antigens.
Genes (Alleles) and Chromosomes
A gene is a segment of deoxyribonucleic acid
(DNA) that encodes a particular protein. A
gene is the basic unit of inheritance of any
trait (defined as a genetically determined
characteristic or condition), including blood
group antigens, that is passed from parents to
offspring. Genes are arranged on chromo-
somes with each gene occupying a specific lo-
cation known as the gene locus. A locus may
be occupied by one of several alternative
forms of the gene called alleles. For example,
the gene that encodes the protein carrying the
Jka antigen is an alternative form (allele) of the
one that encodes the Jkb antigen. The terms
“gene” and “allele” can be used interchange-
ably. Based on internationally accepted gene
and allele terminology, the written gene or al-
lele name is italicized—for example, RHD for
the gene encoding the RhD protein. The name
usually is not italicized when the word “gene”
or “allele” follows the name: for example,
“RHD gene,” or “RHD allele.” The Internation-
al Society of Blood Transfusion (ISBT) Working
Party on Red Cell Immunogenetics and Blood
Group Terminology5,6 has developed allele ter-
minology for use in transfusion medicine. For
alleles encoding polymorphic common anti-
gens, the name is based on the ISBT antigen
name: eg, FY*01 or JK*02 refer to the alleles en-
coding the Fya and Jkb antigens, respectively.
Alternatively, where letters are commonly
used, symbols such as FY*A or JK*B are accept-
able. A genotype may also be written as the
italicized antigen, eg, Fya or Jkb when the geno-
type has been inferred from testing by hemag-
glutination.
Chromosomes are the gene-carrying
structures that are visible during nuclear divi-
sion in the nucleus of the cell; they contain the
genetic material (DNA) necessary to maintain
the life of the cell and the organism. A human
somatic cell contains 46 chromosomes that
make up 23 pairs; each pair has one paternally
and one maternally derived chromosome. In
males and females, 22 of the pairs are homolo-
gous chromosomes (a pair of chromosomes in
which males and females carry equivalent
genes) and are referred to as the autosomes
(any chromosome that is not a sex chromo-
some). The remaining pair is nonhomologous
and consists of the sex chromosomes that de-
termine a person’s sex (gender). The sex-deter-
mining chromosomes of the male are X and Y,
whereas females have two X chromosomes.
The karyotype represents the chromosome
complement of a person; this is written as
“46,XY” and “46,XX” for a normal male and fe-
male, respectively. The hereditary information
carried by the chromosomes is passed from a
parent cell to a daughter cell during somatic
cell division and from parents to offspring
(children) by the gametes during reproduc-
tion.
Chromosomes are best studied during
cell division (mitosis; see “Mitosis” section be-
low) when they become discrete structures in
the nucleus and can be visualized by various
 CHAPTER 11 Blood Group Genetics 䡲 257

microscopic techniques. All chromosomes

have some common morphologic features but
differ in other characteristics, including size,
location of the centromere, and staining prop-
erties. Each chromosome has two sections or
arms that are joined at a central constriction
called the centromere (Figs 11-1 and 11-2).
Chromosomes are distinguished by their
length and the position of the centromere.
These characteristics serve as the basis for
numbering the autosomes 1 through 22 such
that chromosome 1 is the largest and chromo-
some 22 is the smallest. An internationally rec-
ognized terminology is used to describe chro-
mosomes. The chromosomal arms are of
different length, although the difference be-
tween the arms of chromosome 1 is not obvi-
ous (Fig 11-2). The “p” (or petite) arm is the
shorter and, in diagrams, is at the top of the
chromosome. The longer arm is termed the
“q” arm. Thus, the short arm of chromosome 1
is referred to as “1p,” and the long arm of chro-
mosome 4 is “4q.” The terminal portion of a
chromosome is referred to as “ter”; “pter” and
“qter” indicate the terminus of the short (p)
and long (q) arms, respectively.
Staining techniques provide a more de-
tailed means to distinguish individual chro-
mosomes. Selected dyes do not stain chromo-
somes uniformly and, depending on the dye FIGURE 11-2. Morphology and banding pattern of
used, different banding patterns are obtained a Giemsa-stained human chromosome 1. The
so that each human chromosome has a locations of the genes controlling the expression of
unique banding pattern. As shown in Fig 11-2, antigens for the Rh (RH), Scianna (SC), Duffy (FY),
Knops (KN), and Cromer (CROM) blood group
systems are shown.

Giemsa staining reveals a specific pattern of

FIGURE 11-1. Diagram of a metaphase chromosome.
At the metaphase stage of the cell cycle, the
chromosomes have condensed and become visible
by light microscopy. As shown in the diagram,
metaphase chromosomes have replicated in
preparation for cell division so that each chromosome
consists of two sister chromatids connected by the
centromere. The telomere is the end or terminal part
of a chromosome.
dark (G) and light (reverse or R) bands. Quina-
crine, used to stain chromosomal preparations
for fluorescence microscopy, results in fluores-
cent bands equivalent to the dark G bands
seen in light microscopy. The G bands are het-
erochromatin (condensed DNA) and the light-
er bands are euchromatin, which is involved in
the transcription of DNA to mRNA. The bands
are numbered from the centromere outward.
Using chromosome 1 as an example, the re-
gion closest to the centromere on the short or
258 䡲 AABB TECHNICAL MANUAL
long arm is numbered 1p1 or 1q1, respectively.
With greater resolution, there is further
distinction into subbands (eg, 1p11 and 1p12)
that, in turn, can be subdivided (eg, 1p11.1
and 1p11.2). Genes can be individually
mapped to a specific band location (Fig 11-2),
and the chromosomal location of the genes
encoding the 34 red cell systems5-9 are shown
in Table 11-1.
Cell Division
As a cell divides, the chromosomes replicate
and each daughter cell receives a full comple-
ment of genetic material. In somatic cells, this
occurs through mitosis; in reproductive cells a
similar process called “meiosis” takes place. A
feature common to both types of cell division
is that, before the start of the process, each
chromosome replicates to form two identical
daughter chromatids attached to each other
through the centromere (Fig 11-1).
Mitosis
Somatic cells divide for growth and repair by
mitosis (Fig 11-3). Through this process, a sin-
gle cell gives rise to two daughter cells with
identical sets of chromosomes. The daughter
cells, like the parent cell, are diploid (2N); that
is, they contain 46 chromosomes in 23 pairs
and have all the genetic information of the
parent cell.
Meiosis
Meiosis occurs only in germ cells that are in-
tended to become gametes (sperm and egg
cells). Somatic cells are diploid (2N), whereas
gametes are haploid [having half the chromo-
somal complement of somatic cells (1N)]. Mei-
osis is a process of cell division and replication
that leads to the formation of haploid gametes.
During meiosis, diploid cells undergo DNA
replication, followed by two cycles of cell divi-
sion to produce four haploid gametes (see Fig
11-4). Because sperm and egg cells fuse at fer-
tilization, the gametes must be haploid. If each
gamete carried a diploid (2N) set of 46 chro-
mosomes, the resulting zygote would have 92
chromosomes, which would be incompatible
with life.
Meiosis ensures genetic diversity through
two mechanisms: independent assortment
and crossing-over. Through independent as-
sortment, each daughter cell randomly re-
ceives either maternally or paternally derived
homologous chromosomes. Chromosomal
crossing-over involves exchange of genetic
material between homologous chromosome
pairs. Such shuffling of genetic material en-
sures diversity and produces genetically
unique gametes that fuse to produce a unique
zygote.
X Chromosome Inactivation
(Lyonization)
Females have two copies of X-borne genes in
their somatic cells, while males have only one
copy of X-borne genes. Because most X-borne
genes do not have a homolog on the Y chro-
mosome, there is a potential imbalance in the
dosage of X-borne genes between males and
females. This difference is compensated for by
X chromosome inactivation, (also called ly-
onization), a process through which most of
the genes on one of the two X chromosomes in
each female somatic cell are inactivated at a
very early stage of embryonic development.17
It is a matter of chance whether the maternal
or paternal X chromosome is inactivated in
any one cell, but once inactivation has oc-
curred, all descendants of that cell will have
the same inactive X chromosome. Some X-
borne genes escape inactivation; the first gene
found to escape inactivation was XG, the gene
encoding the antigens of the Xg blood group
system. Like XG, most of the genes that escape
inactivation are located on the extreme tip of
the short arm of the X chromosome, but sever-
al are clustered in regions on the short and
long arms of the chromosome.18(pp359-370),19
The XK gene, which encodes the Kx blood
group system, is the only other X-borne gene
known to encode red cell antigens. Changes or
deletions in XK result in McLeod phenotype
red cells that lack Kx antigen and have reduced
expression of Kell antigens.20,21 The XK gene,
unlike XG, is subject to X chromosome inacti-
 CHAPTER 11 Blood Group Genetics 䡲 259

TABLE 11-1. Blood Group Systems

Gene Product and Associated Blood

ISBT System Gene Name ISBT Chromosome Component Name Group Antigens
Name (Number) (HGNC)* Location [CD Number] [Null Phenotype]

ABO ABO 9q34.2 Glycosyltransferase, A; B; A,B; A1

(001) (ABO) carbohydrate [Group O]
MNS MNS 4q31.21 Glycophorin A (GPA) M, N, S, s, U, He, Mia,
(002) (GYPA [CD235a] Vw, and 38 more
[En(a–); U–; MkMk]
GYPB Glycophorin B (GPB)
GYPE) [CD235b]
P1PK P1 22q13.2 Galactosyltransferase, P1, Pk, NOR
(003) (A4GALT) carbohydrate
Rh RH 1p36.11 RhD [CD240D] D, G, Tar
(004) (RHD RhCE [CD240CE] C, E, c, e, V, Rh17,
RHCE) and 45 more
[Rhnull]
Lutheran (005) LU 19q13.32 Lutheran glycoprotein; Lua, Lub, Lu3, Lu4, Aua,
(LU) B-cell adhesion mole- Aub, and 14 more
cule [CD239] [Recessive Lu(a–b–)]
Kell KEL 7q34 Kell glycoprotein K, k, Kpa, Kpb, Ku, Jsa,
(006) (KEL) [CD238] Jsb, and 28 more
[K0 or Knull]
Lewis LE 19p13.3 Fucosyltransferase, Lea, Leb, Leab, Lebh,
(007) (FUT3) carbohydrate ALeb, BLeb
(Adsorbed from [Le(a–b–)]
plasma)
Duffy FY 1q23.2 Duffy glycoprotein Fya, Fyb, Fy3, Fy5, Fy6
(008) (DARC) [CD234] [Fy(a–b–)]
Kidd JK (SLC14A1) 18q12.h3 Human urea trans- Jka, Jkb, Jk3
(009) porter (HUT) [Jk(a–b–)]
Kidd glycoprotein
Diego DI 17q21.31 Band 3, Anion Dia, Dib, Wra, Wrb, Wda,
(010) (SLC4A1) Exchanger 1 Rba, and 16 more
[CD233]
Yt YT 7q22.1 Acetylcholinesterase Yta, Ytb
(011) (ACHE)
Xg XG (XG) Xp22.33 Xga glycoprotein Xga
(012) (MIC2) Yp11.2 CD99 (MIC2 product) CD99
Scianna (013) SC 1p34.2 Erythroid membrane- Sc1, Sc2, Sc3, Rd, and
(ERMAP) associated protein 3 more
(ERMAP) [Sc:–1,–2,–3]

(Continued)
260 䡲 AABB TECHNICAL MANUAL

TABLE 11-1. Blood Group Systems (Continued)

Gene Product and Associated Blood

ISBT System Gene Name ISBT Chromosome Component Name Group Antigens
Name (Number) (HGNC)* Location [CD Number] [Null Phenotype]

Dombrock (014) DO 12p12.3 Do glycoprotein; ART 4 Doa, Dob, Gya, Hy, Joa +
(ART4) [CD297] 3 more
[Gy(a–)]
Colton CO 7p14.3 Aquaporin 1 (AQP1) Coa, Cob, Co3, Co4
(015) (AQP1) [Co(a–b–)]
Landsteiner- LW 19p13.2 LW glycoprotein Intra- LWa, LWab, LWb
Wiener (ICAM4) cellular adhesion mole- [LW(a–b–)]
(016) cule 4 (ICAM4)
[CD242]
Chido/ Rodgers CH/RG (C4A, 6p21.32 Complement compo- Ch1, Ch2, Rg1 + 6
(017) C4B) nent: more
C4A; C4B [Ch–Rg–]
H H 19q13.33 Fucosyltransferase, H
(018) (FUT1) carbohydrate [CD173] [Bombay (Oh)]
Kx XK Xp21.1 XK glycoprotein Kx
(019) (XK) [McLeod phenotype]
Gerbich (020) GE 2q14.3 Glycophorin C (GPC) Ge2, Ge3, Ge4, and 8
(GYPC) [CD236] more
Glycophorin D (GPD) [Leach phenotype]
Cromer (021) CROM 1q32.2 DAF Cra, Tca, Tcb, Tcc, Dra,
(CD55) [CD55] Esa, IFC, and 11 more
[Inab phenotype]
Knops KN 1q32.2 CR1 Kna, Knb, McCa, Sla, Yka,
(022) (CR1) [CD35] and 4 more
Indian IN 11p13 Hermes antigen [CD44] Ina, Inb, and 2 more
(023) (CD44)
Ok OK 19p13.3 Neurothelin, basigin Oka, OKGV, OKGM
(024) (BSG) [CD147]
Raph RAPH (CD151) 11p15.5 Tetraspanin MER2
(025) [CD151] [Raph–]
JMH JMH 15q24.1 Semaphorin 7A JMH and 5 more
(026) (SEMA7A) [CD108] [JMH–]
I GCNT2 6p24.2 Glucosaminyltransfer- I
(027) (IGNT) ase, carbohydrate [I– or i adult]
Globoside (028) GLOB 3q26.1 Transferase, carbohy- P
(B3GALNT1) drate [P–]
(Gb4, globoside)
 CHAPTER 11 Blood Group Genetics 䡲 261

TABLE 11-1. Blood Group Systems (Continued)

Gene Product and Associated Blood

ISBT System Gene Name ISBT Chromosome Component Name Group Antigens
Name (Number) (HGNC)* Location [CD Number] [Null Phenotype]

Gill GIL 9p13.3 Aquaporin 3 (AQP3) GIL

(029) (AQP3) [GIL–]
Rh-associated RHAG 6p21.3 Rh-associated glyco- Duclos, Ola, DSLK†,
glycoprotein protein RHAG4
(030) [CD241] [Rhnull (regulator type)]
Forssman10 FORS 9q34.2 Globoside 3--N- FORS1
(031) (GBGT1) acetylgalactosaminyl-
transferase 1
Forssman glycolipid
JR11,12 JR 4q22.1 Jr glycoprotein Jra
(032) (ABCG2) ATP-binding cassette, [Jr(a−)]
sub-family G, member
2 (ABCG2) [CD338]
Lan13 LAN 2q36 Lan glycoprotein Lan
(033) (ABCB6) ATP-binding cassette, [Lan−]
sub-family B, member
6 (ABCB6)
Vel14-16 VEL 1p36 Small integral mem- Vel
(SMIM1) brane protein 1 [Vel−]
(SMIM1)
*If the genetic information is obtained by blood group typing, the gene name is the italicized form of the of the blood group
system ISBT name. For example, SLC14A1 would be written as JK*A and JK*B or Jka and Jkb.
†
Provisionally numbered by the ISBT because of limited genetic evidence.
ISBT = International Society of Blood Transfusion; HGNC = Human Gene Nomenclature Committee; ATP = adenosine tri-
phosphate.

vation, with the result that a female who is a
carrier (a person who carries one gene for a re-
cessive trait and one normal gene) of a gene
that is responsible for the McLeod phenotype
can have a dual population of Kx– (McLeod
phenotype) and Kx+ (non-McLeod) red cells.
Flow cytometry, using selected Kell antibodies,
shows the weakening of Kell antigens on red
cells of the McLeod phenotype and demon-
strates two red cell populations in carrier fe-
males. This mixed-cell population reflects the
randomness of whether the maternal or pater-
nal X chromosome is inactivated in any single
somatic cell lineage.
Genotype and Phenotype
The genotype of a person is the complement
of genes inherited from his or her parents; the
term is frequently also used to refer to the set
of alleles at a single gene locus. The phenotype
is the observable expression of the genes in-
herited by a person and reflects the biologic
activity of the gene(s). Thus, the presence or
absence of antigens on the red cells, as deter-
mined by serologic testing, represents the phe-
notype. The presence or absence of antigens
on the red cells predicted by DNA-based test-
ing represents the genotype. Sometimes the
262 䡲 AABB TECHNICAL MANUAL
FIGURE 11-3. Diagram showing mitosis.
genotype can be predicted from the pheno-
type; for example, when a person’s red cells are
reactive with anti-Jka and anti-Jkb, which is a
Jk(a+b+) phenotype, a JK*A/JK*B genotype can
be inferred. Frequently, the phenotype pro-
vides only a partial indication of the genotype;
for example, red cells that are group B reflect
the presence of a B gene, but the genotype may
be B/B or B/O. For decades, family studies were
often the only way to determine a person’s
genotype, but now that most antigens and
phenotypes can be defined at the DNA level,
family studies to determine genotype can be
mostly replaced by DNA analysis (see “Blood
Group Genomics” section below).
Alleles
A gene at a given locus on a chromosome may
exist in more than one form, that is, be allelic.
Each person has two alleles for a trait, one that
is maternally derived and another that is pa-
ternally derived. At the simplest level, the ABO
gene locus can be considered to have three al-
leles: A, B, and O. With three alleles, there are
six possible genotypes (A/A, A/O, A/B, B/B, B/O,
and O/O). Depending on the parental contri-
bution, a person could inherit any combina-
tion of two of the alleles and express the corre-
sponding antigens on their red cells. For
example, inheritance of A/A and A/O would
 CHAPTER 11
FIGURE 11-4. Diagram showing meiosis.
result in group A red cells, A/B would result in
group AB red cells, B/B and B/O would result in
group B red cells, and O/O would result in
group O red cells.
When identical alleles for a given locus
are present on both chromosomes, the person
is said to be “homozygous” for the particular
allele. A person who is hemizygous for an al-
lele has only a single copy of an allele instead
of the customary two copies; an example is the
deletion of one RHD in a D-positive pheno-
type. When different (ie, not identical) alleles
are present at a particular locus, the person is
Blood Group Genetics 䡲 263
said to be “heterozygous.” For example, a per-
son with K–k+ red cells is homozygous at the
KEL locus for the allele (KEL*02) encoding the
k antigen. A person who is heterozygous for
KEL*01 and KEL*02 (KEL*01/02 genotype),
would have red cells that are K+k+.
Antigens that are encoded by alleles at the
same locus are said to be “antithetical” (mean-
ing “opposite”); thus, K and k are a pair of anti-
thetical antigens. It is incorrect to refer to red
cells that are K–k+ or Kp(a–b+) as being homo-
zygous for the k or Kpb antigen; rather, it
should be said that the cells have a double
264 䡲 AABB TECHNICAL MANUAL
dose of the antigen and that they are from a
person who is homozygous for the allele.
Genes are allelic whereas antigens are anti-
thetical.
The quantity of antigen expressed (anti-
gen density) is influenced by whether a per-
son is heterozygous or homozygous for an al-
lele; the antigen density is generally greater
when a person is homozygous. In some blood
group systems, this difference in antigen den-
sity is manifested by antibodies giving stron-
ger reactions with cells that have a double
dose of the antigen. Red cells with the Jk(a+b–)
phenotype, encoded by a JK*A/A genotype,
have a double dose of the Jka antigen and often
are more strongly reactive with anti-Jka than
those that are Jk(a+b+) and have a single dose
of the antigen. Similarly, M+N– red cells tend
to be more strongly reactive with anti-M than
are M+N+ red cells. Antibodies that are weakly
reactive may not be detected if they are tested
with red cells expressing a single dose of the
antigen. This observable difference in strength
of reaction, based on homozygosity or hetero-
zygosity for an allele, is termed the “dosage ef-
fect.”
Polymorphism
For blood group genetics, polymorphism re-
fers to the occurrence in the population of ge-
nomes with allelic variation (two or more
alleles at one locus) producing different phe-
notypes, each with appreciable (greater than
1%) frequency. Some blood group systems (Rh
and MNS for example) are highly polymorphic
and have many more alleles at a given locus
than other systems such as Duffy, and Colton.5
An allele that is polymorphic in one popula-
tion is not necessarily polymorphic in all pop-
ulations; for example, the FY allele associated
with silencing of Fyb in red cells (FY*02N.01) is
polymorphic in populations of African ethnici-
ty with a prevalence of greater than 70%, but
this allele is not found in other populations. A
gene polymorphism may represent an evolu-
tionary advantage for a population, and a
polymorphic population is likely to adapt to
evolutionary change more rapidly than if the
population had genetic uniformity. It is not yet
understood what, if any, evolutionary advan-
tages were derived from the extensive poly-
morphism displayed by red cell antigens, but
many publications associate resistance to, or
susceptibility for, a particular disease with a
particular blood type.22
The difference between two alleles is the
result of a permanent change in the DNA. An
event that leads to the production of an altered
gene and a new allele or polymorphism that
did not exist in the biologic parents is referred
to as a “mutation.” The mutation rate of ex-
pressed genes resulting in a new phenotype
has been estimated to be less than 10-5 (<1 in
100,000) in humans, and a mutation has to oc-
cur in the germ cells (gametes) to be inherited.
A mutation can occur spontaneously or
be brought about by agents such as radiation
(eg, ultraviolet rays or x-rays) or chemicals. A
mutation may occur within a gene or in the
intergenic regions. It may be silent—that is,
have no effect on the encoded protein—or it
may alter the gene product and potentially
cause an observable effect in the phenotype.
In the context of an allele that encodes a pro-
tein that carries red cell antigens, any geneti-
cally induced change must be recognized by a
specific antibody before an allele can be said
to encode a new antigen.
Numerous genetic events that generate
red cell antigens and phenotypes have been
identified. The event can occur at the level of
the chromosome (eg, deletion or transloca-
tion of part of a chromosome), the gene (eg,
deletion, conversion, or rearrangement), the
exon (eg, deletion or duplication), or the nu-
cleotide (eg, deletion, substitution, or inser-
tion). The mechanism that has given rise to
most diversity in the human genome is single
nucleotide polymorphism (SNP)—that is, a
single nucleotide change in the DNA.23-25 Ac-
cordingly, the majority of polymorphic blood
group antigens are the result of SNPs.3,26,27 DNA
analysis to predict the red cell phenotype is
discussed in more detail in the section on
“Blood Group Genomics.”
 CHAPTER 11
INHERITANCE OF GENETIC
TR AI TS
A genetic trait is the observed expression of
one or more genes. The inheritance of a trait
(and red cell antigens) is determined by
whether the gene responsible is located on an
autosome or on the X chromosome (sex-
linked) and whether the trait is dominant or
recessive.
Pedigrees
A family study follows the inheritance of a ge-
netic characteristic—for example, an allele en-
coding the expression of a red cell antigen—as
it is transmitted through a kinship. A diagram
that depicts the relationship of family mem-
bers and shows which family members express
(are affected), or do not express, the trait un-
der study is termed a pedigree. A review of a
pedigree should reveal the pattern or type of
inheritance for the trait, or antigen, of interest.
The person who first caused the family to be
investigated is considered the index case and
is often referred to as the proband or proposi-
tus/proposita (male singular form or gender
unknown/female singular form); propositi is
the plural form regardless of gender. Details of
the conventions and the symbols used for the
construction of pedigrees are provided in Figs
11-5 and 11-6.
Autosomal Dominant Inheritance
An antigen (or any trait) that is inherited in an
autosomal dominant manner is always ex-
pressed when the relevant allele is present, re-
gardless of whether a person is homozygous or
heterozygous for the allele. The antigen ap-
pears in every generation and occurs with
equal frequency in both males and females. A
person who carries an autosomal dominant
trait, on average, transmits it to half of his or
her children. The pedigree in Fig 11-7 demon-
strates autosomal dominant inheritance and
shows that the B allele is dominant over O.
Blood Group Genetics 䡲 265
FIGURE 11-5. An example of a pedigree. Males are
denoted by squares and females by circles, and each
different generation in a pedigree is identified by
Roman numerals. Persons in each generation are
identified by Arabic numerals; the numbering is
sequential from left to right, with the eldest child for
each family unit being placed on the left of any series
of siblings. Closed symbols represent family
members affected by the trait, whereas open
symbols are unaffected members.
Autosomal Codominant Inheritance
Blood group antigens that appear to be auto-
somal dominant may be encoded by alleles
that are inherited in a codominant manner—
that is, when two different alleles are present
(the heterozygous condition), the products of
both alleles are expressed. Thus, when red
cells have the S+s+ phenotype, the presence of
one allele encoding S and another allele en-
coding s [or an S/s (GYPB*S/s) genotype] can be
inferred.
Autosomal Recessive Inheritance
A trait with autosomal recessive inheritance is
expressed only in a person who is homozygous
for the allele and has inherited the recessive al-
lele from both parents. When a person inherits
a single copy of a recessive allele in combina-
tion with a silent or deleted (null) allele—that
is, a nonfunctioning allele or one that encodes
a product that cannot be detected—the reces-
sive trait is expressed and the person appears
to be homozygous. It is difficult or impossible
266 䡲 AABB TECHNICAL MANUAL

to distinguish such a combination from homo-

FIGURE 11-6. Symbols, and their significance, used
in the construction of pedigrees.
zygosity for the recessive allele through sero-
logic testing, but DNA-based testing can usu-
ally make this distinction.
A mating between two heterozygous car-
riers results in one in four of the children being
homozygous for the trait. The parents of a
child who is homozygous for a recessive trait
must be obligate carriers of the trait. If the fre-
quency of the recessive allele is low, the condi-
tion is rare and usually found only among sib-
lings (brothers and sisters) of the person and
not in other relatives. The condition is not
found in preceding or successive generations
unless consanguineous mating (ie, between
blood relatives) occurs. When a recessive allele
is rare, the parents of an affected person are
most likely consanguineous because a rare al-
lele is more likely to occur in blood relatives
than in unrelated persons in a random popu-
lation. When a recessive trait is one that is
common, consanguinity is not a prerequisite
for homozygosity; for example, the O allele of
the ABO system, although recessive, is not
rare, and persons who are homozygous for O
are easily found in the random population.

FIGURE 11-7. Autosomal dominant inheritance of the ABO alleles. Based on the ABO groups of his children, I-1
would be expected to have a B/O rather than a B/B genotype (showing that the B allele is dominant over O)
because two of his children (II-6 and II-7) are group O and must have inherited an O allele from their father (I-1)
in addition to the O allele inherited from their mother (I-2). Similarly, II-2 and II-3 are B/O, based on the ABO
type of their children, showing the dominance of B over O.
 CHAPTER 11 Blood Group Genetics 䡲 267

In blood group genetics, a recessive trait Sex-Linked Inheritance

or condition almost always means that red
cells express a null phenotype [eg, the Lu(a–b–)
or Rhnull or O phenotypes] because of homozy-
gosity for a “silent” or “amorphic gene” that
either results in no product or encodes a de-
fective product. The family in Fig 11-8 demon-
strates the inheritance of a recessive silent LU
gene, which in the homozygous state results in
the Lu(a–b–) phenotype. The proband, II-3,
who was multitransfused, was identified be-
cause of the presence of anti-Lu3 (an antibody
to a high-prevalence Lutheran antigen) in his
plasma. Because his Lu(a–b–) phenotype is the
result of recessive inheritance, any potential
donors in the family can be found by testing
his siblings. About 25%, or one in four of the
offspring of the mating between I-1 and I-2, is
expected to have the Lu(a–b–) phenotype; in
this case, only the proband has Lu(a–b–) red
cells.
A sex-linked trait is one that is encoded by a
gene located on the X or Y chromosome. The Y
chromosome carries few functional genes and
discussion of sex-linked inheritance generally
is synonymous with inheritance of X-borne
genes. In females with two X chromosomes the
inheritance of X-borne genes, like the inheri-
tance of genes carried on the autosomes, can
be dominant or recessive. Males, in contrast,
have one X chromosome (always maternally
derived) and one Y chromosome (always pa-
ternally derived) and are hemizygous for
genes on the X or Y chromosome because only
one chromosome (and thus one copy of a
gene) is present. Most X-borne genes do not
have a homolog (a similar sequence of DNA)
on the Y chromosome. As a consequence, in-
heritance of an X-borne dominant trait is the
same in males and females. However, an X-
borne trait that is recessive in females is

FIGURE 11-8. Autosomal recessive inheritance. The offspring of II-3, the Lu(a–b–) proband, and II-4, his
Lu(a+b+) wife, demonstrate that Lu is recessive to Lua and Lub and that the presence of the silent Lu allele is
masked by the product of Lua or Lub at the phenotype level.
268 䡲 AABB TECHNICAL MANUAL

expressed by all males who carry the gene for
the trait. The most striking feature of both
dominant and recessive X-linked inheritance
is that there is no male-to-male transmission;
that is, the trait is never transmitted from fa-
ther to son.
Sex-Linked Dominant Inheritance
A trait encoded by an X-borne allele that has
sex-linked dominant inheritance is expressed
by hemizygous males and by both heterozy-
gous and homozygous females. A male passes
his single X chromosome to all of his daugh-
ters and all daughters will express the condi-
tion or trait. When a female is heterozygous for
an allele that encodes a dominant trait, each of
her children, whether male or female, has a
50% chance of inheriting the trait. When a fe-
male is homozygous for an X-borne allele with
dominant inheritance, the encoded trait is ex-
pressed by all her children.
The Xga antigen (Xg blood group system)
is encoded by an allele on the X chromosome
and is inherited in a sex-linked dominant
manner. The first indication that the Xga anti-
gen is X-borne came from the observation that
the prevalence of the Xg(a–) and Xg(a+) phe-
notypes differed noticeably between males
and females; the Xga antigen has a prevalence
of 89% in females and only 66% in males.5
Figure 11-9 shows the inheritance of the
Xga antigen in a three-generation family. In
generation I, the father (I-1) is Xg(a+) and has
transmitted Xga to all his daughters but to
none of his sons. His eldest daughter (II-2), for
example, must be heterozygous for Xga/Xg; she
received the allele encoding the Xga antigen
from her Xg(a+) father and a silent allele, Xg,
from her Xg(a–) mother. II-2 has transmitted
Xga to half her children, regardless of whether
they are sons or daughters.
Sex-Linked Recessive Inheritance
A trait encoded by an X-borne recessive allele
is carried, but not expressed, by a heterozy-
gous female. A male inherits the trait from his

FIGURE 11-9. Sex-linked dominant inheritance. The Xga antigen is encoded by an allele on the tip of the short
arm of the X chromosome. This family demonstrates the sex-linked dominant inheritance of the Xga antigen.
 CHAPTER 11 Blood Group Genetics 䡲 269

mother, who is usually a carrier (or could be
homozygous for the trait if she is the offspring
of a male who expresses the trait and a carrier
female). An affected male transmits the trait to
all of his daughters, who in turn transmit the
trait to approximately half of their sons. There-
fore, the prevalence of the expression of an X-
borne recessive trait is much higher in males
than in females. A carrier female who mates
with a male lacking the trait transmits the trait
to one-half of her daughters (who will also be
carriers) and to one-half of her sons (who will
be affected). If mating is between an affected
male and a female who lacks the trait, all of the
sons will lack the trait and all of the daughters
will be carriers. If an X-borne recessive trait is
rare in the population, the trait is expressed al-
most exclusively in males.
The XK gene encodes the Kx protein and
demonstrates X-borne recessive inheritance.
Mutations in XK result in red cells with the
McLeod phenotype; such red cells lack Kx and
have reduced expression of Kell antigens
(McLeod syndrome). McLeod syndrome is as-
sociated with late-onset clinical or subclinical
myopathy, neurodegeneration, and central
nervous system manifestations as well as with
acanthocytosis and, frequently, compensated
hemolytic anemia. More than 30 different XK
gene mutations associated with a McLeod
phenotype have been found. Different XK
mutations appear to have different clinical ef-
fects and may account for the variability in the
prognosis.28 Sequencing of XK to determine
the specific type of mutation in individuals
with McLeod phenotypes has clinical prognos-
tic value. McLeod syndrome is an X-linked re-
cessive condition and, as demonstrated by the
family in Fig 11-10, is found only in males.

FIGURE 11-10. Sex-linked recessive inheritance. This family demonstrates that a sex-linked trait that is
recessive in females will be expressed by any male who inherits the trait. Homozygosity for such a trait is
required for it to be expressed in females. The trait skips one generation and is carried through females.
270 䡲 AABB TECHNICAL MANUAL

The Principles of Independent
Segregation and Independent
Assortment
The passing of a trait from one generation to
the next follows certain patterns or principles.
The principle of independent segregation re-
fers to the separation of homologous chromo-
somes and their random distribution to the
gametes during meiosis. Only one member of
an allelic pair is passed on to the next genera-
tion, and each gamete has an equal probability
of receiving either member of a parental ho-
mologous allelic pair; these chromosomes are
randomly united at fertilization and thus seg-
regate independently from one generation to
the next. The family in Fig 11-11 demonstrates
the independent segregation of the ABO alleles
on chromosome 9.
The principle of independent assort-
ment states that alleles determining various
traits are inherited independently from each
other. In other words, the inheritance of one
allele (eg, a B allele, on chromosome 9, encod-
ing B antigens) does not influence the inheri-
tance of another allele (eg, an M allele, on
chromosome 4, encoding M antigens). This is
demonstrated by the family in Fig 11-11.
Linkage and Crossing-Over
Linkage is the physical association between
two genes that are located on the same chro-
mosome and are inherited together. Examples
include RHD and RHCE encoding the antigens
of the Rh system, which are both on chromo-
some 1 and are linked loci that do not assort
independently.
Crossing-over is the exchange of genetic
material between homologous chromosome
pairs (Fig 11-4). In this process, a segment
from one chromatid (and any associated
genes) changes places with the corresponding
part of the other chromatid (and its associated
genes); the segments are rejoined, and some
genes will have switched chromosomes. Thus,
crossing-over is a means to shuffle genetic ma-
terial. Because crossing-over can result in new

FIGURE 11-11. Independent segregation and independent assortment are illustrated by the inheritance of blood
group alleles in one family. Parental ABO alleles were randomly transmitted (independent segregation), and each
child has inherited a different combination. The family also illustrates that the alleles encoding antigens of the
ABO and MNS blood group systems are inherited independently from each other.
 CHAPTER 11 Blood Group Genetics 䡲 271

gene combinations on the chromosomes in-
volved, it is also referred to as recombination,
and the rearranged chromosomes can be re-
ferred to as recombinants. Crossing-over and
recombination, using chromosome 1 as an ex-
ample, are explained in Fig 11-12.
Two gene loci carried by the same chro-
mosome that are not closely linked are re-
ferred to as being syntenic. For example, the
loci for RH and FY, both located on chromo-
some 1, are syntenic because the distance be-
tween them (RH on the short arm and FY on
the long arm) is great enough for them to un-
dergo crossing-over and to assort indepen-
dently.
The frequency of crossing-over involving
two genes on the same chromosome is a mea-
sure of the distance [measured in centimor-
gans (cM)] between them; the greater the dis-
tance between two loci, the greater the
probability that crossing-over (and recombi-
nation) will occur. In contrast, genes located
very close together (linked) tend to be trans-
mitted together with no recombination. The
degree of crossing-over between two genes
can be calculated by analyzing pedigrees of
families informative for the genes of interest
and observing the extent of recombina-
tion.The traditional method of linkage analysis
requires the use of lod (logarithm of the odds)
scores.29 Linkage analysis was the basis
through which chromosomes were mapped
and the relative position and distance between
genes established. Linkage between Lutheran
(LU) and ABH secretion (SE or FUT2) was the
first recognized example of autosomal linkage
in humans and is explained in Fig 11-13.
Although crossing-over occurs readily be-
tween distant genes, rare examples of recom-
bination have been documented for genes that
are very closely linked or adjacent on a chro-
mosome. Such genes include those encoding
the MN (GYPA) and Ss (GYPB) antigens on
chromosome 4 and are reviewed by Dan-
iels.18(pp96-142)
Linkage Disequilibrium
Genes at closely linked loci tend to be inherit-
ed together and constitute a haplotype (a
combination of alleles at two or more closely
linked loci on the same chromosome). The al-
leles encoding the MNS antigens are inherited
as four haplotypes: MS, Ms, NS, or Ns. Because
linked genes do not assort independently, the
antigens encoded by each of these haplotypes
have a different prevalence in the population
than would be expected by random assort-
ment. If M and S were not linked, the expected
prevalence for M+ and S+ in the population
would be 17% (from frequency calculations),
whereas the actual or observed prevalence
(obtained from testing and analyzing families)
of the MS haplotype is 24%.18(pp96-142) This con-
stitutes linkage disequilibrium, which is the
tendency of specific combinations of alleles at
two or more linked loci to be inherited togeth-
er more frequently than would be expected by
chance.

FIGURE 11-12. Crossing-over and recombination. In the diagram, chromosome 1 is used as an example. The
very closely linked RH genes, RHD and RHCE, are located near the tip of the short arm of chromosome 1. The
loci for FY and KN are on the long arm of the chromosome and are not linked. During meiosis, crossing-over
occurs between this homologous chromosome pair, and portions of the chromosome break and become
rejoined to the partner chromosome. Crossing-over of the long arm of chromosome 1 results in recombination
between the loci for FY and KN such that the gene encoding the Fyb antigen now travels with a gene that encodes
the Sl(a–) phenotype of the Knops system.
272 䡲 AABB TECHNICAL MANUAL
FIGURE 11-13. Linkage between LU and SE. I-2 is homozygous for Lub and se and must transmit these alleles to
all her offspring. I-1 is doubly heterozygous (Lua/Lub and Se/se). He has transmitted Lub with Se and Lua with se,
showing linkage between Lu and Se. Several such informative families would need to be analyzed to statistically
confirm linkage.
Gene Interaction and Position Effect
Alleles that are carried on the same chromo-
some are referred to as being in cis position
whereas those on opposite chromosomes of a
homologous pair are in trans position. Alleles
that are in cis and linked are always inherited
together on the same chromosome, whereas
genes in trans segregate independently.
Historically, the Rh blood group system
was used to explain the meaning of cis and
trans. For example, the DCe/DcE genotype was
described as having C and e alleles in cis in the
DCe haplotype, with c and E alleles in cis in the
partner DcE haplotype, whereas C and E and
also c and e are in opposing haplotypes and are
in trans. In this alignment, C and e, for exam-
ple, are always inherited together, but C and E
are not. The preceding explanation, which im-
plies that one gene encodes C and c antigens
while another linked gene encodes E and e an-
tigens, was based on the Fisher-Race theory of
three genes at the RH locus. In contrast, mo-
lecular analysis indicates that only one gene
(RHCE), with four alleles (RHCE*Ce, RHCE*cE,
RHCE*ce, and RHCE*CE), encodes one protein
that carries the CcEe antigens. Thus, for Rh,
DCe is the haplotype, and the RHD allele is in
cis to the RHCE*Ce allele.
The expression of red cell antigens may
be modified or affected by gene or protein
interactions that manifest primarily as re-
duced antigen expression. One example in
which the haplotype on one chromosome af-
fects the expression of the haplotype on the
paired chromosome is commonly referred to
as the “position effect” and can be observed
with Rh antigen expression. When a Ce haplo-
type (note the absence of RHD) is in trans to a
D antigen-encoding haplotype, the expression
of D is dramatically reduced and a weak D
phenotype can result. When the same D-en-
coding haplotype is inherited with either ce or
cE, D antigen is normally expressed. The cause
of this reduced antigen expression is not
known, but may involve differences in gene
expression levels or altered assembly of pro-
teins in the membrane. In the presence of the
Kell system antigen Kpa, expression of other
Kell system antigens encoded by the same al-
lele is suppressed to varying degrees (cis-mod-
ifier effect). This is best observed in persons
who have a silenced K0 (a Kellnull gene) in trans.
The amino acid change that results in the ex-
pression of Kpa adversely affects trafficking of
 CHAPTER 11
the Kell glycoprotein to the red cell surface, so
that the quantity of Kpa-carrying Kell glycopro-
tein that reaches the red cell surface is greatly
reduced.
Suppressor or modifier genes affect the
expression of another gene, or genes. For ex-
ample, KLF1, located on chromosome
19p13.3-p13.12, encodes erythroid Krüppel-
like factor, which is a transcription factor es-
sential for terminal differentiation of red cells.
Singleton et al30 first discovered that heterozy-
gosity for nucleotide changes in KLF1 is re-
sponsible for the dominant Lu(a–b–) pheno-
type,5 which is also known as the In(Lu)
phenotype. This heterozygosity is character-
ized by reduced expression of antigens in the
Lutheran system (Lumod) and for P1, Inb and
AnWj antigens.
In kind, the transcription factor GATA-1,
encoded by the X-borne GATA-1 gene, is es-
sential for erythroid and megakaryocyte differ-
entiation. Changes in this gene were associat-
ed with the X-linked type of Lumod that initially
presented as an Lu(a–b–) phenotype.31 Sero-
logic differentiation of these Lumod phenotypes
from the true Lu(a–b–) (Lunull) phenotype can
be challenging, yet has clinical value. Now that
the molecular basis of these phenotypes is un-
derstood, sequencing of the relevant genes can
be used to make the distinction.
In the past, independent, unidentified
modifier or regulator genes were postulated to
be the basis of several null or variant pheno-
types when a silent or inactive (nonfunction-
ing) gene was not evident. For example, the
regulator type (as opposed to the amorph
type) of Rhnull was shown through family stud-
ies to result from a gene not at the RH locus.
This phenotype is now known to result from
various silencing changes in RHAG, a gene lo-
cated on chromosome 6 (and thus indepen-
dent of RH). RHAG encodes the Rh-associated
glycoprotein RhAG, which is required in the
red cell membrane for Rh antigen expression.
Similarly, molecular analysis indicates that the
modifying gene that causes the Rhmod pheno-
type is a mutated RHAG.5
Several red cell antigens require the inter-
action of the products of two or more indepen-
dent genes for their expression. Assignment of
Blood Group Genetics 䡲 273
the high-prevalence antigen Wrb to the Diego
blood group system took many years because
the only red cells that lacked Wrb were rare
MNS null phenotypes [En(a–) and MkMk] and
variants, yet Wra, the antigen that is antitheti-
cal to Wrb was clearly independent of MNS.
This anomaly was resolved when it was under-
stood that GPA (or more precisely, amino acids
75 to 99), which carries MN antigens, must be
present in the red cell membrane for Wrb ex-
pression. The Wra/Wrb polymorphism is en-
coded by DI and carried on band 3, whereas
GPA is the product of GYPA, a gene that is in-
dependent of DI. An absence of RhD and RhCE
protein (Rhnull) results in red cells that lack LW
antigens and lack or have reduced expression
of U and S or s antigens, again demonstrating
the interaction in the membrane of the prod-
ucts of two or more independent blood group
genes.
The sequential interaction of genes at
several loci is required for the expression of
ABO, H, Lewis, and I antigens on red cells and
in secretions. These antigens are carbohydrate
determinants carried on glycoproteins or gly-
colipids, and the genetics of these antigens is
more complex than that of protein-based anti-
gens. The carbohydrate antigens are carried
on oligosaccharide chains that are assembled
by the stepwise addition of monosaccharides.
ABO genes and the genes encoding the other
carbohydrate-based antigens do not encode
membrane proteins but do encode an enzyme,
a glycosyltransferase, that catalyzes the se-
quential transfer of the appropriate immuno-
dominant monosaccharide. Each monosac-
charide structure is transferred by a separate
glycosyltransferase, such that two genes are re-
quired for a disaccharide, three for a trisaccha-
ride, and so on. An inactivating change at one
locus can prevent or modify the expression of
the other gene products. The product encoded
by the H gene is the biosynthetic precursor for
A and B antigen production; if the H gene is si-
lenced, A or B antigens cannot be produced. A
mutated A or B allele may result in a glycosyl-
transferase that is inactive or that causes more
or less antigen to be expressed. Details on the
biosynthesis of the ABO, H, Lewis, and I anti-
gens may be found in Chapter 12.
274 䡲 AABB TECHNICAL MANUAL
P O P U L AT I O N G E NE T IC S
Population genetics is the study of the distri-
bution patterns of genes and of the factors that
maintain or change gene (or allele) frequen-
cies. A basic understanding of population ge-
netics, probability, and the application of sim-
ple algebraic calculations is important for
relationship (identity) testing. In transfusion
medicine, the knowledge can be applied to
clinical situations such as predicting the likeli-
hood of finding compatible blood for a patient
who has made antibody(ies) to red cell anti-
gens. It may be helpful to define three com-
monly used words so that their appropriate
use is understood. “Frequency” is used to de-
scribe prevalence at the genetic level—that is,
the occurrence of an allele (gene) in a popula-
tion. “Prevalence” is used to describe the oc-
currence of a permanent inherited character-
istic at the phenotypic level—for example, a
blood group—in any given population. “Inci-
dence” is used when describing the rate of oc-
currence in a population of a condition that
changes over time, such as a disease, and is
thus not suitable for use with blood groups.
Phenotype Prevalence
The prevalence of a blood group antigen or
phenotype is determined by testing red cells
from a large random sample of people of the
same race or ethnicity with a specific antibody
and calculating the percentage of positive and
negative reactions. The larger the cohort being
tested, the more statistically significant is the
result. The sum of the percentages for the
prevalence of the phenotypes should equal
100%. For example, in the Duffy blood group
system, the prevalence in a random popula-
tion of African ethnicity for the Fy(a+b–),
Fy(a–b+), Fy(a+b+), and Fy(a–b–) phenotypes
is 9%, 22%, 1%, and 68%, respectively; togeth-
er, these percentages total 100%. If the red cells
from 1000 donors of European ethnicity are
tested with anti-c, and 800 of the samples are
positive and 200 are negative for the Rh anti-
gen c, the prevalence of the c+ phenotype is
80% and that of the c– phenotype is 20%. Thus,
in this donor population, approximately 20%
of ABO-compatible units of blood, or 1 in 5,
should be compatible with serum from a pa-
tient who has made anti-c.
Calculations for Antigen-Negative
Phenotypes
When blood is provided for a patient with anti-
bodies directed at one or more red cell anti-
gens, a simple calculation can be used to esti-
mate the number of units that need to be
tested to find the desired antigen combina-
tion. To calculate the prevalence of the com-
bined antigen-negative phenotype, the preva-
lences of each of the individual antigens are
multiplied together because the antigens are
inherited independently of each other. An ex-
ception occurs when the antigens are encoded
by alleles that are closely linked and are inher-
ited as haplotypes (M, N, S, s) or reside on the
same carrier protein (C, c, E, e). If a patient
with antibodies to K, S, and Jka antigens re-
quires 3 units of blood, for example, the preva-
lence of the antigen-negative phenotype and
the number of units that need to be tested to
find them can be calculated as follows:
䡲 The prevalence of: K– donors = 91%; S– do-
nors = 48%; Jk(a–) donors = 23%
䡲 The percentage of donors negative for each
antigen is expressed as a decimal and mul-
tiplied: 0.91(K–) × 0.48(S–) × 0.23[Jk(a–)] =
0.10
䡲 0.10 expressed as a % = 0.10 × 100% = 10%
䡲 10% expressed as occurrence = 10%/100% =
1/10
䡲 Thus, approximately 1 in 10 ABO-compati-
ble RBC units are expected to be K– S– Jk(a–)
The patient in question requires 3 units,
so on average, 30 units would need to be tested
to fulfill the request. Based on these calcula-
tions, a hospital transfusion service would be
able to determine the likelihood of having the
requested units in house.
The prevalence of a particular antigen (or
phenotype) can vary with race,5 and the preva-
lence for a combined antigen-negative pheno-
type calculation should be selected on the ba-
sis of the predominant race found in the donor
population.
 CHAPTER 11 Blood Group Genetics 䡲 275

Allele (Gene) Frequency
The allele frequency is the proportion of one
allele relative to all alleles at a particular gene
locus in a given population at a given time.
This frequency can be calculated from the
prevalence of each phenotype observed in a
population. The sum of allele frequencies at
any given locus must equal 100% (or 1 in an al-
gebraic calculation) in the population sample
tested. The genotype frequency in a popula-
tion is the number of individuals with a given
genotype divided by the total number of indi-
viduals in population.
The Hardy-Weinberg Equilibrium
Gene frequencies tend to remain constant
from generation to generation in any relatively
large population unless they are influenced by
factors such as selection, mutation, migration,
or nonrandom mating, any of which would
have to be rampant to have a discernible ef-
fect. According to the principles proposed by
the British mathematician Hardy and the Ger-
man physician Weinberg, gene frequencies
reach equilibrium. This equilibrium can be ex-
pressed in algebraic terms by the Hardy-Wein-
berg formula or equation:
p2 + 2pq + q2 = 1
If two alleles, classically referred to as A
and a, have gene frequencies of p and q, the
homozygotes and heterozygotes are present in
the population in the following proportions:
AA = p2 Aa = 2pq aa = q2
In such a two-allele system, if the gene
frequency for one allele, say p, is known, q can
be calculated by p + q = 1.
The Hardy-Weinberg equation permits
the estimation of genotype frequencies from
the phenotype prevalence in a sampled popu-
lation and, reciprocally, allows the determina-
tion of genotype frequency and phenotype
prevalence from the gene frequency. The
equation has a number of applications in
blood group genetics, and its use is demon-
strated below. In a population of European
ethnicity, the frequencies of the two alleles en-
coding K or k can be determined as follows:
Frequency of the K allele = p
Frequency of the k allele = q
Frequency of the KK genotype = p2
Frequency of the Kk genotype = 2pq
Frequency of the kk genotype = q2
The K antigen is expressed on the red cells of
9% of people of European ethnicity; there-
fore:
p2 + 2pq = the frequency of people who carry K
and are K+
Thus, p2 + 2pq = 0.09
q2 = 1 – (p2 + 2pq) = the frequency of people
who carry kk and are K–
q2 = 1 – 0.09
q2 = 0.91
q= 0.91
q = 0.95 = the frequency of k
Because the sum of the frequencies of both al-
leles must equal 1.00:
p+q=1
p=1–q
p = 1 – 0.95
p = 0.05 = the frequency of K
Once the allele frequencies for K and k have
been calculated, it is possible to calculate the
percentage of k+ (both K+k+ and K–k+) and K+
(both K+k– and K+k+) people:
Prevalence of k+ = 2pq + q2
= 2(0.05 × 0.95) + (0.95)2
= 0.9975 × 100
= a calculated preva-
lence of 99.75% (the
observed prevalence
of the k+ phenotype is
99.8%)
276 䡲 AABB TECHNICAL MANUAL

Prevalence of K+ = 2pq + p2 when applied to a two-allele situation, are rel-

= 2(0.05 × 0.95) + (0.05)2
= 0.0975
= 0.0975 × 100 = a calcu-
lated prevalence of K+
of 9.75% (the obser-
ved prevalence of the
K+ phenotype is 9%)
The Hardy-Weinberg equation also can be
used to calculate the frequencies of the three
possible genotypes KK, Kk, and kk from the
gene frequencies K (p) = 0.05 and k (q) = 0.95:
p2 + 2pq + q2 = 1
Frequency of KK = p2 = 0.0025
Frequency of Kk = 2pq = 0.095
Frequency of kk = q2 = 0.9025
If antibodies are available to test for the
products of the alleles of interest (in this exam-
ple, anti-K and anti-k), the allele frequencies
also can be obtained by direct counting as
demonstrated in Table 11-2. The allele fre-
quencies obtained by direct testing are the ob-
served frequencies for the population being
sampled, whereas those obtained by gene fre-
quency calculations (above) are the expected
frequencies. The various calculations above,
atively simple; the calculations for three or
more alleles are much more complex and be-
yond the scope of this chapter.
For a given population, if the prevalence
of one genetic trait, such as a red cell antigen,
is known, the Hardy-Weinberg equation can
be applied to calculate allele and genotype fre-
quencies. The Hardy-Weinberg equilibrium
principle is valid when the population is suffi-
ciently large that chance alone cannot alter an
allele frequency and when the mating is ran-
dom. A lack of selective advantage or disad-
vantage of a particular trait and other influ-
encing factors, such as mutation or migration
in or out of the population, are assumed to be
absent when the Hardy-Weinberg equilibrium
principle is applied. When all of these condi-
tions are met, the gene pool is in equilibrium
and allele frequencies do not change from one
generation to the next. If the conditions are
not met, changes in allele frequencies may oc-
cur over a few generations and may explain
many of the differences in allele frequencies
between populations.
R E L ATI O N S HI P T E ST I NG
Polymorphisms are inherited characteristics
or genetic markers that can distinguish be-
tween people. The blood groups with the

TABLE 11-2. Allele Frequencies of K and k Calculated Using Direct Counting (assuming the absence
of null alleles)

Phenotype No. of Persons No. of Kk Alleles K k

K+k– 2 4 4
K+k+ 88 176 88 88
K–k+ 910 1820 0 1820
Totals 1000 2000 92 1908
Allele frequency 0.046 0.954
A random sample of 1000 people tested for K and k antigens, have a total of 2000 alleles at the Kk locus
because each person inherits two alleles, one from each parent. Therefore, the two persons with a K+k– phe-
notype (each with two alleles) contribute a total of four alleles. To this are added 88 K alleles from the K+k+
group, for a total of 92 K alleles or an allele frequency of 0.046 (92 ÷ 2000). The frequency of the k allele is
0.954 (1908 ÷ 2000).
 CHAPTER 11
greatest number of alleles (greatest polymor-
phism) have the highest power of discrimina-
tion and are the most useful for determining
relationships. Blood is a rich source of inherit-
ed characteristics that can be detected, includ-
ing red cell, HLA, and platelet antigens. Red
cell and HLA antigens are easily identifiable,
are polymorphic, and follow Mendelian laws
of inheritance. The greater the polymorphism
of a system, the less chance there is of finding
two people who are identical. The extensive
polymorphism of the HLA system alone allows
the exclusion of over 90% of falsely accused
men in cases of disputed paternity.
Serologic methods of identity testing have
been surpassed and replaced by DNA-based
assays32 (referred to as DNA fingerprinting,
DNA profiling, or DNA typing) that were pio-
neered by Jeffreys and colleagues.33,34 Tandem-
ly repeated sequences of DNA of varying
lengths occur predominantly in the noncoding
genomic DNA, and they are classified into
groups depending on the size of the repeat re-
gion. The extensive variation of these tandem-
ly repeated sequences between individuals
makes it unlikely for the same number of re-
peats to be shared by two individuals, even if
these individuals are related. Minisatellite
[also referred to as variable number of tandem
repeats (VNTR)] loci have tandem repeat units
of nine to 80 base pairs, whereas microsatellite
[also referred to as short tandem repeat [STR)]
loci consist of two to five base pair tandem re-
peats.35 Microsatellites and minisatellites are
reviewed by Bennett.36
Assays for VNTR and STR sequences in-
volve the electrophoretic separation of DNA
fragments according to size. DNA profiling in-
volves amplification of selected, informative
VNTR and STR loci using locus-specific oligo-
nucleotide primers with the subsequent mea-
surement of the size of the PCR products.
Hundreds of STR loci have been mapped
throughout the human genome, and many
have been applied to identity testing. Analysis
of different STR loci (usually at least 12) is used
to generate a person’s DNA profile that is virtu-
ally guaranteed to be unique to that person (or
to two identical twins). DNA fingerprinting is a
powerful tool not only for identity testing and
Blood Group Genetics 䡲 277
population genetics but also for monitoring
chimerism after marrow transplantation.37,38
STR analysis also has been used to monitor pa-
tients for graft-vs-host disease after organ
transplantation, particularly after a liver trans-
plant.39
In a case of disputed paternity, if an al-
leged father cannot be excluded from paterni-
ty, the probability of his paternity can be
calculated. The calculation compares the
probability that the alleged father transmitted
the paternal obligatory genes with the proba-
bility that any other randomly selected man
from the same racial or ethnic group transmit-
ted the genes. The result is expressed as a like-
lihood ratio (paternity index) or as a percent-
age. The AABB has developed standards and
guidance documents for laboratories that per-
form relationship testing.40
B LO OD G RO U P G E N E M A P PI N G
Gene mapping is the process through which a
gene locus is assigned to a location on a chro-
mosome. The initial mapping of blood group
genes was accomplished by testing many fam-
ilies for selected red cell antigens. Pedigrees
were analyzed for evidence of recombination
between the genes of interest to rule out or es-
tablish linkage of a blood group with another
marker, with a known chromosomal location.
The gene encoding the antigens of the
Duffy blood group system was the first to be
assigned to a chromosome, by showing that
the gene is linked to an inherited deformity of
chromosome 1. More recently, recombinant
DNA methods were used to establish the phys-
ical locations of genes, and today, with se-
quencing of the human genome, determining
the location of a gene involves a computer da-
tabase sequence search. The Human Genome
Project (http://www.ornl.gov/sci/tech resourc
es/Human_Genome/home.shtml) has result-
ed in construction of a physical gene map in-
dicating the position of gene loci and the dis-
tance between loci is expressed by the number
of base pairs of DNA.
Currently, 34 blood group systems are rec-
ognized by the ISBT.6 The genes for all of them
have been cloned and assigned to their respec-
278 䡲 AABB TECHNICAL MANUAL
tive chromosomes (see Table 11-1). Tradition-
ally, genes were mapped to chromosomes ac-
cording to the metaphase banding patterns
produced through staining with Giemsa (G
banding) or quinacrine (Q banding). Other
methods used in chromosome mapping have
included deletion mapping (partial or total
loss of a chromosome related to the presence
or absence of a gene), somatic cell hybridiza-
tion (based on the fact that human-rodent hy-
brid cell lines randomly shed human chromo-
somes, permitting the association of a trait
with the presence or absence of a chromo-
some), in-situ hybridization (use of fluores-
cent DNA probes with a preparation of intact
chromosomes), and chromosome walking (a
technique to clone a gene from its known clos-
est markers). Details on gene mapping proce-
dures are beyond the scope of this chapter, but
reviews are available.7
Advances in genetic technology and in-
formation generated through the Human Ge-
nome Project make it feasible to construct a
physical gene map of the absolute positions of
gene loci in which the distance between loci is
expressed by the number of base pairs of DNA.
CHIMERISM
The observation that a sample gives mixed-
field agglutination is not unusual in a labora-
tory. Often this is the result of artificially in-
duced chimerism through the transfusion of
donor red cells or a stem cell transplant. On
rare occasions, the observation of mixed-field
agglutination identifies a true chimera, that is,
a person with a dual population of cells de-
rived from more than one zygote. Indeed, the
first example of a human chimera was a female
blood donor discovered through mixed-field
agglutination during antigen typing. Most hu-
man chimeras can be classified as either twin
chimeras or tetragametic (dispermic) chime-
ras. Chimerism is not a hereditary condition.41
Twin chimerism occurs through the for-
mation of placental blood vessel anastomo-
ses, which results in the mixing of blood be-
tween two fetuses. This vascular bridge allows
hematopoietic stem cells to migrate to the
marrow of the opposite twin. Each twin may
have two distinct populations of cells (red cells
and leukocytes), that of their true genetic type
and that of their twin. The percentage of the
two cell lines in each twin tends to vary; the
major cell line is not necessarily the autolo-
gous cell line, and the proportions of the two
cell lines may change throughout life. Chime-
ric twins have immune tolerance; they do not
make antibody against the A or B antigens that
are absent from their own red cells but are
present on the cells of the engrafted twin. This
tolerance extends beyond red cells to negative
mixed lymphocyte cultures and the mutual ac-
ceptance of skin grafts.
In twin chimeras, the dual cell popula-
tion is strictly confined to blood cells. Tetraga-
metic or dispermic chimeras present chime-
rism in all tissues and are more frequently
identified because of infertility than because
of mixed populations of red cells. The mecha-
nism(s) leading to the development of tetraga-
metic chimeras are unknown, but they arise
through the fertilization of two maternal nu-
clei by two sperm followed by fusion of the two
zygotes and development into one person
containing two cell lineages.
More commonly, chimeras occur through
medical intervention and arise from the trans-
fer of actively dividing cells, such as through
hematopoietic transplantation.41 However,
chimerism may be more prevalent than once
thought. DNA analysis to confirm the Rh-neg-
ative status of Northern European blood do-
nors identified a donor with a dual red cell
population of which 95% was Rh negative and
5% was Rh positive. The donor was confirmed
to be a chimera. Chimerism was found to be
the cause of a discrepancy observed when
ABO was determined, and news headlines
were made by a case of disputed maternity
when a woman was falsely excluded as the
mother of her children because of chime-
rism.42,43
B LO OD G RO U P T E R M I N OLO G Y
Antigens were originally named using an al-
phabetical (eg, A/B, C/c) notation or they were
named after the proband whose red cells car-
ried the antigen or who made the first known
 CHAPTER 11
antibody (eg, Duclos). A symbol with a super-
script letter (eg, Lua, Lub; Jka, Jkb) was used, and
a numerical (eg, Fy3, Jk3, Rh32) terminology
was introduced. In blood group systems, anti-
gens are named using more than one scheme
(eg, the Kell blood group system: K, k, Jsa, Jsb,
K11, K17, TOU).
In 1980, the ISBT established its Working
Party on Terminology for Red Cell Surface An-
tigens. The working party was charged to de-
velop a uniform nomenclature that would be
“both eye and machine readable” and “in
keeping with the genetic basis of blood
groups.” A blood group system consists of one
or more antigens under the control of a single
gene locus or of two or more homologous
genes that are so closely linked that virtually
no recombination occurs between them.
Thus, each blood group system is genetically
independent from every other blood group
system. The failure of an antibody to be reac-
tive with red cells of a particular null pheno-
type is not sufficient for assignment of the cor-
responding antigen to a system. Some null
phenotypes are the result of inhibitor or modi-
fying genes that may suppress the expression
of antigens from more than one system [eg,
the Rhnull phenotype lacks not only Rh anti-
gens but also LW system antigens, Fy5 antigen
(Duffy system), and sometimes U (MNS sys-
tem) antigen]. Similarly, a blood group antigen
must be shown to be inherited through family
studies, or the expression of the antigen must
be demonstrated to be associated with a varia-
tion in the nucleotide sequence of the gene
controlling the system, to be assigned antigen
status by the ISBT terminology working party.
A blood group antigen must be defined sero-
logically by an antibody; a polymorphism that
is detectable only by DNA analysis and for
which there is no corresponding antibody can-
not be called a blood group antigen.
The working party established a terminol-
ogy consisting of uppercase letters and Arabic
numerals to represent blood group systems
and antigens.6,44 Each system can also be iden-
tified by a set of numbers (eg, ABO system =
001; Rh system = 004). Similarly, each antigen
in the system is assigned a number (eg, A anti-
gen = 001; B antigen = 002; D antigen = 001).
Blood Group Genetics 䡲 279
Thus, 001001 identifies the A antigen and
004001 identifies the D antigen. Alternatively,
the sinistral zeros may be omitted so that the A
antigen becomes 1.1 and the D antigen be-
comes 4.1. Each system also has an alphabeti-
cal abbreviation (Table 11-1); thus KEL is the
ISBT symbol for the Kell system, the Rh ISBT
system symbol is RH, and an alternative name
for the D antigen is RH1. This alphanumeric
terminology, which was designed primarily for
computer use, is not ideal for everyday com-
munication. To achieve uniformity, a recom-
mended list of user-friendly alternative names
was compiled.45
The ISBT working party meets periodical-
ly to assign names and numbers to newly
discovered antigens. For terminology criteria;
tables listing the systems, antigens, and phe-
notypes; and other information see ISBT Red
Cell Immunogenetics and Blood Group Termi-
nology web resources.6 A comprehensive re-
view of the terminology and its usage is found
in Garratty et al.45
The working party is charged to develop,
maintain, and monitor a terminology for
blood group genes and their alleles.6 The ter-
minology takes into account the guidelines for
human gene nomenclature published by the
Human Genome Organization (HUGO), which
is responsible for naming genes based on the
International System for Human Gene No-
menclature.46 For information regarding the
current status of gene and allele terminology
see ISBT Red Cell Immunogenetics and Blood
Group Terminology web resources.6 An exam-
ple of the traditional and current ISBT termi-
nology as it applies to alleles, genotypes, phe-
notypes, and antigens is shown in Table 11-3.
B LO OD G RO U P G E NO M I C S
As discussed in earlier sections of this chapter,
the antigens expressed on red cells are the
products of genes and can be detected directly
by hemagglutination techniques (as long as
relevant antisera are available). Their detec-
tion is an important aspect of the practice of
transfusion medicine because an antigen can,
if it is introduced into the circulation of an
280 䡲 AABB TECHNICAL MANUAL
TABLE 11-3. Example of Allele, Genotype, Phenotype and Antigen Terminology
Duffy System Traditional
Allele Fy a, Fy b, Fy
Genotype/haplotype Fy a/Fy b
Phenotype Fy(a+b+)
Antigen Fya, Fyb
ISBT = International Society of Blood Transfusion; N denotes “null”; FY*01N or FY*02N indicates that the
null allele is on a FY*A or FY*B background, respectively.
individual who lacks that antigen, elicit an im-
mune response.
It is the antibody from such an immune
response that causes problems in clinical
practice, such as patient/donor blood transfu-
sion incompatibility, maternal-fetal incompat-
ibility, and the reason why antigen-negative
blood is required for safe transfusion in these
patients. Hemagglutination is simple, quick,
and relatively inexpensive. When carried out
correctly, it has a specificity and sensitivity
that is appropriate for most testing. However,
hemagglutination has limitations; for exam-
ple, it is difficult and often impossible to ob-
tain an accurate phenotype for a recently
transfused patient or to type red cells that are
coated with IgG, and some typing reagents are
in short supply or not available. Because the
genes encoding the 34 known blood group sys-
tems have been cloned and sequenced and the
molecular bases of most blood group antigens
and phenotypes are known, DNA-based meth-
ods (genotyping) are increasingly being used
as an indirect method to predict a blood group
phenotype. This approach has introduced
blood group genomics, often referred to as
“molecular immunohematology,” into the
practice of transfusion medicine. Prediction of
a blood group antigen by testing DNA is sim-
ple and reliable for the majority of antigens be-
cause most result from SNPs that are inherited
in a straightforward Mendelian manner. For
example, the antithetical antigens S and s arise
from GYPB alleles that differ by one nucleo-
tide—143T for S and 143C for s—and the re-
ISBT
FY*01 or FY*A, FY*02 or FY*B, FY*N or FY*01N or
FY*02N
FY*A/FY*B or FY*01/FY*02
FY:1,2
FY1, FY2
sulting proteins differ by one amino acid, me-
thionine at residue 48 for S and threonine for s
(designated c.143T>C p.Met48Thr). As a re-
sult, assay design and interpretation are fairly
straightforward for the prediction of most phe-
notypes.
However, detailed serologic and molecu-
lar studies have shown that there are far more
alleles than phenotypes for some systems, es-
pecially ABO and Rh. More than 100 different
alleles encoding the glycosyltransferases re-
sponsible for the four ABO types have been
identified, and a single nucleotide change in
an A or B allele can result in an inactive trans-
ferase and a group O phenotype (see Chapter
12). Testing for the common Rh antigens D,
C/c, and E/e is uncomplicated for most popu-
lations, but antigen expression is more com-
plex in some ethnic groups. There are more
than 200 RHD alleles encoding weak D or par-
tial D phenotypes, and more than 100 RHCE
alleles encoding altered, or novel, hybrid Rh
proteins, some of which result in weakened
antigen expression (see Chapter 13). RH geno-
typing, particularly in minority populations,
requires sampling of multiple regions of the
gene(s) and algorithms for interpretation.
The basis of DNA assays is the amplifica-
tion of a target gene sequence through the
polymerase chain reaction (PCR), followed by
manual, semi-automated, or automated
downstream analysis. Commonly used meth-
ods are sequence-specific PCR (SSP-PCR) and
allele-specific PCR (AS-PCR). For manual
methods, gel electrophoresis is used to sepa-
 CHAPTER 11
rate the PCR products for fragment size deter-
mination. As an alternative, the assay may in-
clude digestion of the PCR products with a
restriction fragment length polymorphism
(RFLP), followed by electrophoresis and visu-
alization of the fragments. Semi-automated
approaches include real-time PCR using fluo-
rescent probes with quantitative and qualita-
tive automated read-out. Manual methods are
labor-intensive, and each assay is performed
separately on each sample. Automated DNA
arrays allow for higher throughput with a larg-
er number of target alleles in the PCR reaction,
which makes possible the determination of
numerous antigens in a single assay. Most
available platforms are based on fluorescent
bead technology or mass spectrometry. Rou-
tine ABO and RhD testing is not currently
available on most automated platforms be-
cause the expression of these antigens is com-
plex and further development is required.
To resolve discrepancies and identify new
alleles, specialty referral laboratories use
methods that are similar to those used for
high-resolution HLA typing, ie, gene-specific
amplification of coding exons followed by se-
quencing, or gene-specific cDNA amplifica-
tion and sequencing. These methods are used
to investigate new alleles and resolve serologic
and molecular discrepancies. The application
of these methods has been reviewed by several
groups.47-49
Clinical Application of the Prediction
of Blood Groups by DNA Analysis
A major use of DNA-based assays is to predict
the red cell phenotype of a fetus, or of a patient
who has been transfused, or when red cells are
coated with IgG. Additional applications in-
clude the resolution of discrepancies in the
ABO and Rh systems and identification of the
molecular basis of unusual serologic results.
DNA analysis also affords the capability of dis-
tinguishing alloantibodies from autoantibod-
ies. This section gives an overview of some of
the major applications of DNA-based analysis
that are currently employed in patient and do-
nor testing. These, and additional clinical ap-
plications, are summarized in Table 11-4.
Blood Group Genetics 䡲 281
DNA-Based Assays to Predict the Red
Cell Phenotype: Recently Transfused
Patients
In patients receiving chronic or massive trans-
fusions, the presence of donor red cells often
makes typing by hemagglutination inaccurate.
Time-consuming and cumbersome cell sepa-
ration methods that are often unsuccessful in
isolating and typing the patient’s reticulocytes
can be avoided when DNA typing is used. PCR-
based assays mostly use DNA extracted from
WBCs isolated from a sample of peripheral
blood. Interference from donor-derived DNA
is avoided by targeting and amplifying a region
of the gene that is common to all alleles so that
the minute quantity of donor DNA is not de-
tected. This approach makes possible reliable
blood group determination with DNA pre-
pared from a blood sample collected after
transfusion. DNA isolated from a buccal smear
or urine sediment is also suitable for testing. In
transfusion-dependent patients who produce
alloantibodies, an extended antigen profile is
important to determine additional blood
group antigens to which the patient can be-
come sensitized.
In the past, when a patient with autoim-
mune hemolytic anemia was transfused be-
fore the patient’s red cell phenotype for minor
antigens was established, time- and resource-
consuming differential allogeneic absorptions
were required to determine the presence or
absence of alloantibodies underlying the auto-
antibody. Establishing the patient’s most prob-
able phenotype through DNA-based assays
makes it possible to match the antigen profile
of the absorbing red cells to that of the patient,
thereby reducing the number of cell types re-
quired for absorption. This approach also al-
lows matching of the antigen profile of the do-
nor to that of the patient for the most clinically
significant, common antigens (eg, Jka, Jkb; S, s)
when transfusion is required. This matching
avoids the use of “least incompatible” blood
for transfusion, and allows transfusion of units
that are “antigen-matched for clinically signifi-
cant blood group antigens” to prevent delayed
transfusion reactions and circumvent addi-
tional alloimmunization.
282 䡲 AABB TECHNICAL MANUAL

TABLE 11-4. Applications of DNA-Based Assays for Patient and Donor Testing

To predict a patient’s red cell phenotype:

䡲 After a recent transfusion.

– Aid in antibody identification and RBC unit selection.
– Select RBCs for adsorption.

䡲 When antibody is not available (eg, anti-Doa, -Dob, -Jsa, -V, -VS).

䡲 Distinguish an alloantibody from an autoantibody (eg, anti-e, anti-Kpb).

䡲 Help identify alloantibody when a patient’s type is antigen-positive and a variant phenotype is possible (eg,
anti-D in a D-positive patient, anti-e in an e-positive patient).

䡲 When the patient’s red cells are coated with immunoglobulin (DAT+).
– When direct agglutinating antibodies are not available.
– When the antigen is sensitive to the IgG removal treatment (eg, antigens in the Kell system are denatured
by EDTA-glycine-acid elution).
– When testing requires the indirect antiglobulin test and IgG removal techniques are not effective at
removing cell-bound immunoglobulin.
– When antisera are weakly reactive and reaction is difficult to interpret (eg, anti-Doa, anti-Dob, anti-Fyb).

䡲 After allogeneic stem cell transplantation.

– If an antibody problem arises, test stored DNA samples from the patient and the donor(s).

䡲 To detect weakly expressed antigens (eg, Fyb with the FyX phenotype); where the patient is unlikely to make
antibodies to transfused antigen-positive RBCs.

䡲 Identify molecular basis of unusual serological results, especially Rh variants.

䡲 Resolve discrepancies, eg, A, B, and Rh.

䡲 Aid in the resolution of complex serologic investigations, especially those involving high-prevalence anti-
gens when reagents are not available.

䡲 Identify if a fetus is or is not at risk for hemolytic disease of the fetus and newborn.
– Predict if the partner of a prospective mother with anti-D is homozygous or heterozygous for RHD.

To predict the donor’s red cell phenotype:

䡲 Screen for antigen-negative donors.

䡲 When antibody is weak or not available (eg, anti-Doa, -Dob; -Jsa, -Jsb; -V/VS).

䡲 Mass screening to increase antigen-negative inventory.

䡲 Find donors whose red cells lack a high-prevalence antigen.

䡲 Resolve blood group A, B, and Rh discrepancies.

䡲 Detect genes that encode weak antigens.

䡲 Type donors for reagent red cells for antibody screening cells and antibody identification panels (eg, Doa,
Dob, Jsa, V, VS).

䡲 Determine zygosity of donors on antibody detection/identification reagent panels, especially D, S, Fya, and
Fyb.
 CHAPTER 11
DNA-Based Assays to Predict the Red
Cell Phenotype: When Red Cells Are
Coated with IgG
In patients with or without autoimmune he-
molytic anemia, the presence of immunoglob-
ulin bound to the red cells [positive result on
direct antiglobulin testing (DAT)] often makes
antigen typing results by serologic methods in-
valid. Certain methods, such as treatment of
the red cells with chloroquine diphosphate or
EDTA-glycine acid (EGA) may be employed to
remove the red-cell-bound IgG. These meth-
ods are not always successful, the antigen of
interest may be denatured by the treatment
(eg, EGA destroys antigens of the Kell blood
group system), and direct agglutinating anti-
bodies for the antigen of interest may not be
available. DNA testing allows determination of
an extended antigen profile to select antigen-
negative red cell units for transfusion.
DNA-Based Assays to Distinguish
Alloantibody from Autoantibody
When an antibody specificity is found in a pa-
tient whose red cells express the correspond-
ing antigen, it is essential to know whether the
antibody is an allo- or autoantibody and a
DNA-based investigation is helpful for transfu-
sion management. If DNA typing predicts the
red cells to be antigen positive, further investi-
gation by high-resolution gene sequencing
should be considered because the sample may
have a novel amino acid change in the protein
carrying the blood group antigen. These novel
amino acid changes result in new epitopes and
altered (weakened or partial) expression of the
conventional antigen.
This situation is especially relevant in pa-
tients with sickle cell disease (SCD) or thalas-
semia who require long-term transfusion sup-
port and are at risk of alloimmunization that is
often complicated by the presence of autoanti-
bodies. In patients of African ancestry who
have SCD, partial expression of common Rh
antigens (D, C, c, and e) is prevalent. Such pa-
tients frequently present with a combination
of anti-D, -C, and -e and yet their red cells type
serologically as D+, C+, and e+. Although such
Blood Group Genetics 䡲 283
patients may make alloantibodies to these an-
tigens, autoantibody production with Rh-
related specificity is prevalent, and distin-
guishing between the two is critical for safe
transfusion practice to avoid hemolytic trans-
fusion reactions.50,51 Delayed hemolytic trans-
fusion reaction in particular, places patients
with SCD at risk for life-threatening anemia,
pain crisis, acute chest syndrome, and/or
acute renal failure. Patients may also experi-
ence hyperhemolysis, in which hemoglobin
levels drop below pretransfusion levels due to
bystander hemolysis of the patients’ own anti-
gen-negative red cells. RH genotyping has re-
vealed that many of these patients have vari-
ant RHD and/or RHCE alleles that encode
amino acid changes in Rh proteins that result
in altered or partial antigens. For details on
RHD and RHCE alleles that encode partial an-
tigen, refer to Chapter 13.
Reports of autoantibodies to Jka and Jkb
are not uncommon. With the discovery of vari-
ant JK alleles that encode partial Jka and Jkb an-
tigens, it is probable that some previously
identified autoantibodies were alloantibodies
(see Chapter 14). DNA analysis for JK variants
is helpful to clarify the situation. As in other
blood group systems, Kidd system genetic di-
versity is higher in populations of African an-
cestry.
DNA-Based Testing in Prenatal
Practice
DNA-based testing has affected prenatal prac-
tice in the areas that are discussed below.
Hemagglutination, including antibody titers,
gives only an indirect indication of the risk and
severity of hemolytic disease of the fetus and
newborn (HDFN). Antigen prediction by DNA-
based assays can be used to identify the fetus
who is not at risk of HDFN (ie, who is predicted
to be antigen-negative) so that the mother
need not be aggressively monitored. Testing of
fetal DNA should be considered when a moth-
er's serum contains an IgG alloantibody that
has been associated with HDFN and the fa-
ther's antigen status for the corresponding an-
tigen is heterozygous or indeterminable, or he
is not available for testing.
284 䡲 AABB TECHNICAL MANUAL
To Identify a Fetus at Risk for Anemia of
the Neonate
The first application of DNA-based assays for
the prediction of blood group phenotype oc-
curred in the prenatal setting and was report-
ed by Bennett et al52 who tested fetal DNA for
the presence of RHD. Because of the clinical
significance of anti-D, RHD is probably the
most frequent target gene, but DNA-based as-
says can be used to predict the antigen type of
the fetus for any antigen if the molecular basis
is known. When the implicated IgG antibody
in the maternal circulation is not anti-D, it is
prudent, when possible, to also test the fetal
DNA for RHD to preempt unnecessary re-
quests for D– blood for intrauterine transfu-
sion; this is particularly relevant to avoid the
use of rare r'r' or r''r'' blood when anti-c or
anti-e is the implicated antibody.
PCR analyses for the prediction of fetal D
phenotype are based on detecting the pres-
ence or absence of specific portions of RHD. In
populations of European ancestry, the molec-
ular basis of the D-negative phenotype is usu-
ally associated with deletion of the entire RHD,
but several other molecular bases have been
described. In populations of Asian ancestry
15% to 30% of D-negative people have an in-
tact but inactive RHD, while others with red
cells that are nonreactive with anti-D have the
Del phenotype. Approximately a quarter of D-
negative people of African ethnicity have an
RHD pseudogene (RHD), which does not en-
code the D antigen, and many others have a
hybrid RHD-CE-D gene (eg, the rS phenotype).
Predicting the D type by DNA analysis requires
probing for multiple nucleotide changes. The
choice of assays depends upon the patient’s
ethnicity and the degree of discrimination de-
sired. Establishing the fetal KEL genotype is
also of great clinical value in determining
whether a fetus is at risk for severe anemia, be-
cause the strength of the mother's anti-K anti-
body often does not correlate with the severity
of the infant’s anemia. The same is true for
anti-Ge3.53
Amniocytes, harvested from amniotic flu-
id, are the most common source of fetal DNA.
Chorionic villus sampling and cordocentesis
are not favored because of their more invasive
nature and associated risk to the fetus. A non-
invasive sample source is the cell-free fetal
DNA that is present in maternal plasma as ear-
ly as 5 weeks of gestation; the amount of DNA
increases with gestational age and reliable re-
sults in DNA-based assays are obtained start-
ing at about 15 weeks of gestation (sometimes
earlier, depending on the gene of interest).54,55
These assays are particularly successful for D
typing because the D-negative phenotype in
the majority of samples is due to the absence
of the RHD gene.
Testing for the presence or absence of a
gene is less demanding than testing for a sin-
gle gene polymorphism or SNP to predict, for
example, the K/k antigen status. Cell-free fetal
DNA from the maternal plasma is routinely
tested in Europe for the presence of a fetal
RHD gene to eliminate the unnecessary
administration of antepartum RhIG to the ap-
proximately 40% of D-negative women who
are carrying a D-negative fetus. In the United
States, due to intellectual property ownership,
testing of cell-free fetal DNA is limited to wom-
en with active anti-D and is available only
commercially.
DNA-Based Testing for D in Pregnant
Women
Serologic typing for D cannot easily distin-
guish women whose red cells lack some epi-
topes of D (partial D) and are at risk for D im-
munization, from those with a weak D
phenotype who are not at risk for D immuni-
zation. Red cells with partial D type as D+,
some in direct tests and others by indirect
tests. These women might benefit from receiv-
ing RhIG prophylaxis if they deliver a D+ fetus.
RHD genotyping can distinguish weak D from
partial D to guide RhIG prophylaxis and blood
transfusion recommendations.
DNA-Based Testing of Paternal Samples
The father’s red cells should be tested for the
antigen corresponding to the antibody in the
maternal plasma. If the red cells are negative,
the fetus is not at risk. If the father is positive
 CHAPTER 11
for the antigen, zygosity testing can determine
whether the father is homozygous or heterozy-
gous for the gene encoding the antigen, partic-
ularly when there is no allelic antigen or no an-
tisera to detect the allelic product.
Zygosity testing of paternal samples is
most often performed when testing for possi-
ble HDFN due to anti-D or anti-K. If the pater-
nal red cells are K+ and the mother has anti-K,
they can be tested serologically for expression
of the allelic k antigen. However, many centers
do not have a licensed reagent available and
genetic counselors often request DNA testing.
If the paternal red cells are K–, the maternal
anti-K is mostly likely the result of immuniza-
tion through transfusion.
For maternal anti-D, DNA testing of RHD
zygosity is the only method to determine the
paternal gene copy number. Several different
genetic events cause a D-negative phenotype,
and multiple assays must be conducted to ac-
curately determine RHD zygosity, especially in
minority ethnic groups. If the father is RHD
homozygous, all of his children will be D+ and
any pregnancy in his partner needs to be mon-
itored. If the father is heterozygous, the fetus
has a 50% chance of being at risk. The D type
of the fetus should be determined to prevent
invasive and unnecessary testing so the moth-
er need not be aggressively monitored or re-
ceive immune-modulating agents.
DNA-Based Testing for Antigen-
Negative Blood Donors
DNA-based typing to predict donor antigen
profiles in the search for antigen-negative
units is now a standard procedure for blood
centers, especially when suitable antibodies
are not available. Because red cell typing for
Dombrock antigens is notoriously difficult,
one of the most frequent approaches is to type
for Doa and Dob. Many other antibody specific-
ities are unavailable for mass donor screening.
These specificities include anti-Hy, -Joa, -Jsa,
-Jsb, -CW, -V, and -VS. Even specificities consid-
ered to be common, such as anti-S and anti-
Fyb, are not always readily available.
DNA arrays can be used to screen for mul-
tiple minor antigens in a single assay format
Blood Group Genetics 䡲 285
and have the potential to be used for mass
screening of donors. This practice not only in-
creases the antigen-negative inventory by ex-
panding combinations of the minor antigens
and some high-prevalence antigens, but also
allows consideration of the possibility of pro-
viding donor components that are DNA
matched to the patient’s type. Although DNA
methods are not yet licensed by the Food and
Drug Administration for the labeling of donor
units in the United States, DNA testing is a
valuable screening tool and only negative test
results need to be confirmed using a licensed
reagent when one is available.
DNA-Based Testing to Confirm D Type
of Donors
Donor centers must test donors for weak D to
avoid labeling a product as D-negative that
might result in anti-D in response to trans-
fused RBCs. Some donor red cells with very
weak D expression (weak D type 2, and espe-
cially those with the Del phenotype) are not
typed as D+ using current methods and are la-
beled as D–. The prevalence of weak D red cells
not detected by serologic reagents is approxi-
mately 0.1% (but may vary depending on the
test method and population). Although the
clinical significance has not been established,
donor red cells with weak D expression have
been associated with alloimmunization. RHD
genotyping would improve donor testing by
confirming D– phenotypes,56 but a high-
throughput and cost-effective platform is not
yet available.
Discrepancies between Serologic
(Phenotype) and DNA (Genotype)
Testing
Differences between serologic and DNA test-
ing results do occur and must be investigated.
Often, these discrepancies lead to interesting
discoveries such as the presence of a novel al-
lele or genetic variant, particularly when peo-
ple of diverse ethnicities are tested. Causes of
discrepancies include recent transfusions,
stem cell transplantation, and natural chime-
rism. Stem cell transplantation and natural
286 䡲 AABB TECHNICAL MANUAL

chimerism also may cause results of testing
DNA from somatic cells to differ from results
of testing DNA from extracted from peripheral
WBCs. Thus, when using DNA testing, it is im-
portant to obtain an accurate medical history.
Many genetic events can cause apparent dis-
crepant results between hemagglutination and
DNA test results and the genotype does not al-
ways predict the phenotype (see Table 11-5 for
examples).3,5,27
Silenced or Nonexpressed Genes
DNA testing interrogates a single SNP or a few
SNPs associated with antigen expression and
cannot sample every nucleotide in the gene.
Although a gene may be detected by DNA test-
ing, there are times when the gene product is
not expressed on the red cells because of a
mutation that silences the gene. Such changes
result in discrepancies in the typing of patients
and donors. Homozygosity (or compound het-
erozygosity) for a silenced gene results in a
null phenotype and most null phenotypes
have more than one molecular basis.5
With donor typing, the presence of a
grossly normal gene whose product is not ex-
pressed on the red cell surface results in the
donor being falsely typed as antigen-positive.
Although this situation means loss of an anti-
gen-negative donor, it does not jeopardize the
safety of blood transfusion. However, if a
grossly normal gene is detected in a patient
but the gene is not expressed, the patient
could produce an antibody if he or she re-
ceives a transfusion of antigen-positive blood.

TABLE 11-5. Examples of Some Molecular Events Where Analysis of Gene and Phenotype Do Not
Agree

Molecular Event Mechanism Observed Blood Group Phenotype

Transcription Nt change in GATA box Fy(b–)

Alternative splicing
Premature stop codon
Amino acid change
Reduced amount of
protein
Hybrid genes
Interacting protein
Modifying gene
Nt = nucleotide; aas = amino acids.
Nt change in splice site: Partial/
complete skipping of exon
Deletion of nt(s)
Deletion of nt(s)  frameshift
Insertion of nt(s)  frameshift
Nt change
Missense nucleotide change
Missense nucleotide change
Cross-over
Gene conversion
Absence of RhAG
Absence of Kx
Absence of aas 75 to 99 of GPA
Absence of protein 4.1
In(Jk)
S–s–; Gy(a–)
Dr(a–)
Fy(a–b–); D–; c−E−; Rhnull; Gy(a–);
GE:–2,–3,–4; K0; McLeod
D–; Co(a–b–)
Fy(a–b–); r; Gy(a–); K0; McLeod
D–; Rhnull; K0; McLeod
FyX; Co(a–b–)
GP.Vw; GP.Hil; GP.TSEN
GP.Mur; GP.Hop; D– –; R0Har
Rhnull
Weak expression of Kell antigens
Wr(b–)
Weak expression of Ge antigens
Jk(a–b–)
 CHAPTER 11 Blood Group Genetics 䡲 287

To avoid misinterpretation, routine as-
says must include appropriate tests to detect a
change that silences gene expression if preva-
lent in the population tested. Silenced alleles
can be specific to a particular ethnic group.
For example, in the Duffy blood group system,
a single nucleotide change (–67T>C) within
the promoter region (GATA box) of FY prevents
transcription of FY*A and/or FY*B in red cells
but not in other tissues. Although silencing of
FY*A is rare, silencing of FY*B is frequent in
persons of African ethnicity where homozy-
gosity for the –67T>C change in FY*B results in
the Fy(a–b–) phenotype, which has a preva-
lence of 60% or higher. To ensure accuracy,
testing for the GATA box mutation must be in-
cluded in typing for Duffy in persons of African
ethnicity.
When the assay is used to predict the
presence or absence of D antigen, particularly
in populations of African ancestry, it is essen-
tial to include a test for the complete but inac-
tive RHD pseudogene (RHD), which has a
37-bp sequence duplication. If the assay tests
for GYPB*S (S antigen), additional testing
should be performed to detect a C>T change at
nucleotide 230 in GYP*B exon 5 or a change in
intron 5 (+5g>t); both changes prevent expres-
sion of S antigen when testing persons of Afri-
can ancestry. Homozygosity, or compound
heterozygosity, for the 230T, or the +5g>t
change, results in the S–s–U+W phenotype.
Other common causes of discrepancies
include the presence in the sample of an
altered FY*B allele that encodes an amino acid
change causing an FyX phenotype with greatly
reduced expression of Fyb antigen. The red
cells type as Fy(b–) with most serologic re-
agents. The prevalence of the allele encoding
the FyX phenotype in people of European an-
cestry is as high as 2%, and the allele has been
found in persons of African ancestry also. Si-
lencing mutations associated with the loss of
Kidd antigen expression occur more often in
people of Asian ancestry, whereas nucleotide
changes encoding amino acid changes that
weaken Kidd expression occur in people of Af-
rican ethnicity.
The routine detection of some blood
group polymorphisms by DNA analysis is
complex and not practical at this time. This in-
cludes situations where 1) a large number of
alleles encode one phenotype (eg, ABO, Rh,
and null phenotypes in many blood group sys-
tems), 2) a phenotype results from alleles with
a large deletion (eg, GE:–2,3 and GE:–2,–3,–4),
or 3) a phenotype results from hybrid alleles
(eg, in the Rh and MNS systems). In addition,
there is a high probability that not all alleles in
all ethnic populations are known.
Blood group genomics has become an es-
sential component of the practice of transfu-
sion medicine.57 Genomics has provided a
greater understanding of genetic blood group
variants, including the complexity of the
molecular basis of Rh variants, such as those
that encode the Hr–hrS– and hrB–HrB–
phenotypes58,59 and the associated partial Rh
antigens that are a daily challenge for the man-
agement of patients with SCD.50,51 RH genotyp-
ing expands and extends matching for Rh in
this patient population. High-throughput plat-
forms provide a means to test relatively large
numbers of donors, thereby opening up the
possibility of changing the way antigen-nega-
tive blood is provided to patients to prevent
immunization or to eliminate transfusion re-
actions for those who are already immunized.

KEY PO I NT S

1. Genetics is the study of heredity, that is, the mechanisms by which a particular characteris-
tic, such as a blood group, is passed from parents to offspring.
2. A gene is a segment of DNA and is the basic unit of inheritance; it occupies a specific loca-
tion on a chromosome (the gene locus). Alleles are alternative forms of a gene at the same
gene locus (eg, alleles JK*A and JK*B encode the Jka and Jkb antigens, respectively).
288 䡲 AABB TECHNICAL MANUAL

3. A human somatic (body) cell is diploid, containing 46 chromosomes in 23 pairs: 22 pairs are
alike (homologous) in males and females and are termed autosomes. The remaining pair is
the sex chromosomes: X and Y for males, or two X chromosomes for females.
4. Somatic cells divide for growth and repair by mitosis. Mitosis replicates the chromosomes
and produces two identical nuclei in preparation for cell division. The new cells are diploid,
like the parent cell, and have all the genetic information of the parent cell.
5. Meiosis is the process by which germ cells divide to become gametes (sperm and egg cells);
diploid cells undergo DNA replication and two divisions to form four gametes, each of
which is haploid and has half the chromosomal complement of the parent somatic cell.
6. The term “genotype” traditionally refers to the complement of genes inherited by each per-
son from his or her parents; the term is also used to refer to the set of alleles at a single gene
locus. Whereas the genotype of a person is his or her genetic constitution, the phenotype is
the observable expression of the genes and reflects the biologic activity of the gene(s). Thus,
the presence or absence of antigens on the red cells, as determined by serologic testing, rep-
resents the phenotype.
7. When identical alleles for a given locus are present on both chromosomes, a person is ho-
mozygous for the particular allele, whereas when nonidentical alleles are present at a par-
ticular locus, the person is heterozygous. Antigens encoded by alleles at the same locus are
said to be antithetical. Thus, genes are allelic but not antithetical, whereas antigens are anti-
thetical but not allelic.
8. The expression of blood group antigens on the red cell may be modified or affected by gene
interaction. Homozygosity (or compound heterozygosity) for a silenced gene results in a
null phenotype and most null phenotypes have more than one molecular basis.
9. A blood group system consists of one or more antigens under the control of a single gene lo-
cus (eg, KEL encodes the Kell blood group antigens) or of two or more homologous genes
(eg, RHD and RHCE encode the Rh blood group antigens) so closely linked that virtually no
recombination occurs between them; thus, each blood group system is genetically indepen-
dent. Currently, 34 blood group systems are recognized.
10. The genes encoding the 34 blood group systems have been sequenced and the molecular
bases of most antigens and phenotypes are known so that DNA-based methods (genotyp-
ing) can be used to predict a blood group phenotype.
11. DNA-based assays (blood group genotyping) have major applications in patient and donor
testing. They can be used to predict the red cell phenotype of a fetus, or of a patient who was
transfused, or when red cells are coated with IgG; they can be used to resolve ABO and Rh
discrepancies and to identify the molecular basis of unusual serologic results. DNA analysis
aids in distinguishing alloantibodies from autoantibodies and is being applied to high-
throughput screening of donors.

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 C h a p t e r
ABO, H, and Lewis Blood Groups
and Structurally Related Antigens
Laura Cooling, MD, MS
T H E A N T I G E N S O F the ABO, H, Lew-
is, I, and P blood group systems are de-
fined by small carbohydrate epitopes on glyco-
proteins and glycolipids. Because the epitopes
represent posttranslational modifications, the
synthesis of those antigens requires the action
of several enzymes known as “glycosyltrans-
ferases.” Glycosyltransferases reside in the
Golgi apparatus and are responsible for add-
ing specific sugars, in a specific sequence and
steric or anomeric linkage (-linked or -
linked), to growing oligosaccharide chains on
glycolipids and glycoproteins.
Transcriptional regulation coupled with
the enzymatic specificity of these glycosyl-
transferases for both sugar donors [nucleotide
sugars; eg, uridine diphosphate (UDP)-galac-
tose] and acceptor substrates (eg, Type 1 chain
vs Type 2 chain) is responsible for the tissue-
specific distribution of many blood group an-
tigens.1,2 Because they are widely distributed
on many tissues, including embryonic stem
cells, such antigens are considered to be histo-
blood-group antigens.2-5 Several studies have
shown that these antigens have a role in devel-
Laura Cooling, MD, MS, Associate Professor, Department of Pathology, and Associate Medical Director, Trans-
fusion Medicine, University of Michigan, Ann Arbor, Michigan
The author has disclosed no conflicts of interest.
1 2
opment, cell adhesion, malignancy, and infec-
tious disease.1,6,7
A B O S Y S T EM
The ABO system, initially described by Karl
Landsteiner in 1900, remains the most impor-
tant blood group system in transfusion and or-
gan transplantation medicine.7 In blood, ABO
antigens are found on red cells, platelets, and
many circulating proteins. As histo-blood-group
antigens, ABO antigens are also present on
many tissues, including those of the endotheli-
um, kidney, heart, bowel, pancreas, and lung.2
Transfusion of ABO-incompatible blood
can be associated with acute intravascular he-
molysis, renal failure, and death. Likewise,
transplantation of ABO-incompatible organs
is associated with acute humoral rejection. 7
Because of the dire clinical consequences as-
sociated with ABO incompatibilities, ABO typ-
ing and ABO compatibility testing remain the
foundation of pretransfusion testing and an
important component of typing before trans-
plantation.
291
292 䡲 AABB TECHNICAL MANUAL
The ABO system contains four major ABO
phenotypes: A, B, O, and AB. The four pheno-
types are determined by the presence or ab-
sence of two antigens (A and B) on red cells
(see Table 12-1). The ABO system is also char-
acterized by the presence or absence of natu-
rally occurring antibodies, termed “isohemag-
glutinins,” directed against missing A and B
antigens. As shown in Table 12-1, an inverse
reciprocal relationship exists between the
presence of A and/or B antigens on red cells
and the presence of anti-A, anti-B, or both, in
sera. For example, group O individuals, who
lack A and B antigens on red cells, possess
both anti-A and anti-B. It is believed that the
immunizing sources for such naturally occur-
ring antibodies are gut and environmental
bacteria, such as the Enterobacteriaceae, which
have been shown to possess ABO-like struc-
tures on their lipo-polysaccharide coats.8,9
Biochemistry
The A and B antigens are defined by three sug-
ar terminal epitopes on glycolipids and glyco-
proteins.7 As shown in Fig 12-1, H antigen is
characterized by a terminal 12 fucose; it is
the immediate biosynthetic precursor for both
A and B antigens and is required for their ex-
pression. In group A individuals, an N-acetyl-
galactosamine is added, in an 13 linkage,
TABLE 12-1. Routine ABO Grouping
Reaction of Red Cells Reaction of Serum with
with Antisera Reagent Red Cells
(Red Cell Grouping) (Serum Grouping)
Anti-A Anti-B A1 Cells B Cells
0 0 + +
+ 0 0 +
0 + + 0
+ + 0 0
0 0 + +
*H null phenotype (see section on H antigen).
+ = agglutination; 0 = no agglutination.
to the subterminal galactose of the H antigen
to form A antigen. In group B individuals, an
13 galactose is added to the same subter-
minal galactose to form B antigen. In group AB
individuals, both A and B structures are syn-
thesized. In group O individuals, neither A nor
B antigens are synthesized as a result of a mu-
tation in the ABO gene.7,10 As a consequence,
group O individuals express only H antigen. A
and B antigens are also absent in the very rare
Bombay phenotype because of the absence of
the H-antigen precursor (see section below on
the H system).
As terminal motifs, the A and B antigens
can be displayed on a number of oligosaccha-
ride scaffolds that differ in their size, composi-
tion, linkages, and tissue distribution. On red
cells, ABH sites are present on N-linked (65%-
75%) and O-linked (5%-15%) glycoproteins,
polyglycosylceramides (10%-15%), and sim-
pler glycosphingolipids (Fig 12-2). ABO anti-
gens are subclassified by the carbohydrate se-
quence immediately upstream of the ABO
motif. In humans, ABH is expressed predomi-
nantly on four different oligosaccharide back-
bones (see Table 12-2). On human red cells,
the majority of endogenous ABH antigen syn-
thesized is present on Type 2 chain structures.
The ability to synthesize and use different
carbohydrate chains is genetically determined
and can contribute to antigenic differences in
Interpre- Prevalence (%) in
tation US Population
European African
O Cells ABO Group Ethnicity Ethnicity
0 O 45 49
0 A 40 27
0 B 11 20
0 AB 4 4
+ Bombay* Rare Rare
 CHAPTER 12
FIGURE 12-1. GalNAc added to the subterminal Gal
confers A activity to the sugar; Gal added to the
subterminal Gal confers B activity. Unless the fucose
moiety that determines H activity is attached to the
number 2 carbon, galactose does not accept either
sugar on the number 3 carbon.
weak ABO subgroups.10-12 For instance, Type 3
(repetitive A) and Type 4 (globo-A) A antigens
are present on A1, but not A2, red cells. Differ-
ences in cis carbohydrate sequences upstream
of the terminal ABO motif can also influence
antibody reactivity.12 As an example, ABO anti-
gens on Type 1 chain substrates can be recog-
nized by antibodies directed against both ABO
and Leb antigen (see “Lewis System” section
below).10,13
ABO in Development and Aging
ABO antigens can be detected on red cells of
embryos as early as 5 to 6 weeks of gestation.13
The quantity of ABO antigens on umbilical
cord red cells is less than that of adults as the
ABO, H, and Lewis Blood Groups 䡲 293
result of the immaturity of Type 2 chain pre-
cursors on cord red cells (see “I and i Antigens”
section below).14 With increasing age, precur-
sor chains become increasingly branched,
thereby allowing more A and B antigen to be
expressed. Adult levels of ABO expression are
generally present by age 2 to 4 years.13,14
Anti-A and anti-B are not present at birth
or, if present, are of maternal origin. Endoge-
nous synthesis of anti-A and anti-B can devel-
op as early as age 3 to 6 months, with nearly all
children displaying the appropriate isohemag-
glutinins in their sera at 1 year of age.13,15 Titers
of anti-A and anti-B continue to increase dur-
ing early childhood and achieve adult levels
within 5 to 10 years.
Among healthy adults, ABO titers can nat-
urally vary from 4 to 2048 or higher.13,15,16 High-
titer ABO antibodies can be present in group O
multiparous women and in patients taking
certain bacteria-based nutritional supple-
ments.7,9,13 Although older reports indicated a
fall in isoagglutinin titers in the elderly, more
recent studies have disputed these findings.15
In industrialized countries, isoagglutinin ti-
ters have generally decreased with increasing
consumption of processed foods.16
Genetics
The ABO gene is located on chromosome 9q34
and is fairly large, consisting of seven exons
spread over 18 kb.7 The open reading frame of
the protein is located primarily in exons 6 and
7. A study of the promoter region indicates
that ABO gene expression is transcriptionally
regulated by several mechanisms, including
methylation, tissue-specific transcription-
factor-binding proteins, antisense RNA, and,
possibly, a minisatellite enhancer region 4 kb
upstream of exon 1.7 ABO expression is also
regulated by the H gene, which is responsible
for the synthesis of H antigen substrate, the
precursor of A and B antigens. The H gene is
tightly regulated in a tissue-specific manner
through tissue-specific transcription factors
and promoters.17 In the absence of H, no A or B
antigen is expressed regardless of ABO geno-
type (Bombay or Oh phenotype).7,10
294 䡲 AABB TECHNICAL MANUAL

FIGURE 12-2. Schematic representation of the red cell membrane showing antigen-bearing glycosylation of
proteins and lipids.
GPI = glycosylphosphatidylinositol. (Courtesy of ME Reid)

A series of elegant studies over the past i d s ( A  B ; G l y 2 3 5 Se r, L e u 2 6 6 Me t , a n d

decade has identified the molecular basis for
A, B, O, cis-AB, and weak ABO subgroups.7,10,18
Fundamentally, three common ABO alleles are
responsible for the A, B, and O phenotypes.
The A and B consensus alleles are autosomal
codominant and differ by only seven nucleo-
tides and four amino acids.7,18 Three amino ac-
Gly268Ala) are critical in determining whether
the glycosyltransferase uses UDP-N-acetyl-D-
galactosamine or UDP-D-galactose donor sug-
ars to synthesize A or B antigens, respective-
ly.7,10 The rare cis-AB phenotype is a chimeric
enzyme with a mix of A-specific and B-specific
amino acids at those or other amino acid posi-

TABLE 12-2. Chain Variants of A Antigen in Humans

Antigen Oligosaccharide Sequence*

A epitope GalNAc1-3(Fuc1-2)Gal1-R
Type 1 A GalNAc1-3(Fuc1-2)Gal1-3GlcNAc1-3-R
Type 2 A GalNAc1-3(Fuc1-2)Gal1-4GlcNAc1-3-R
Type 3 A GalNAc1-3(Fuc1-2)Gal1-3GalNAc1-3(Fuc1-2)Gal1-4GlcNAc1-3-R
(repetitive A)
Type 4 A GalNAc1-3(Fuc1-2)Gal1-3GalNAc1-4Gal1-4Gal1-4Glc-Cer
(globo-A)
*Underlined sequences denote the critical differences between Type 1, 2, and 4 chains. Linkages and anomery ( or 
linked) of the A antigen galactose are in bold. Bracketed sequences denote repetitive A antigen characteristic of Type 3 chain
A antigen.
Cer = ceramide; Fuc = fucose; Gal = galactose; GalNAc = N-acetyl-galactosamine; Glc = glucose; GlcNAc = N-acetyl-glucosa-
mine; R = upstream oligosaccharide.
 CHAPTER 12 ABO, H, and Lewis Blood Groups
tions. 18 A plethora of mutations associated
with weak A and B subgroups has been de-
scribed. As an example, group A 2 (a weak A
subgroup) is commonly the result of a nucleo-
tide deletion and frameshift, resulting in an
enzyme with an additional 21 amino acids at
the C-terminus of the molecule.10,18
The O allele is an amorph, encoding a
nonfunctional enzyme. The group O pheno-
type, therefore, is an autosomal recessive trait,
representing inheritance of two nonfunction-
al ABO genes. Overall, more than 50 O alleles
have been identified.7,18 The two most com-
mon O alleles (O01 and O02) contain a nucleo-
tide deletion and frameshift, leading to a trun-
cated, 117-amino-acid protein. Another
common O allele is O03 (or O 2 ), a group of
nondeletional O alleles that contains a muta-
tion at amino acid 268 (Gly268Arg), which is a
critical residue for donor binding (UDP-galac-
tose or UDP-N-acetylgalactosamine). One
German study found that O03 and a related al-
lele (Aw08) were responsible for 25% of all ABO
typing discrepancies caused by reverse-group-
ing problems in healthy donors.19 It was specu-
lated that weak anti-A and anti-B could reflect
weak residual glycosyltransferase activity;
however, a later study was unable to demon-
strate A antigen or enzyme activity in individu-
als with the O03 allele.20
ABO Subgroups
ABO subgroups are phenotypes that differ in
the amount of A and B antigen carried on red
cells and present in secretions. In general, A
subgroups are more common than B sub-
groups. Clinically, the two most common sub-
groups encountered are A1 and A2. A1 repre-
sents the majority of group A donors (80%)
and is characterized by approximately 1 mil-
lion A antigen epitopes per red cell. A2 is the
second most common subgroup (20%) and
possesses only one fifth (2.2 × 105) the number
of A antigen sites as A1. Both A1 and A2 are
strongly agglutinated by reagent anti-A in rou-
tine direct testing. A1 can be distinguished
from A2 by the lectin Dolichos biflorus, which
agglutinates A1 red cells but not A2 red cells. In
addition, 1% to 8% of individuals with A2 and
䡲 295
22% to 35% of individuals with A2B possess an
alloanti-A1 in their sera. Because the A2 pheno-
type reflects the inefficient conversion of HA
antigen, A2 red cells have increased reactivity
with the anti-H lectin Ulex europaeus. Enzyme
studies comparing A1 and A2 glycosyltransfer-
ase activity show that the A1 enzyme is 5 to 10
times more active than the A2 enzyme, result-
ing in quantitative and qualitative differences
in A antigen expression.7,10 The latter includes
the synthesis of unusual Type 3 and Type 4
chain A antigen on A1 red cells that is not pres-
ent on A2 or weaker A subgroups.10,11
In addition to A2 , several weaker A sub-
groups have been described (eg, A3, Ax, Am, and
Ael). The extremely weak A (and B) subgroups
are infrequently encountered and are usually
recognized by apparent discrepancies be-
tween red cell (forward) and serum (reverse)
grouping results. Most weak A and B sub-
groups were originally described before the
advent of monoclonal typing reagents and
were based on reactivity with human poly-
clonal anti-A, anti-B, and anti-A,B reagents.
Weak A subgroups are frequently nonreactive
with human polyclonal anti-A (see Table 12-3)
and can show variable reactivity with human
polyclonal anti-A1, anti-A,B, and murine
monoclonal antibodies (not shown).10,13,18 The
degree of reactivity with commercial murine
monoclonal reagents is clone dependent;
however, most commercially available anti-A
agglutinates A3 red cells. Because of the recip-
rocal relationship between H and synthesis of
A and B antigens, nearly all weak A and B sub-
groups have higher H expression.7 In clinical
practice, it is seldom necessary to identify a
patient’s specific A or B subgroup.
When performed, classification of weak A
subgroups is typically based on the following:
1. Degree of red cell agglutination by anti-A
and anti-A1.
2. Degree of red cell agglutination by human
and some monoclonal anti-A,B.
3. Degree of H antigen (anti-H lectin and
Ulex europaeus) expression.
4. Presence or absence of anti-A1 (Method 2-
9).
5. Presence of A and H in saliva.
296 䡲 AABB TECHNICAL MANUAL

TABLE 12-3. Serologic Reactions Observed in A and B Subgroups

Red Cell Reactions with Serum Reactions with

Antisera or Lectins Reagent Red Cells
Red Cell Saliva
Phenotype Anti-A Anti-B Anti-A,B Anti-H A1 Cells B Cells O Cells (Secretors)

A1 4+ 0 4+ 0 0 4+ 0 A&H
A2 4+ 0 4+ 2+ 0/2+* 4+ 0 A&H
mf mf
A3 2+ 0 2+ 3+ 0/2+* 4+ 0 A&H
Ax 0/ 0 1-2+ 4+ 0/2+ 4+ 0 H
Ael 0 0 0 4+ 0/2+ 4+ 0 H
B 0 4+ 4+ 0 4+ 0 0 B&H
mf mf
B3 0 1+ 2+ 4+ 4+ 0 0 B&H
Bx 0 0/ 0/2+ 4+ 4+ 0 0 H
B(A) /2+ †
4+ 4+ 0 4+ 0 0 B&H
*The occurrence of anti-A1 is variable in these phenotypes.
†
Most often detected with anti-A clones containing the MHO4 clone.
1+ to 4+ = agglutination of increasing strength;  = weak agglutination; mf = mixed-field agglutination; 0 = no agglutination.

6. Adsorption and elution studies. addition to UDP-galactose, resulting in detect-

7. Family (pedigree) studies.
B(A) and A(B) Phenotypes
The B(A) phenotype is an autosomal dominant
phenotype characterized by weak A expression
on group B red cells.13,21 Serologically, red cells
from B(A)-phenotype individuals are strongly
reactive with anti-B and weakly reactive with
monoclonal anti-A (<2+), and they possess a
strong anti-A that is reactive with both A1 and
A2 red cells in their sera. B(A) red cells can
show varying reactivity with monoclonal anti-
A reagents; however, most cases are detectable
with monoclonal typing reagents containing
the MHO4 clone. In general, the agglutination
is weak with fragile, easily dispersed aggluti-
nates. Testing the sample with polyclonal anti-
A or a different monoclonal anti-A should
resolve the discrepancy. Amino acid polymor-
phisms have been identified in some B(A) in-
dividuals near (Pro234Ala) or at (Ser235Gly)
critical amino acids.10,18 The B glycosyltransfer-
ase in these individuals has an increased ca-
pacity to use UDP-N-acetylgalactosamine in
able A antigen synthesis.
An A(B) phenotype has also been de-
scribed with monoclonal anti-B. The A(B) phe-
notype was associated with elevated H antigen
and plasma H-transferase activity. 13 It is hy-
pothesized that the increased H precursor on
these cells may permit the synthesis of some B
antigen by the A glycosyltransferase.
Acquired B Phenotype
The acquired B phenotype phenomenon is a
transient serologic discrepancy in group A in-
dividuals that is a cause of red cell grouping
discrepancies.22 Acquired B should be suspect-
ed when a patient or donor who has historical-
ly typed as group A now presents with weak B
expression on forward or red cell grouping. Se-
rologically, the acquired B phenotype shows
strong agglutination with anti-A, shows weak
agglutination (2+ or less) with monoclonal
anti-B, and contains a strong anti-B in serum.
Despite reactivity of the patient’s red cells with
reagent anti-B, the patient’s serum is not reac-
tive with autologous red cells.
 CHAPTER 12 ABO, H, and Lewis Blood Groups
Chemically, acquired B is the result of
deacetylation of the A antigen’s N-acetyl-
galactosamine, yielding a B-like galactosamine
sugar.23,24 In patients’ samples, acquired B is
often present in the setting of infection by gas-
trointestinal bacteria. Many enteric bacteria
possess a deacetylase enzyme capable of con-
verting A antigen to a B-like analog.24 Identifi-
cation of the acquired B phenotype can also be
influenced by reagent pH and specific mono-
clonal anti-B typing reagents. 22 In the past,
anti-B reagents containing the ES-4 clone were
associated with an increased incidence of ac-
quired B.
To resolve a patient’s true red cell type
and confirm the presence of acquired B, red
cells should be retyped using a different
monoclonal anti-B or acidified (pH 6.0) hu-
man anti-B. Acidified human anti-B does not
react with acquired B antigen. The ability of
monoclonal anti-B to recognize acquired anti-
B should be noted in the manufacturer’s insert.
ABO Antibodies
Anti-A and Anti-B
Immune globulin M (IgM) is the predominant
isotype found in group A and group B individ-
uals, although small quantities of IgG antibody
can be detected. In group O serum, IgG is the
major isotype for anti-A and anti-B. As a con-
sequence, hemolytic disease of the fetus and
newborn (HDFN) is more common among the
offspring of group O mothers than of mothers
with other blood types because IgG can cross
the placenta but IgM cannot.
Both IgM and IgG anti-A and anti-B pref-
erentially agglutinate red cells at room tem-
perature (20 to 24 C) or cooler, and both can
efficiently activate complement at 37 C. The
complement-mediated lytic capability of
these antibodies becomes apparent if serum
testing includes an incubation phase at 37 C.
Hemolysis caused by ABO antibodies should
be suspected when either the supernatant se-
rum is pink to red or the cell button is smaller
or absent. Hemolysis must be interpreted as a
positive result. The use of plasma for testing or
of reagent red cells suspended in solutions
䡲 297
that contain EDTA prevents complement ac-
tivation and hemolysis.
Anti-A,B
Sera from group O individuals contain an anti-
body known as “anti-A,B” because it is reactive
with both A and B red cells. Such anti-A and
anti-B reactivity cannot be separated by differ-
ential adsorption, suggesting that the anti-
body recognizes a common epitope shared by
the A and B antigens.7 Saliva containing secret-
ed A or B substance can inhibit the activity of
anti-A,B against both A and B red cells.
Anti-A1
Anti-A1 is present as an alloantibody in the se-
rum of 1% to 8% of A2 individuals and 22% to
35% of A2B individuals. Anti-A1 is sometimes
present in the sera of individuals with other
weak A subgroups. Group O serum contains a
mixture of anti-A and anti-A1.24 Anti-A1 can
cause ABO discrepancies during routine test-
ing and lead to incompatible crossmatches
with A1 and A1B red cells. Anti-A1 is usually of
IgM isotype, reacting best at room tempera-
ture or below, and is usually considered clini-
cally insignificant. Anti-A1 is considered clini-
cally significant if reactivity is observed at
37 C.24 Group A2 patients with an anti-A1 that
is reactive at 37 C testing should be transfused
with group O or A2 red cells only; group A2B pa-
tients should receive group O, A2, A2B, or B red
cells.
Routine Testing for ABO
Donor blood samples are routinely typed for
ABO at the time of donation and on receipt of
Red Blood Cell (RBC) units in the hospital
transfusion service (confirmatory typing). Re-
cipient samples are typed before transfusion.
ABO grouping requires both antigen typing of
red cells for A and B antigen (red cell grouping
or forward type) and screening of serum or
plasma for the presence of anti-A and anti-B
isoagglutinins (serum grouping or reverse
type). Both red cell and serum or plasma
grouping are required for donors and patients
because each grouping serves as a check on
298 䡲 AABB TECHNICAL MANUAL
the other. Reverse or serum grouping is not re-
quired in two circumstances: 1) for confirma-
tion testing of labeled, previously typed donor
red cells and 2) in infants younger than 4
months of age. As previously discussed, isoag-
glutinins are not present at birth and develop
only after 3 to 6 months of age.
Commercially available anti-A and anti-B
for red cell typing are extremely potent and ag-
glutinate most antigen-positive red cells di-
rectly, even without centrifugation. Most
monoclonal typing reagents have been formu-
lated to detect many weak ABO subgroups (see
manufacturers’ inserts for specific reagent
characteristics). Additional reagents (anti-A1
and anti-A,B) and special techniques to detect
weak ABO subgroups are not necessary for
routine testing but are helpful for resolving
ABO-typing discrepancies.
In contrast to commercial ABO typing re-
agents, human anti-A and anti-B in the sera of
patients and donors can be relatively weak, re-
quiring incubation and centrifugation. Tests
for serum grouping, therefore, should be per-
formed using a method that adequately de-
tects human anti-A and anti-B. Several meth-
ods are available for determining ABO group,
including slide, tube, microplate, and column
agglutination techniques.
ABO Discrepancies
Table 12-1 shows the results and interpreta-
tions of routine red cell and serum tests for
ABO. A discrepancy exists when the results of
red cell tests do not agree with those of serum
tests, usually due to unexpected negative or
positive results in either the forward or reverse
typing (see Table 12-3). ABO discrepancies
may arise from intrinsic problems with either
red cells or serum or from technical errors in
performing the test (see Table 12-4 and section
on Resolving ABO Discrepancies).
When a discrepancy is encountered, the
discrepant results must be recorded, but inter-
pretation of the ABO group must be delayed
until the discrepancy has been resolved. If the
specimen is from a donor unit, the unit must
be quarantined and cannot be released for
transfusion. When an ABO discrepancy is
identified in a patient, it may be necessary to
transfuse group O red cells pending an investi-
gation. It is important to obtain a sufficient
pretransfusion blood sample from the patient
to complete any additional studies that may be
required.
Red Cell Testing Problems
ABO testing of red cells may give unexpected
results for many reasons, including the follow-
ing:
1. Weak ABO expression that results from
inheritance of a weak ABO subgroup.
Some patients with leukemia and other
malignancies can also show weakened
ABO expression.25
2. Mixed-field agglutination with circulating
red cells of more than one ABO group fol-
lowing out-of-group red cell transfusion
or hematopoietic progenitor cell (HPC)
transplantation (eg, group O to group A).
Mixed-field agglutination is also present
in some ABO subgroups (A3), blood
group chimerism in fraternal twins, and
very rare cases of mosaicism arising from
dispermy.
3. Neutralization of anti-A and anti-B typing
reagents by high concentrations of A or B
blood group substance in serum, result-
ing in unexpected negative reactions with
serum- or plasma-suspended red cells.
4. Spontaneous agglutination or autoagglu-
tination of serum- or plasma-suspended
red cells caused by heavy coating of red
cells by potent autoagglutinins.
5. Nonspecific aggregation of serum- or
plasma-suspended red cells caused by
abnormal concentrations of serum pro-
teins or infused macromolecular solu-
tions.
6. False-positive reactions caused by a pH-
dependent autoantibody, a reagent-
dependent antibody (eg, EDTA or para-
ben), or rouleaux.
7. Anomalous red cell grouping resulting
from acquired B, B(A), or A(B) pheno-
types.
 CHAPTER 12 ABO, H, and Lewis Blood Groups 䡲 299

TABLE 12-4. Possible Causes of ABO Typing Discrepancies

Category Causes

Weak/missing red cell reactivity ABO subgroup

Leukemia/malignancy

Transfusion

Intrauterine fetal transfusion

Transplantation

Excessive soluble blood group substance

Extra red cell reactivity Autoagglutinins/excess protein coating red cells

Unwashed red cells: plasma proteins

Unwashed red cells: antibody in patient’s serum to reagent constituent

Transplantation

Acquired B antigen

B(A) phenomenon

Out-of-group transfusion

Mixed-field red cell reactivity Recent transfusion

Transplantation

Fetomaternal hemorrhage

Twin or dispermic (tetragametic) chimerism

Weak/missing serum reactivity Age related (<4-6 months old, elderly)

ABO subgroup

Hypogammaglobulinemia

Transplantation

Extra serum reactivity Cold autoantibody

Cold alloantibody

Serum antibody to reagent constituent

Excess serum protein

Transfusion of plasma components

Transplantation

Infusion of intravenous immune globulin

300 䡲 AABB TECHNICAL MANUAL
8. Polyagglutination (eg, T activation)
resulting from inherited or acquired
abnormalities of the red cell membrane,
with exposure of “cryptic autoantigens.”24
Because all human sera contain naturally
occurring antibodies to such cryptic anti-
gens, those abnormal red cells are agglu-
tinated by sera from group A, B, and AB
individuals. Monoclonal anti-A and anti-
B reagents do not detect polyagglutina-
tion.
Problems with Serum or Plasma Testing
Problems may arise during ABO testing of se-
rum or plasma, including the following:
1. Small fibrin clots in plasma or incom-
pletely clotted serum that can be mis-
taken for red cell agglutinates.
2. Lack of detectable isoagglutinins in
infants younger than 4 to 6 months. Chil-
dren do not develop isoagglutinins until 3
to 6 months of age. Isoagglutinins present
at birth are passively acquired from the
mother.
3. Unexpected absence of ABO agglutinins
caused by a weak A or B subgroup (see
Table 12-3).
4. Unexpected absence in children of anti-B
agglutinins that are sterile, free of bacte-
ria, and result from long-term parenteral
and enteral nutrition.26
5. Unexpected absence of anti-A agglutinins
in patients receiving equine-derived
immunoglobulins.27
6. ABO-incompatible HPC transplantation
with induction of tolerance. For example,
a group A patient receiving a group O
marrow transplant will have circulating
group O red cells but will produce only
anti-B in serum. (Refer to Chapter 25 for
more information on ABO-mismatched
transplants.)
7. Severe hypogammaglobulinemia second-
ary to inherited immunodeficiency or dis-
ease therapy. Hypogammaglobulinemia
with dilution of isoagglutinins can also
occur after several courses of plasma
exchange with albumin replacement.
8. Cold alloantibodies (eg, anti-M) or auto-
antibodies (eg, anti-I) reactive with
reverse-grouping cells, leading to unex-
pected positive reactions.
9. Antibodies directed against constituents
in the diluents used to preserve reagent A1
and B red cells.24
10. Nonspecific aggregation or agglutination
caused by high-molecular-weight plasma
expanders, rouleaux, high serum-protein
concentrations, or altered serum-protein
ratios.
11. Recent transfusion of out-of-group
plasma-containing components (eg, a
group A patient transfused with platelets
from a group O donor, causing unex-
pected anti-A in the patient’s plasma).
12. Recent infusion of intravenous immuno-
globulin, which can contain ABO isoag-
glutinins.
Technical Errors
Technical problems with a sample or during
testing can also lead to problems in ABO
grouping, including:
1. Specimen mix up.
2. Too heavy or too light red cell suspen-
sions.
3. Failure to add reagents.
4. Missed observation of hemolysis.
5. Failure to follow the manufacturer’s
instructions.
6. Under- or overcentrifugation of tests.
7. Incorrect interpretation or recording of
test results.
Resolving ABO Discrepancies
The first step in resolving an apparent serolog-
ic testing discrepancy should be to repeat the
test with the same sample to exclude the pos-
sibility of a technical error during testing. Ad-
ditional studies may include testing washed
red cells; testing a new sample; testing for un-
expected red cell alloantibodies; and reviewing
 CHAPTER 12 ABO, H, and Lewis Blood Groups
the patient’s medical record for conditions,
medications, or recent transfusions that may
have contributed to the conflicting test results
(Method 2-4). Samples with apparent weak or
missing ABO antigens and/or antibodies may
require tests using methods that enhance
antigen-antibody binding, including incubat-
ing red cells at 4 C (Method 2-5), using
enzyme-treated red cells (Method 2-6), and
conducting adsorption and elution studies
(Method 2-7). In some instances, it may be
necessary to test for the secretion of ABO anti-
gens in saliva (Method 2-8). Patients with sus-
pected B(A), acquired B, or A(B) phenotypes
should be retested using different monoclonal
and human polyclonal reagents.
ABO discrepancies caused by unexpected
serum reactions are not uncommon. Com-
monly encountered causes of serum-group-
ing discrepancies include cold autoantibodies,
rouleaux, cold-reacting alloantibodies (eg,
anti-M), and weak A subgroups with an anti-
A1. To resolve an ABO discrepancy caused by
an anti-A1 in a group A individual, red cells
should be tested with Dolichos biflorus lectin,
which agglutinates group A 1 but not A 2 and
weaker A subgroups. The presence of an anti-
A1 should be confirmed by testing serum
against A 1 , A2, and O red cells (Method 2-9).
Reverse-grouping problems resulting from
either a cold alloantibody (Method 2-10) or
autoantibody can be identified with a room-
temperature antibody detection test and a di-
rect antiglobulin test. Techniques to identify
ABO antibodies in the presence of cold auto-
antibodies include testing at 37 C without cen-
trifugation (Method 2-11) and cold autoad-
sorption (Method 4-5). Serum or plasma
properties can induce rouleaux formation that
resembles agglutination with A1 and B red
cells. Saline replacement or saline dilution
(Method 3-7) can be used to distinguish rou-
leaux from agglutination and identify ABO an-
tibodies.
Cold autoantibodies can cause autoag-
glutination of red cells and unexpected reac-
tions during red cell typing. Red cells heavily
coated with autoantibodies can spontaneous-
ly agglutinate and cause false-positive reac-
tions in tests with anti-A and anti-B. Usually,
䡲 301
false-positive reactions caused by autoanti-
bodies can be eliminated by washing red cells
with warm saline (Method 2-17). Autoaggluti-
nation caused by IgM can also be inhibited or
dispersed by incubating red cells in the pres-
ence of either dithiothreitol or 2-aminoethyl-
isothiouronium bromide (Method 3-16).
These reagents reduce the disulfide bonds on
IgM molecules, decreasing their polyvalency
and ability to directly agglutinate red cells.
T HE H SYS TE M
H antigen is ubiquitously expressed on all red
cells except the rare Bombay phenotype. Be-
cause H antigen serves as the precursor to
both A and B antigens, the amount of H anti-
gen on red cells depends on an individual’s
ABO type. H antigen is highly expressed on
group O red cells because group O individuals
lack a functional ABO gene. In group A and B
individuals, the amount of H antigen is con-
siderably less because H is converted to the A
and B antigens, respectively. The amount of H
antigen on red cells, based on agglutination
with the anti-H lectin Ulex europaeus, is repre-
sented thus: O>A2>B>A1>A1B. H antigen is
present on HPCs, red cells, megakaryocytes,
and other tissues.2,3,28 H antigen has been im-
plicated in cell adhesion, normal hematopoi-
etic differentiation, and several malignan-
cies.6,7,29
Biochemistry and Genetics
H antigen is defined by the terminal disaccha-
ride fucose 12 galactose. Two different fu-
cosyltransferase (FUT) enzymes are capable of
synthesizing H antigen: FUT1 (H gene) and
FUT2 (secretor gene). FUT1 specifically fu-
cosylates Type 2 chain oligosaccharides on red
cell glycoproteins and glycolipids to form Type
2 chain H. In contrast, FUT2 or secretor recog-
nizes Type 1 chain precursors to form Type 1
chain H and Leb antigens in secretions (Fig 12-
3).10 Secretion of Type 1 chain ABH antigens in
saliva and other fluids requires a functional
FUT2 gene. FUT2 is not expressed in red cells
but is expressed in salivary glands, gastrointes-
tinal tissues, and genitourinary tissues.1,7,10
 302
䡲
AABB TECHNICAL MANUAL

FIGURE 12-3. Synthesis of Type 1 chain ABH and Lewis antigens by Secretor (FUT2), Lewis (FUT3), and ABO enzymes. Type 2 chain ABH antigen synthesis is also shown
for comparison.
Fuc = fucose; Gal = galactose; GalNAc = N-acetylgalactosamine; GlcNAc = N-acetylglucosamine; R = other upstream carbohydrate sequence. Reproduced with permission
from Cooling.30
 CHAPTER 12 ABO, H, and Lewis Blood Groups
Type 1 chain ABH antigen present on red cells
is passively adsorbed from circulating glyco-
lipid antigen present in plasma (see “Lewis
System” section).24
Null Phenotypes
Bombay (Oh ) Phenotype
Originally described in Bombay, India, the Oh
or Bombay phenotype is a rare, autosomal re-
cessive phenotype characterized by the ab-
sence of H, A, and B antigens on red cells and
in secretions. Genetically, Oh individuals are
homozygous for nonfunctional H (hh) and se-
cretor (sese) genes, resulting in a complete ab-
sence of Type 1 and Type 2 chain H, A, and B.
Oh red cells type as H negative with the anti-H
lectin Ulex europaeus and monoclonal anti-H.
Because these individuals lack a functional
secretor gene necessary for Leb synthesis, Oh
individuals also type as Le(b–) (see “Lewis
System” section). Genotyping studies have de-
scribed a wide range of inactivating mutations
in both the H and secretor genes in Oh individ-
uals.10,18 The Oh phenotype is also present in
leukocyte adhesion deficiency 2 (LAD2) due to
a mutation in the GDP-fucose transporter
gene.25
Because they lack all ABH antigens, Oh in-
dividuals possess natural isohemagglutinins to
A, B, and H (see Table 12-1). In routine ABO
typing, these individuals initially type as group
O. The Oh phenotype becomes apparent dur-
ing antibody-detection tests with group O red
cells, which are rich in H antigen. The anti-H
present in Oh individuals strongly agglutinates
all group O red cells and sometimes demon-
strates in-vivo hemolysis. The Oh phenotype
can be confirmed by demonstrating an ab-
sence of H antigen on red cells and the pres-
ence of an anti-H in serum that is reactive with
group O red cells but not with Oh red cells from
other individuals.
Para-Bombay Phenotype
Individuals with the para-Bombay phenotype
are H-deficient secretors.7,10 Genetically, these
individuals are homozygous for a nonfunc-
tional H gene (hh), but they have inherited at
䡲 303
least one functional secretor gene (Se). The red
cells from H-deficient secretors lack serologi-
cally detectable H antigen but can carry small
amounts of A and/or B antigen because, unlike
persons with classic Bombay phenotype, para-
Bombay persons express Type 1 chain ABH an-
tigens in their secretions and plasma (Method
2-8).24 Type 1 chain A or B antigen in plasma is
then passively adsorbed onto red cells, result-
ing in weak A or B antigen expression. Para-
Bombay can also occur in group O individuals,
as evidenced by trace Type 1 chain H on their
red cells and in their secretions. Red cells from
para-Bombay individuals are designated “Ah,”
“Bh,” and “ABh.”
In laboratory testing, red cells from para-
Bombay individuals may (or may not) have
weak reactions with anti-A and anti-B re-
agents. In some cases, A and B antigens may
be detected only after adsorption and elution.
Para-Bombay red cells are nonreactive with
anti-H lectin, monoclonal anti-H, and human
anti-H from Oh individuals. The sera of para-
Bombay individuals contain anti-H, anti-HI,
or both and, depending on their ABO type,
anti-A and anti-B.13,24
Anti-H
Alloanti-H (Bombay and Para-Bombay)
The anti-H in Bombay and para-Bombay phe-
notypes is clinically significant and associated
with acute hemolytic transfusion reactions.
The antibody is predominantly of IgM isotype
and exhibits a broad thermal range (4 to 37 C)
with all red cells except Oh red cells. As with
anti-A and anti-B, alloanti-H is capable of acti-
vating complement and causing red cell he-
molysis.
Autoanti-H and Autoanti-HI
Autoantibodies to H and HI antigens can be
encountered in healthy individuals. When
present, these autoantibodies are most com-
mon in A1 individuals, who have very little H
antigen on their red cells. Autoanti-H and
autoanti-HI are usually of IgM isotype and are
reactive at room temperature.
304 䡲 AABB TECHNICAL MANUAL
Transfusion Practice
Alloanti-H is highly clinically significant and is
capable of fixing complement and causing he-
molytic transfusion reactions. As a result, pa-
tients with alloanti-H caused by a Bombay or
para-Bombay phenotype must be transfused
with H-negative (Oh) RBCs.
In contrast, autoantibodies against H and
HI are generally clinically insignificant. In
most patients, transfused group-specific or
group O RBCs should have normal in-vivo sur-
vival. Occasionally, autoanti-HI can result in
decreased red cell survival and hemolytic
transfusion reactions after transfusion of
group O RBCs.13,24 In most instances, hemoly-
sis follows transfusion of group O RBCs to a
group A1 or B patient with an unusually potent
high-titer anti-HI that is reactive at 37 C.24 In
such patients, it may be advisable to transfuse
group-specific (A1, B, or AB) RBCs.
TH E LEW I S SY ST E M
The Lewis blood group system consists of two
main antigens, Lea (LE1) and Leb (LE2), and
three common phenotypes, Le(a+b–), Le(a–
b+), and Le(a–b–). Four additional Lewis anti-
gens represent composite reactivity between
Lea, Leb, and ABO antigens: Leab (LE3), LebH
(LE4), ALeb (LE5), and BLeb (LE6).18,25 In addi-
tion to being present on red cells, Lewis anti-
gens are widely expressed on platelets,
endothelium, and the kidney as well as on
genitourinary and gastrointestinal epithelium.
Lewis antigens are not synthesized by red
cells but are passively adsorbed onto red cell
membranes from a pool of soluble Lewis gly-
colipid present in plasma.25 The gastrointesti-
nal tract, which is rich in Lewis-active glyco-
lipid and glycoprotein, is thought to be the
primary source of Lewis glycolipid in plasma.
Because Lewis antigens are passively adsorbed
onto red cell membranes, they can be eluted
from red cells after transfusion or by increases
in plasma volume and increased circulating li-
poproteins, which also adsorb Lewis glycolip-
id. For example, Lewis antigen is often de-
creased on red cells during pregnancy, with
some women transiently typing as Le(a–b–).
The latter is attributed to an increase in circu-
lating plasma volume and a fourfold increase
in lipoprotein.24 Leb expression and immuno-
reactivity are also influenced by ABH type as a
result of the synthesis of hybrid structures with
both Lewis and ABH activity (Fig 12-3).2,10,28
Biochemistry and Synthesis
Lewis antigen synthesis depends on the inter-
action of two distinct fucosyltransferases (Fig
12-3): Lewis (FUT3) and secretor (FUT2).25,30
Unlike H or FUT1, which is relatively specific
for Type 2 chain substrates, Lewis and secretor
preferentially fucosylate Type 1 chain sub-
strates. Secretor (FUT2) can add a terminal
12 fucose to a Type 1 chain precursor to
form Type 1 chain H antigen. The Lewis gene
encodes an 13/4 fucosyltransferase that
transfers a fucose, in an 14 linkage, to the
penultimate N-acetyl-glucosamine of Type 1
chain precursor (“Lewis c”) to form Lea anti-
gen. Lewis (FUT3) can also add a second fu-
cose to Type 1 H antigen to form Leb antigen.
Note that Leb cannot be formed from Lea be-
cause the presence of a subterminal fucose on
Lea sterically inhibits binding by the secretor
enzyme.1
In individuals with both Lewis and secre-
tor enzymes, Type 1 chain H is favored over Lea
synthesis. As a result, most of the Lewis anti-
gen synthesized is Leb [Le(a–b+) phenotype].
In group A1 and B individuals, Leb and Type 1
chain H can be further modified by ABO glyco-
syltransferases to form LE5, LE6, Type 1 A, and
Type 1 B antigens.2,30 In group A1 individuals,
the majority of Lewis antigen in plasma is ac-
tually ALeb.31
Genetics and Lewis Phenotypes
The three Lewis phenotypes commonly en-
countered represent the presence or absence
of Lewis and secretor enzymes (see Table 12-
5). Le(a+b–) individuals have inherited at least
one functional Lewis gene (Le) but are homo-
zygous for nonfunctional secretor alleles (se-
se). As a result, such individuals synthesize and
secrete Lea antigen but lack Leb and Type 1
chain ABH antigens.
 CHAPTER 12 ABO, H, and Lewis Blood Groups 䡲 305

The Le(a–b+) phenotype reflects inheri- Lewis Expression in Children

tance of both Le and Se alleles, leading to the
synthesis of Le a, Le b, and Type 1 chain ABH.
Because most Type 1 chain precursor is con-
verted to Leb in those individuals, they appear
to type as Le(a–). An Le(a+b+) phenotype is
transiently observed in infants because secre-
tor activity increases with developmental age.
An Le(a+b+) phenotype is also present in 16%
of Japanese individuals as a result of inheri-
tance of a weak secretor gene (Sew ).18 In the ab-
sence of a functional Lewis gene (lele), neither
Lea nor Leb is synthesized, leading to an Le(a–
b–) or null phenotype. Type 1 chain ABH anti-
gens may still be synthesized and secreted in
individuals who have inherited at least one
functional Se allele (Method 2-8). The Le(a–
b–) phenotype is significantly more common
in persons of African ethnicity. Although rare,
an Le(a-b-) phenotype is also present in indi-
viduals with LAD2 due to defects in fucose
transport.25
Several inactivating mutations have been
identified in both Lewis and secretor genes.18
Many of the mutations are geographically and
racially distributed, with many populations
displaying a few predominant alleles. Of the
nonfunctional Le alleles described, most have
more than one mutation.10,18
Table 12-5 shows the distribution of Lewis
types in adults. In contrast, most newborns
type as Le(a–b–) with human anti-Lewis typing
reagents. Approximately 50% of newborns
subsequently type as Le(a+) after ficin treat-
ment. The prevalence of Leb antigen, however,
is low in newborns compared to in adults be-
cause of developmental delays in secretor
(FUT2) activity. An Le(a+b+) phenotype can be
transiently present in children as the level of
secretor activity approaches adult levels. A val-
id Lewis phenotype is not developed until age
5 or 6.13
Lewis Antibodies
Antibodies against Lewis antigens are general-
ly of IgM isotype and occur naturally. Clinical-
ly, Lewis antibodies are most often encoun-
tered in the sera of Le(a–b–) individuals and
may contain a mixture of anti-Lea, anti-Leb,
and anti-Leab, an antibody capable of recog-
nizing both Le(a+) and Le(b+) red cells. Be-
cause small amounts of Lea are synthesized in
the Le(a–b+) phenotype, Le(a–b+) individuals
do not make anti-Lea. Anti-Leb is present infre-
quently in the Le(a+b–) phenotype. Lewis anti-
bodies, accompanied by a transient Le(a–b–)

TABLE 12-5. Adult Phenotypes and Prevalence in the Lewis System

Red Cell Reactions Prevalence (%) Genotype*

Anti-Lea Anti-Leb Phenotype Whites Blacks Lewis Secretor Saliva†

+ 0 Le(a+b–) 22 23 Le sese Lea

0 + Le(a–b+) 72 55 Le Se Lea, Leb, ABH
0 0 Le(a–b–) 6 22 lele sese Type 1 precursor
lele Se Type 1 ABH
+ + Le(a+b+)‡ Rare Rare Le Sew Lea, Leb
*Probable genotype at the Lewis (FUT3) and Secretor (FUT2) loci.
†
Type 1 chain antigens present in saliva and other secretions.
‡
Le(a+b+) is present in 16% of Japanese individuals and is also transiently observed in infants.
Le = gene encoding functional Lewis enzyme; lele = homozygous for gene encoding an inactive enzyme; Se = gene encoding
sactive secretor enzyme; sese = homozygous for gene encoding an inactive enzyme; Se w = gene encoding weak secretor
enzyme.
306 䡲 AABB TECHNICAL MANUAL
phenotype, are present during pregnancy. Fi-
nally, anti-Leb can demonstrate ABO specifici-
ty (anti-LebH, anti-ALeb, and anti-BLeb), and is
preferentially reactive with Le(b+) red cells of a
specific ABO group.24,28 Anti-LebH, the most
common reactivity, is more strongly reactive
with Le(b+) group O and A2 red cells than with
group A1 and B red cells, which have low H an-
tigen levels. Anti-LebL is strongly reactive with
all Le(b+) red cells, regardless of ABO group.
Most examples of Lewis antibodies are sa-
line agglutinins that are reactive at room tem-
perature. Unlike ABO, the agglutination is rela-
tively fragile and easily dispersed, requiring
gentle resuspension after centrifugation. Ag-
glutination is sometimes observed after 37 C
incubation, but the reaction is typically weaker
than that at room temperature. On occasion,
Lewis antibodies can be detected in the anti-
human globulin (AHG) phase. Such detection
may reflect either IgG or bound complement
(if polyspecific AHG reagent is used). Very
rarely, Lewis antibodies can cause in-vitro he-
molysis. Hemolysis occurs more often when
fresh serum containing anti-Lea or anti-Leab is
tested, particularly against enzyme-treated red
cells.
Transfusion Practice
In general, Lewis antibodies are not consid-
ered clinically significant. Red cells that are
compatible in tests at 37 C, regardless of Lewis
phenotype, are expected to have normal in-
vivo survival. It is not necessary to transfuse
antigen-negative RBCs in most patients. Un-
like ABO antigens, Lewis antigens are extrinsic
glycolipid antigens that are readily eluted and
shed from transfused red cells within a few
days of transfusion.25 Furthermore, Lewis anti-
gens in transfused plasma can neutralize Lew-
is antibodies in the recipient. For these rea-
sons, hemolysis is rare following transfusion of
either Le(a+) or Le(b+) red cells.
Lewis antibodies are not a cause of HD-
FN.13 Lewis antibodies are predominantly of
IgM isotype and do not cross the placenta. In
addition, Lewis antigens are poorly expressed
on neonatal red cells, with many newborns
typing as Le(a–b–) with human Lewis antibod-
ies (see above).
Disease Associations
The Leb and H antigens are receptors for Heli-
cobacter pylori, a gram-negative bacterium
implicated in gastritis, peptic ulcer disease,
gastric carcinoma, mucosa-associated lym-
phoma, and idiopathic thrombocytopenia. Leb
and Type 1 H antigens are also receptors for
noroviruses, common causes of acute gastro-
enteritis. An Le(a–b–) phenotype is associated
with increased susceptibility to infections by
Candida and uropathogenic Escherichia coli,
cardiovascular disease, and possibly de-
creased graft survival after renal transplanta-
tion.6,25
I AND i ANTIGENS
The I and i antigens are ubiquitous, structural-
ly related antigens present on all cell mem-
branes. The minimum epitope common to
both i and I is a terminal repeating lactos-
amine (Gal14GlcNAc) or Type 2 chain pre-
cursor. The minimum i antigen is a linear,
nonbranched structure containing at least two
successive lactosamine structures.14 The I anti-
gen is a polyvalent, branched glycan derived
from the i antigen (Fig 12-4). Both i and I serve
as substrate and scaffold for the synthesis of
ABO, Lewis X [Gal1-4(Fuc1-3) GlcNAc],
and other Type 2 chain antigens.1,2,14 On red
cells, i and I antigens are present on N-linked
glycoproteins and glycolipids.
Phenotypes
Two phenotypes are recognized according to
the presence or absence of I antigen: I and i (I–).
The i phenotype is characteristic of neonatal
red cells, whereas I+ is the common pheno-
type in adults. With increasing age, there is a
gradual increase in I antigen accompanied by
a reciprocal decrease in i antigen; most chil-
dren develop an adult I+ phenotype by age 2.14
An increase in i antigen can occur in people
with chronic hemolytic disorders and is a sign
of stressed erythropoiesis.32
 CHAPTER 12 ABO, H, and Lewis Blood Groups 䡲 307

FIGURE 12-4. Structure of I gene (GCNT2) (above) and synthesis of I antigen from i antigen (below).
Gal = galactose; GlcNAc = N-acetylglucosamine; R = other upstream carbohydrate sequence.

Two genetic disorders are associated with
an increase in i antigen.14 The iadult phenotype
(I–i+) is an autosomal recessive phenotype
caused by mutations in the I gene (GCNT2 or
IGnT). In populations of Asian ancestry, the
i adult phenotype can be associated with con-
genital cataracts. Increased i antigen levels are
also present in people with congenital dys-
erythropoietic anemia Type 2 (also known as
hereditary erythroblastic multinuclearity with
positive acidified serum lysis test). The latter
is a genetic defect in Golgi transport associat-
ed with abnormal glycosylation, chronic he-
molysis, splenomegaly, and erythroid multi-
nuclearity.
Genetics
The I gene (GCNT2) encodes a 16 N-acetyl-
glucosaminyltransferase that converts the lin-
ear i antigen into the branched I antigen.14,18
The gene resides on chromosome 6p24 and
contains five exons, including three tissue-
specific exons (exons 1A, 1B, and 1C). As a re-
sult, three slightly different mRNA transcripts
308 䡲 AABB TECHNICAL MANUAL
can be synthesized, depending on which exon
1 is used.
In the iadult phenotype without cataracts,
there is a mutation in exon 1C that is specific
for I antigen synthesis in red cells. As a conse-
quence, I antigen is missing on red cells but is
still synthesized in other tissues that use either
exon 1A or exon 1B. In the iadult phenotype with
cataracts, there is a loss of I antigen synthesis
in all tissues caused by either gene deletion or
mutations in exons 2 and 3.
Antibodies
Anti-I
Anti-I is common in the serum of healthy indi-
viduals. Anti-I is usually of IgM isotype and is
strongly reactive at 4 C with titers of <64. Sam-
ples with higher titers may also be detectable
at room temperature. Anti-I is identified by
strong reactions with adult red cells but weak
or no agglutination with cord red cells (see Ta-
ble 12-6). Anti-I can be enhanced by 4 C incu-
bation, the presence of albumin, or use of
enzyme-treated red cells. An alloanti-I can be
seen in the iadult phenotype.
Some examples of anti-I can demon-
strate complex reactivity and are more strong-
ly reactive with red cells of specific ABO, P1, or
Lewis phenotypes. Many of those antibodies
appear to recognize branched oligosaccha-
rides that have been further modified to ex-
press additional blood group antigens. Anti-IH
is commonly present in the serum of A1 indi-
viduals. Anti-IH is more strongly reactive with
TABLE 12-6. Comparative Serologic Behavior of the I/i Blood Group Antibodies with Saline Red Cell
Suspensions
Temperature Cell Type
4C I adult
i cord
i adult
22 C I adult
i cord
i adult
group O and group A2 red cells, which are rich
in H antigen, than with group A1 red cells. Anti-
IH is suspected when serum from a group A in-
dividual directly agglutinates all group O red
cells but is compatible with most group A
donor blood tested. Other examples of com-
plex reactivity include anti-IA, -IP1, -IBH, and
-ILebH.24
Anti-i
Autoanti-i is a relatively uncommon cold ag-
glutinin in sera from healthy individuals. Like
anti-I, anti-i is primarily of IgM isotype but is
weakly reactive at 4 to 10 C. Anti-i is most
strongly reactive with cord and iadult red cells
and more weakly reactive with I+ adult red
cells (Table 12-6). Patients with infectious
mononucleosis often have transient but po-
tent anti-i. As with anti-I, complex reactivity
can sometimes occur (eg, anti-iH).
Cold Agglutinin Syndrome
Autoanti-I and autoanti-i are pathologically
significant in cold agglutinin syndrome (CAS)
and mixed-type autoimmune hemolytic ane-
mia. In those disorders, autoanti-I (or anti-i)
behaves as a complement-binding antibody
with a high titer and wide thermal range. Pri-
mary CAS occurs with lymphoproliferative dis-
orders (eg, Waldenström macroglobulinemia,
lymphoma, and chronic lymphocytic leuke-
mia). A potent autoanti-I can also occur in the
setting of infection. Mycoplasma pneumoniae
infections are a common cause of autoanti-I
and can be accompanied by a transient hemo-
Anti-I Anti-i
4+ 0-1+
0-2+ 3+
0-1+ 4+
2+ 0
0 2-3+
0 3+
 CHAPTER 12 ABO, H, and Lewis Blood Groups 䡲 309

lysis. (See Chapter 17 for additional informa- P BL OOD GROU PS/GLOB

tion on CAS.)
The specificity of the autoantibody in CAS
may not be apparent when undiluted samples
are tested. Titration and thermal amplitude
studies may be required to discern the speci-
ficity of the autoantibodies and their potential
clinical significance. Table 12-6 illustrates the
serologic behavior of anti-I and anti-i at 4 C
and 22 C. (See Chapter 17 and Method 4-7 for
additional information regarding titration and
thermal amplitude studies.)
Transfusion Practice
Autoanti-I can interfere with ABO typing, anti-
body screening, and compatibility testing. In
laboratory testing, these antibodies can be re-
active in the antiglobulin phase of testing, par-
ticularly when polyspecific AHG is used. Such
reactions rarely indicate antibody activity at 37 C
but are the consequence of antibody binding,
followed by complement binding, at low tem-
peratures. Usually, avoiding room-tempera-
ture testing and using anti-IgG-specific AHG
prevents detection of nuisance cold autoanti-
bodies. For stronger antibody samples, auto-
antibody can be removed from serum by cold
autoadsorption techniques (see Method 4-5).
Cold autoadsorbed serum can also be used for
ABO testing.
CO L LE CT IO N
The first antigen of the P blood group sys-
tem was discovered by Landsteiner and
Levine in 1927 in a series of experiments
that also led to the discovery of M and N an-
tigens. Several related glycosphingolipid an-
tigens belong to P1PK (P1, Pk, NOR), GLOB
(P, PX2), and 209 collection (LKE).18 Pk, P, and
LKE are high-incidence antigens expressed
on nearly all red cells except rare null phe-
notypes, which lack P (Pk phenotype) or
both P and Pk antigens (p phenotype) (see
Table 12-7). Red cells are particularly rich in
P antigen, which makes up nearly 6% of to-
tal red cell lipid. Pk and P antigens are also
widely expressed on nonerythroid cells, in-
cluding lymphocytes; platelets; and plasma,
kidney, lung, heart, endothelium, placenta,
and synovium cells.33 In contrast, P1 antigen
is uniquely expressed on red cells.33
Phenotypes
More than 99% of donors have the P1 (P1+) or
P2 (P1–) phenotype (see Table 12-7). Both phe-
notypes synthesize Pk and P antigens and dif-
fer only in the expression of the P1 antigen.
Three rare, autosomal recessive phenotypes
have been identified (p, P1k, P2k) as well as weak

TABLE 12-7. Phenotypes and Prevalence in the P1PK and GLOB Group

Red Cell Reactions with Antisera Prevalence (%)

Antibodies European African

Anti-P1 Anti-P Anti-Pk Anti-PP1Pk in Serum Phenotype Ethnicity Ethnicity

+ + 0 + None P1 79 94

0 + 0 + P1* P2 21 6

0 0† 0 0 PP1Pk (Tja) p Rare Rare

+ 0 + + P P1k Rare Rare

0 0 + + P P2k Rare Rare

*An anti-P1 is detected in approximately 25% of P2 individuals.

†
Usually negative. Some examples may be weakly positive as a result of crossreactivity of anti-P with X2 and sialosyl-X2
glycolipid on p red cells.
310 䡲 AABB TECHNICAL MANUAL

variants.34 Analogous to the ABO system, the
rare p and Pk phenotypes are associated with
the presence of naturally occurring antibodies
against missing antigens (anti-P1, anti-P, and
anti-PP1Pk).
Biochemistry
The synthesis of the Pk, P, and P1 antigens
proceeds through the stepwise addition of
sugars to lactosylceramide, a ceramide dihex-
ose (CDH). (See Fig 12-5 and Table 12-8.) The
first step in this process is the synthesis of the
Pk antigen, the ultimate precursor of all globo-
type glycosphingolipids. To make the Pk anti-
gen, 1,4 galactosyltransferase 1 (A4GALT1)
adds a terminal galactose, in an 14 linkage,
to CDH. The Pk antigen can then serve as a
substrate for 1,3 N-acetylgalactosaminyl-
transferase I (B3GALNACT1), which adds a
13 N-acetylgalactosamine to the terminal
galactose of Pk (Gb3) to form P antigen. In
some cells, including red cells, the P antigen is
further elongated to form additional, globo-
family antigens, such as Luke (LKE), Type 4

GlcCer

Neolacto Family LacCer Globo Family

(CDH)
A4GALT1

Lc3 Pk
(Gb3)

B3GalNAcT1
A4GALT1
Paragloboside P NOR
(PG, nLc4) (Gb4)
A4GalT1
B3GalT5

X2 SPG P1 Gb5
ST3GAL2 FUT1/2

Sialosyl-X2 LKE Globo-H

FIGURE 12-5. Synthesis of P1, Pk, P, and related glycosphingolipid antigens. Carbohydrate structures are
shown in Table 12-8.
 CHAPTER 12 ABO, H, and Lewis Blood Groups 䡲 311

TABLE 12-8. Structures of P1, GLOB, and Related Glycosphingolipids

Family* Name Oligosaccharide Structure

CDH Gal1-4Glc1-1Cer

Globo (Gb) Gb3, Pk Gal1-4Gal1-4Glc1-1Cer

Gb4, P GalNAc1-3Gal1-4Gal1-4Glc1-1Cer

Gb5 Gal1-3GalNAc1-3Gal1-4Gal1-4Glc1-1Cer

NOR1 Gal1-4GalNAc1-3Gal1-4Gal1-4Glc1-1Cer

Globo-H Fuc1-2Gal1-3GalNAc1-3Gal1-4Gal1-4Glc1-1Cer

LKE NeuAc2-3Gal1-3GalNAc1-3Gal1-4Gal1-4Glc1-1Cer

NOR2 Gal1-4GalNAc1-3Gal1-4GalNAc1-3Gal1-4Gal1-4Glc1-1Cer

Neolacto (nLc) Lc3 GlcNAc1-3Gal1-4Glc1-1Cer

nLc4, PG Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer

P1 Gal1-4Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer

SPG NeuAc2-3Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer

X2 GalNAc1-3Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer

Sialosyl-X2 NeuAc2-3GalNAc1-3Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer
*Glycosphingolipid family. Note: Neolacto are type 2 chain glycosphingolipids.
CDH = ceramide dihexose or lactosylceramide; Cer = ceramide; Gal = galactose; GalNAc = N-acetylgalactosamine; Glc = glu-
cose, GlcNAc = N-acetylglucosamine; NeuAc = N-acetylneuramanic acid (sialic acid); PG = paragloboside; SPG = sialosyl-
paragloboside.

chain ABH antigens (globo-ABH), and NOR. Molecular Biology

NOR, a rare polyagglutinable red cell pheno-
type, is the result of unusual globo-family anti-
gens characterized by the addition of an 14
galactose to the terminus of P and related long-
chain globo-glycolipids (see Table 12-8).35
Unlike Pk and P antigens, the P1 antigen is
not a globo-glycosphingolipid but is a member
of the neolacto family (Type 2 chain glyco-
sphingolipids). In P 1 individuals, A4GALT1
adds an 14 galactose to the terminus of
paragloboside. The P1 antigen is not expressed
on red cell glycoproteins.36 The weak P-like ac-
tivity on p red cells is believed to be X2 (PX2), a
related Type 2 chain glycolipid. Recent studies
have shown that B3GALNACT1 is capable of
synthesizing PX2-active structures.37
Several inactivating mutations have been identi-
fied in both 4GALT1 and 3GALNACT1.18,38
The p phenotype is the consequence of muta-
tions in the protein-coding sequence of
A4GALT1. In the absence of A4GALT1 activity,
there is a loss of all globo-family and P1 anti-
gens. These patients have a compensatory
increase in Type 2 chain glycolipid synthesis,
as evidenced by increased paragloboside,
sialoparagloboside, and PX2.37 Mutations in
B3GalNACT1 give rise to the Pk phenotype,
which is characterized by a loss of P and LKE
antigens and by increased Pk expression. The
P2 phenotype arises from a point mutation
that introduces an alternate start codon. It is
hypothesized that this alternate A4GALT1
312 䡲 AABB TECHNICAL MANUAL
transcript or peptide may downregulate
A4GAT1 transcription with low enzyme levels
and decreased affinity for paragloboside. In-
terestingly, individuals with weak P1 expres-
sion are heterozygous for P1P 2 alleles.34
P Blood Group Antibodies
Anti-P1
Anti-P1 is a present in the sera of one-quarter
to two-thirds P2 donors.24 Anti-P1 is a naturally
occurring antibody of IgM isotype and is often
detected as a weak, room-temperature aggluti-
nin. In rare cases, anti-P1 is reactive at 37 C or
shows in-vitro hemolysis. Because anti-P1 is
nearly always IgM, anti-P1 does not cross the
placenta and has not been reported to cause
HDFN. Anti-P1 has only rarely been reported
to cause in-vivo hemolysis. Anti-P1 titers are
often elevated in patients with hydatid cyst
disease or fascioliasis (liver fluke) and in bird
handlers. It is believed that P1-like substance
in bird excrement can stimulate anti-P1 lev-
els.25 Some people with anti-P1 also have I
blood group specificity (anti-IP1).24
P1 expression varies in strength among in-
dividuals and has been reported to decrease
during in-vitro storage. 24 As a consequence,
anti-P 1 may not be reactive with all P 1 + red
cells tested. Anti-P1 can be enhanced by incu-
bation at low temperatures (eg, 4 C) or by test-
ing serum against enzyme-treated red cells.
Anti-P1 reactivity can be inhibited in the pres-
ence of hydatid cyst fluid or P1 substance de-
rived from pigeon eggs. Inhibiting P1 activity
may be helpful when testing sera containing
multiple antibodies.
Alloanti-PP1Pk and Alloanti-P
Anti-PP1Pk (historically known as anti-Tja) is a
separable mixture of anti-P, anti-P1, and anti-
Pk in the sera of p individuals. Alloanti-P is
present in the sera of P1k and P2k individuals,
occurs naturally, and is predominantly of IgM
isotype or a mixture of IgM and IgG (see Table
12-7). The antibodies are potent hemolysins
and are associated with hemolytic transfusion
reactions and, occasionally, HDFN. There is an
association between anti-PP1Pk and early
spontaneous abortion. The placenta, which is
of fetal origin, is rich in Pk and P antigen and is
a target for maternal cytotoxic IgG antibodies.
Autoanti-P (Donath-Landsteiner)
An autoantibody with P specificity is present
in patients with paroxysmal cold hemoglobin-
uria (PCH), a clinical syndrome that most
commonly occurs in children following viral
infection. In PCH, autoanti-P is an IgG bipha-
sic hemolysin capable of binding red cells at
colder temperatures, followed by intravascular
hemolysis at body temperature. This charac-
teristic can be demonstrated in vitro in the
Donath-Landsteiner test (see Chapter 17 and
Method 4-11 for further details).
Transfusion Practice
Alloanti-PP1Pk and alloanti-P are clinically sig-
nificant antibodies associated with acute he-
molytic transfusion reactions and spontane-
ous abortion. Rare individuals of p and Pk
phenotypes should be provided with antigen-
negative, crossmatch-compatible RBCs for
transfusion.
In general, anti-P1 is a clinically insignifi-
cant, room-temperature agglutinin. Patients
with anti-P 1 , which is reactive only at room
temperature or below, can be safely trans-
fused with P1+ RBCs, which results in normal
red cell survival. It is not necessary to provide
antigen-negative units to these patients. Very
rarely, anti-P1 can cause decreased red cell sur-
vival and hemolytic transfusion reactions.
Anti-P1 that is capable of fixing comple-
ment at 37 C and is strongly reactive in the
AHG phase of testing is considered potentially
clinically significant. In such rare instances,
units selected for transfusion should be nonre-
active at 37 C and in an indirect antiglobulin
test with either polyspecific AHG or anti-C3.24
Disease Associations
The Pk antigen is a receptor for Shiga toxins,
the causative agent of shigella dysentery, and
Escherichia coli-associated hemolytic uremic
syndrome.33 The Pk antigen is also a receptor
for Streptococcus suis, a species of bacteria that
 CHAPTER 12 ABO, H, and Lewis Blood Groups 䡲 313

triggers zoonotic illness capable of causing
bacterial meningitis.33 Pk expression may also
modulate host resistance to human immuno-
deficiency virus.39 The P antigen is a receptor
for parvovirus B19, the etiologic agent of ery-
thema infectiosum (fifth disease). In some pa-
tients, B19 can cause a transient anemia or
aplastic crisis. P1, Pk, P, and LKE antigens can
all serve as receptors for P-fimbriated uro-
pathogenic E. coli, a cause of chronic urinary
tract infections.33 LKE is a common oncofetal
marker present on tumors and pluripotent
embryonic, mesenchymal, and very small em-
bryonic-like stem cells.4,34

KEY PO I NT S

1. The antigens of the ABO, H, Lewis, I, and P blood group systems are defined by small car-
bohydrate epitopes on glycoproteins and glycolipids. They are widely distributed on many
tissues, including embryonic stem cells, and are considered histo-blood-group antigens.
2. The ABO system contains four major ABO phenotypes: A, B, O, and AB. The four phenotypes
are determined by the presence or absence of two antigens (A and B) on red cells. An inverse
reciprocal relationship exists between the presence of A and B antigens on red cells and the
presence of anti-A, anti-B, or both, in sera.
3. ABO grouping requires both antigen typing of red cells for A and B antigen (red cell group-
ing or forward type) and screening of serum or plasma for the presence of anti-A and anti-B
isoagglutinins (serum grouping or reverse type).
4. H antigen is ubiquitously expressed on all red cells, except the rare Bombay phenotype.
5. H antigen is the precursor to both A and B antigens; thus, the amount of H antigen on red cells
depends on the person’s ABO group. H antigen is highly expressed on group O red cells be-
cause group O persons lack a functional ABO gene. In group A and B persons, the amount of H
antigen is considerably less because H is converted to the A and B antigens, respectively.
6. Lewis antigens are not synthesized by red cells but are passively adsorbed onto red cell
membranes from a pool of soluble Lewis glycolipid present in plasma.
7. The three common Lewis phenotypes indicate the presence or absence of Lewis and secre-
tor enzymes.
8. With increasing age, there is a gradual increase in I antigen accompanied by a reciprocal de-
crease in i antigen. Most children develop an adult I+ phenotype by age 2.
9. Autoanti-I and autoanti-i are pathologically significant in cold agglutinin syndrome and
mixed-type autoimmune hemolytic anemia.
10. More than 99% of donors have the P1 (P1+) or P2 (P1–) phenotype. Both phenotypes synthe-
size Pk and P antigens and differ only in the expression of the P1 antigen.

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 C h a p t e r 1 3

The Rh System

Gregory A. Denomme, PhD, FCSMLS(D), and

Connie M. Westhoff, PhD, SBB

R H I S T H E most complex of the 34 hu- antigens—antithetical C and c, and E and e—

man blood group systems. The clinical
importance of Rh is underscored by the D anti-
gen, which is considered to be the most immu-
nogenic of all antigens. Anti-D is present in
more than 50% of Rh-negative recipients
transfused with Rh-positive blood, and the
prevalence of alloimmunization in Rh-nega-
tive females with an Rh-positive fetus was a
significant risk prior to the availability of Rh
Immune Globulin (RhIG) prophylaxis. A his-
torical account of the Rh system and its confu-
sion with the LW system has been described by
Rosenfeld.1 It was in 1939, when Levine and
Stetson made key observations that the serum
of a pregnant woman agglutinated 80% of
samples within her ABO blood group, that a
new blood group antigen was suspected to ex-
ist. These authors also proposed that “prod-
ucts of the disintegrating fetus” and an adverse
transfusion reaction in the mother to a blood
transfusion from her husband were related.2
Today, the terms “Rh positive” and “Rh
negative” refer to the presence or absence, re-
spectively, of the D antigen. Four additional Rh
are the system’s principal antigens. The anti-
gens were named by Fisher using the next
available letters of the alphabet. Thus, the five
principal Rh antigens—D, C, c, E, and e—are
responsible for the majority of clinically signif-
icant Rh antibodies among the 61 Rh antigens
that have been characterized (Table 13-1).
A true success story in transfusion medi-
cine therapy, the development of RhIG pro-
phylaxis arose from the observation that ABO
incompatibility between a mother and fetus
had a partial protective effect against immuni-
zation to D.4 The administration of IgG anti-D
obtained from human plasma was effective in
the prevention of hemolytic disease of the fe-
tus and newborn (HDFN).5 Beginning in the
mid-1960s, alloimmunization to D in pregnan-
cy was reduced to about 1 in 2000 live births.
CHARACTER IZ ATION OF RH
Rh proteins, unlike most membrane proteins,
are neither glycosylated nor phosphorylated.6,7
The use of immunoprecipitation followed by

Gregory A. Denomme, PhD, FCSMLS(D), Director of Immunohematology and Transfusion Services, Diagnos-
tic Laboratories, BloodCenter of Wisconsin, Milwaukee, Wisconsin, and Connie M. Westhoff, PhD, SBB,
Director of Immunohematology, Genomics, and Rare Blood, New York Blood Center, New York, New York
G. Denomme disclosed no conflicts of interest. C. Westhoff has disclosed financial relationships with Novar-
tis, BioArray, and Grifols.

317
318 䡲 AABB TECHNICAL MANUAL

TABLE 13-1. Terminology for Rh Antigens

ISBT Antigen or Numerical Antigen or

Designation Symbol(s) Prevalence Designation Symbol(s) Prevalence
#
RH1 D 85% Whites RH32 1% Blacks RN
92% Blacks with DBT
RH2 C 68% Whites RH33 R0Har, DHAR 0.01% Germans
27% Blacks
RH3 E 29% Whites RH34** HrB High
22% Blacks
RH4 c 80% Whites RH35 Low
96% Blacks
RH5 e 98% RH36 Bea Low
RH6 ce or f 65% Whites RH37 Evans Low (several D/CE or
92% Blacks CE/D hybrids)
RH7 Ce or rhi 68% Whites RH39 C-like High
27% Blacks
RH8 CW Low, 2% Whites RH40 Tar Low (DVII)
X
RH9 C Low, 1.8% Finns RH41 Ce-like 70% Whites
RH10 V 30% Blacks RH42 CeS 2% Blacks
W
RH11 E Low RH43 Crawford 0.1% Blacks
RH12* G 84% Whites RH44 Nou High
92% Blacks
RH17† Hr0 High RH45 Riv Low
RH18‡ Hr High RH46 Sec High
§ s
RH19 hr 98% RH47 Dav High
RH20 VS 32% Blacks RH48 JAL Low
G
RH21 C 68% Whites RH49 ††
STEM 6% Blacks
RH22 CE <1% (DCE, CE) RH50 FPTT Low (DFR, R0Har)
RH23|| DW Low (DVa) RH51 MAR High
RH26 c-like High (most c+) RH52 ||
BARC Low (DVI)
RH27 cE 28% Whites RH53 JAHK Low
22% Blacks
RH28 hrH Low RH54|| DAK Low (DIIIa, DOL, RN)
RH29¶ total Rh 100% RH55 LOCR Low
a
RH30 ||
Go Low RH56 CENR Low
§ B
RH31 hr High RH57 CEST High (antithetical to
JAL)
 CHAPTER 13 The Rh System 䡲 319

TABLE 13-1. Terminology for Rh Antigens (Continued)

ISBT Antigen or Numerical Antigen or

Designation Symbol(s) Prevalence Designation Symbol(s) Prevalence

RH58 CELO High (antithetical to RH60 PARG Low

Crawford)
RH59 CEAG High RH61 CEVF
Note: RH13 through RH16, RH24, RH25, and RH38 are obsolete.
*Present on red cells expressing C or D antigen.
†
Antibody made by individuals with D-deletion phenotypes D– –, Dc–, and DCw–.
‡
Antibody made by individuals with altered e and/or D phenotypes prevalent in groups of African ethnicity.
§
Absent from red cells with DcE/DcE (R2R2) phenotype or variant e found in groups of African ethnicity.
||
Low-prevalence antigen associated with the partial D indicated.
¶
Antibody made by individuals with Rhnull red cells.
#
Low-prevalence antigen expressed by red cells with RN or the partial DBT antigen.3
**Antibody made by individuals with altered C, E, and/or D phenotypes prevalent in groups of African ethnicity.
††
Associated with 65% of hrS– Hr– and 30% of hrB– HrB– red cells.

sodium dodecylsulfate polyacrylamide gel
electrophoresis led to the discovery that Rh
proteins have a molecular weight of 30,000 to
32,000 kDa.8-10 N-terminal amino acid se-
quencing of Rh was accomplished in the late
1980s.11 The findings led to the cloning of the
RHCE gene by Cartron12 and colleagues in
1990 and of RHD in 1992.13,14 The different
RHCE alleles responsible for the C or c and E or
e antigens were determined in 1994.15
T E R M I N O LO G Y
Early Rh nomenclature reflects the differences
in opinion concerning the number of genes
that encode DCE antigens. The Fisher-Race
terminology was based on the premise that
three closely linked genes, C/c, E/e, and D, were
responsible. In contrast, the Wiener nomen-
clature (Rh-Hr) was based on the belief that a
single gene encoded several blood group fac-
tors. There are actually two genes, RHD and
RHCE, as proposed by Tippett.16
The Fisher-Race CDE terminology is often
preferred for written communication, but a
modified version of Wiener’s nomenclature
makes it possible to identify the Rh antigens
present on one chromosome, that is, a haplo-
type, using a single term (Table 13-2). In the
modified Wiener’s nomenclature, “R” indi-
cates that D is present, and a subscripted
number or letter indicates the C/c and E/e an-
tigens: R1 for Ce, R2 for cE, Ro for ce, and RZ for
CE. In addition, “r” indicates haplotypes that
lack D, and the C/c and E/e antigens are indi-
cated using superscripted symbols: r for Ce, r
for cE, and ry for CE (Table 13-2).
The International Society of Blood Trans-
fusion (ISBT) Working Party on Terminology
for Red Cell Surface Antigens adopted six-digit
numbers to indicate red cell antigens. The first
three numbers represent the system, and the
remaining three digits refer to the antigenic
specificity; the Rh system was assigned Num-
ber 004. In 2008, the ISBT committee recog-
nized the antigens of the Rh-associated glyco-
protein (RHAG) as a new blood group system
(Number 30).17
RH GENES AND R h PROT EINS
Two genes (RHD and RHCE) are closely linked
on chromosome 1 and encode 416 amino ac-
ids. One gene encodes the D antigen, and the
other encodes CE antigens in four combina-
tions (ce, cE, Ce, or CE) [Fig 13-1(A)]. Each
320 䡲 AABB TECHNICAL MANUAL

TABLE 13-2. Prevalence of the Principal Rh Haplotypes

Modified Prevalence (%)

Fisher-Race Wiener
Haplotype Haplotype White Black Asian

Rh positive
DCe R1 42 17 70
DcE R2 14 11 21
Dce R0 4 44 3
DCE RZ <0.01 <0.01 1
Rh negative
ce ra 37 26 3
Ce r 2 2 2
cE r 1 <0.01 <0.01
CE ry <0.01 <0.01 <0.01

FIGURE 13-1. (A) RHD and RHCE genes. The inverted gene orientation, the antigens they encode, and the
deletion of RHD that results in the D-negative red cell phenotype are shown. (B) Predicted 12-transmembrane
domain model of the RhD and RhCE proteins. The amino acid differences between RhD and RhCE are shown as
grey circles. The zigzag lines represent the location of possible palmitoylation sites. Positions 103 and 226 in
RhCE critical for C or c and E or e expression are indicated as open circles.18
 CHAPTER 13
gene has 10 exons, and the two genes share
97% identity. The two proteins created by
these genes differ by 32 to 35 amino acids [Fig
13-1(B), shown as circles on RhD], depending
on whether D is compared to C or c.
The last decade has witnessed the devel-
opment of an abundance of information on
the genetic diversity of the RH locus, and the
antigens identified by molecular testing have
far exceeded the number of antigens identified
by serology. More than 275 RHD and 50 RHCE
alleles have been documented. A directory of
RHD alleles is maintained on a Rhesus-data-
base,19 and RHCE and RHD alleles are listed on
the National Center for Biotechnology Infor-
mation human blood group mutation web-
site.19,20 The ISBT Working Party on Red Cell
Immunogenetics and Blood Group Terminolo-
gy maintains, names, and catalogs new al-
leles.21
Most D-negative (Rh-negative) pheno-
types are the result of complete deletion of the
RHD gene [Fig 13-1(A)]. These phenotypes
provide the immunological rationale for why
the transfusion of Rh-positive blood to a D-
negative individual often results in produc-
tion of anti-D. The immunogenicity of a pro-
tein correlates with the degree of foreignness
to the host, and the large number of amino
acid differences in D explains why exposure
can result in a potent immune response.
RHCE [found in all but rare D– – individu-
als, where the dashes represent missing anti-
gens] encodes both C/c and E/e antigens on a
single protein. C and c antigens differ by four
amino acids, but only the serine to proline at
position 103 is predicted to be extracellular
[Fig 13-1(B)]. The E and e antigens differ by
one amino acid, a proline to alanine at amino
acid position 226, located on the fourth extra-
cellular loop of the protein. The RH genes and
proteins shown in Fig 13-1(B) are typical of the
majority of individuals. Commercial antibody
reagents are available to detect the expression
of the principal Rh antigens—D, C, c, E, and e
(Table 13-3).
The five principal antigens are responsi-
ble for the majority of Rh incompatibilities, al-
though the Rh system is more complex, with
61 antigens identified to date (Table 13-1).
The Rh System 䡲 321
New antigens may result from single nucleo-
tide polymorphisms (SNPs) to major gene re-
arrangements. For example, the genetic ex-
changes between RHD and RHCE can create
hybrid proteins that express an RhD protein
with a portion of RhCE, or vice versa. Rear-
rangements are common and thought to be fa-
cilitated by the inverted orientation of the RH
genes [Fig 13-1(A)].18 The structure promotes
hairpin loop formation and subsequent genet-
ic exchange via template conversion; one
member acts as a donor template during repli-
cation but remains unchanged in the process.
The donated region can span several base
pairs, single exons, or multiple exons.
A NT I G E N S
Routine donor and patient typing tests only for
D. Testing for other common Rh antigens is
performed primarily to resolve or confirm an-
tibody identification. Exceptions include pa-
tients receiving chronic transfusion, such as in
some sickle cell disease (SCD) programs, to
provide antigen-matched donor units to mini-
mize alloimmunization.
Rh Phenotypes
Testing of red cells with the five principal Rh
antisera has been used to predict the RH geno-
type (Table 13-3). Some haplotypes are more
common in certain ethnic groups. The fre-
quencies of the predicted RH genotypes are
uncertain (eg, the frequencies of RoRo vs Ror in
persons of African ancestry), and frequencies
are more uncertain in people of mixed ethnici-
ty. Serologic testing cannot determine whether
red cells are from a homozygous (D/D) or het-
erozygous (D/–) person because anti-D sel-
dom shows any difference in reactivity be-
tween red cells with a single or a double dose
of D antigen. RHD zygosity can be determined
by DNA molecular testing for the presence of a
RHD deletion or an inactive RHD.18
The Rh haplotype influences the level of
D antigen expression. Less D is expressed in
the presence of C, a phenomenon called the
“Ceppellini effect.”22 Red cells from a DCe/DCe
(R1R1) individual express significantly fewer D
322 䡲 AABB TECHNICAL MANUAL

TABLE 13-3. Results of Tests with Five Principal Rh Antisera with Phenotype and Predicted RH
Genotype

Antisera
Predicted Alternative
Anti-D Anti-C Anti-E Anti-c Anti-e Phenotype Genotype Genotype

Rh positive*
+ + 0 + + D, C, c, e R1r R1R0
DCe/ce DCe/Dce
R0 r
Dce/Ce
+ + 0 0 + D, C, e R1R1 R1 r
DCe/DCe DCe/Ce
+ + + + + D, C, c, E, e R1R2 R1 r
DCe/DcE DCe/cE
R2 r
DcE/Ce
RZ r
DCE/ce
R0 RZ
Dce/DCE
+ 0 0 + + D, c, e R0 r R0 R0
Dce/ce Dce/Dce
+ 0 + + + D, c, E, e R2 r R2 R0
DcE/ce DcE/Dce
R0 r
Dce/cE
+ 0 + + 0 D, c, E R2 R2 R2 r
DcE/DcE DcE/cE
+ + + 0 + D, C, E, e R1 RZ RZ r
DCe/DCE DCE/Ce
+ + + + 0 D, C, c, E R2 RZ RZ r
DcE/DCE DCE/cE
+ + + 0 0 D, C, E RZ RZ RZ r y
DCE/DCE DCE/CE

TABLE 13-3. Results of Tests with Five Principal Rh Antisera with Phenotype and Predicted RH
Genotype (Continued)
Antisera
Anti-D Anti-C Anti-E
0 0 0 +
0 + 0 +
0 0 + +
0 + + +
0 y 1 y 2 y
*Rare genotypes (R r , R r , and R r ) not shown (prevalence of <0.01%).
†
Rare genotypes (rry, rry, rry, and ryry) not shown (prevalence of <0.01%).
antigen sites than red cells from a DcE/DcE
(R2R2) individual. Therefore, it is important to
choose a consistent haplotype when titrating
anti-D in the antenatal setting because signifi-
cantly different titers can be obtained.
D Antigen
The D antigen is composed of numerous epit-
opes (designated by “epD”) that were original-
ly defined by antibodies from D-positive peo-
ple who made anti-D. Subsequent studies with
monoclonal antibodies defined 30 or more
epitopes designated as “epD1” to “epD9.”23
Each epitope has additional subdivisions (eg,
epD6.1). D epitopes are highly conformational
and consist of more than simple linear amino
acid residues. Amino acid changes in intracel-
lular regions of the protein may alter D epit-
opes.
D-Positive (Rh-Positive) Phenotypes
Most D-positive red cell phenotypes have a
conventional RhD protein [Fig 13-1(B)]. How-
ever, more than 275 different RHD alleles that
CHAPTER 13 The Rh System 䡲 323
Predicted Alternative
Anti-c Anti-e Phenotype Genotype Genotype
Rh negative†
+ c, e rr
ce/ce
+ C, c, e r r
Ce/ce
+ c, E, e rr
cE/ce
+ C, c, E, e rr
Ce/cE
encode proteins with amino acid changes
have been reported. These alleles can cause
numerous variations in the expression of D,
and red cells with some form of altered D ex-
pression are encountered in routine transfu-
sion practice. An estimated 1% to 2% of indi-
viduals of European ethnicity carry RHD
alleles that encode altered D antigens, and the
incidence in individuals of African ethnicity is
higher. Altered D is organized into four groups:
weak D, partial D (including category D), Del,
and nonfunctional RHD.24-27
WEA K D. Traditionally, weak D red cells were
defined as having a reduced amount of D anti-
gen (formerly called “Du”) that required an in-
direct antiglobulin test (IAT) for detection.
However, the number of samples identified as
being weak D depended on the typing reagent
and method used, which have changed over
the years. In 1999, Wagner and Flegel proposed
a system to classify altered D red cells on the
basis of their nucleotide substitutions (re-
viewed in Flegel and Denomme28). Weak D
types are the result of an SNP that encodes a
single amino acid change predicted to be
324 䡲 AABB TECHNICAL MANUAL

located in the intracellular or transmembrane
region of the protein, rather than on the outer
surface of the red cell (Fig 13-2). The amino
acid changes may affect the insertion of the
protein in the membrane and, thus, reflect the
reduced number of D antigen sites on the red
cells.
Uniquely different SNPs cause weak D
Types 1-84.19 The most common is weak D
Type 1, which has a valine-to-glycine amino
acid substitution at position 270. Types 1, 2,
and 3 represent approximately 90% of the
weak D types in persons of European ethnici-
ty.25 Weak D types can be further weakened
when C is present in trans to a weak D type (eg,
r in trans with weak D Type 2).
PARTIAL D. Red cells with “category D” have
historically been classified by epitope studies.
Category D individuals type as D positive, but
can make anti-D when they are exposed to
conventional D antigen. Category D pheno-
types are now classified as partial D. The ma-
jority of partial D phenotypes are due to hy-
brid genes in which portions of RHD are
replaced by corresponding portions of RHCE
(Fig 13-3). The novel sequences of the hybrid
protein resulting from regions of RhD joined to
RhCE can result in the loss of D epitopes and
also generate new antigens. For example, DVI
red cells carry the BARC antigen. A few partial
D phenotypes are the result of multiple nucle-
otide changes. Some partial D types are de-
tected only by the IAT. In contrast to weak D
types, partial changes are predicted to be lo-
cated on the exterior membrane surface (Fig
13-2).29
D EL . Red cells that express extremely low lev-
els of D antigen that cannot be detected by
routine serologic methods are designated as
“D-elution” or Del. Red cells have enough D an-
tigens to adsorb and elute anti-D. Del cells are
found in 10% to 30% of D-negative people of
Asian ancestry and result from several differ-
ent RHD mutations that severely reduce RhD
expression in the membrane. Del cells are
much less common in individuals of European
ancestry (0.027%) and carry different
nucleotide substitutions than those of Asian
ancestry.26,27 Del red cells can usually be char-
acterized by RHD genotyping and careful ad-
sorption-elution studies.

FIGURE 13-2. Structural models of weak D and partial D. The locations of amino acid changes are shown as
solid circles in the plasma membrane or on the interior. Weak D Types 1 and 2 (shown by the arrows) are found
in approximately 80% of people with weak D phenotypes. Partial D types are encoded by single amino acid
changes that are generally present on the exterior of the cell.
 CHAPTER 13 The Rh System 䡲 325

FIGURE 13-3. RHD and RHCE genes. The 10 exons of RHD and RHCE are depicted as white and grey boxes, re-
spectively. Also shown are some examples of RHD encoding partial D and of RHCE with mutations often found in
people of African ethnicity. These mutations complicate transfusions in patients with sickle cell disease.

NONFUNCTI ONAL RHD. RHD genes that
cannot produce a full-length polypeptide are
deemed nonfunctional and have been given
allele designations.21
D EPITOPES ON RHCE (RHCE). Expression
of D epitopes by the protein product of the
RHCE gene, in the absence of RHD, further
complicates serologic determination of D sta-
tus. Several Rhce (RHCE) proteins have D-spe-
cific amino acids result in epitopes that are re-
active with some monoclonal anti-D. They are
more often found in a specific population. Ex-
amples include DHAR, which is found in indi-
viduals of German ancestry, and Crawford (ce-
CF), found in individuals of African ancestry.
These two examples are notable because the
red cells show strong reactivity with some
monoclonal reagents but are nonreactive with
others, including polyclonal anti-D reagents
(Table 13-4). DHAR and Crawford phenotypes
can cause D typing discrepancies. Less dra-
matic are changes in Rhce (RHCE) proteins
that mimic a D-epitope structure encoded by
alleles designated as “ceRT ” and “ceSL.” 30,31
The red cells are weakly reactive with some,
but not all, monoclonal anti-D. Most impor-
tantly, individuals with DHAR and Crawford lack
RhD and can be sensitized to D.32,33
ELEVATED D. Several rare deletion pheno-
types, designated as “D– –,” “Dc–,” and “DCw–,”
have an enhanced expression of D antigen and
no, weak, or altered C/c and E/e antigens.34
These variants are the converse of partial D
(discussed above) and result from the replace-
ment of portions of RHCE by RHD. The addi-
tional RHD sequences in RHCE result in the
additional expression of (hybrid) D along with
TABLE 13-4. Reactivity of FDA-Licensed Anti-D Reagents with Some D Variant Red Cells
326

Crawford
DVI DBT DHAR (Whites) (Blacks)
䡲

Reagent IgM Monoclonal IgG IS/AHG* IS/AHG* IS/ AHG* IS/AHG* ceRT ceSL

Gammaclone GAMA401 F8D8 Neg/Pos Pos Pos Pos†

monoclonal
Immucor Series 4 MS201 MS26 Neg/Pos Pos Pos Neg Weakly pos Neg
monoclonal
Immucor Series 5 Th28 MS26 Neg/Pos Pos Pos Neg Weakly pos Weakly
monoclonal pos
Ortho BioClone MAD2 Polyclonal Neg/Pos Neg/Pos Neg/Neg Neg
Ortho Gel (ID-MTS) MS201 Neg Pos Pos Neg Weakly pos Neg
Biotest RH1 BS226 Neg Pos Neg
Biotest RH1 Blend BS221 BS232 Neg/Pos Pos† Neg
H4111B7
AABB TECHNICAL MANUAL

Alba Bioscience alpha LDM1 Neg Pos Neg

Alba Bioscience beta LDM3 Neg Pos Neg
Alba Bioscience delta LDM1 Neg Pos Neg
ESD1-M
Alba blend LDM3 ESD1 Neg/Pos Pos Neg
Polyclonal Neg/Pos Neg/Pos Neg/Neg Neg/Neg Weakly pos‡ Neg
*Result following slash denotes anti-D test result by the indirect antiglobulin test (IAT).
†
Test results will be negative if an IAT is performed.
‡
Enzyme-treated cells.
FDA = Food and Drug Administration; IgM = immune globulin M; IS = immediate spin; AHG = antihuman globulin; pos = positive; neg = negative.
 CHAPTER 13
normal D, which explains the enhanced D ex-
pression and reduced or missing C/c and E/e
antigens.
D-Negative (Rh-Negative) Phenotype
The D-negative phenotype is most common in
people of European ancestry (15-17%), is less
common in people of African ethnicity (3-5%),
and is rare in people of Asian ancestry
(<0.1%).35 The D-negative phenotype has arisen
multiple times, as evidenced by the different
nonfunctional alleles responsible for the lack
of D expression in various ethnic groups.
In most people of European ancestry, the
D-negative phenotype results from a deletion
of the entire RHD gene.36 There are exceptions,
however, and red cell samples with uncom-
mon haplotypes [r (Ce) or r (cE)] are more
likely to carry a nonfunctional RHD. In other
ethnic groups, D-negative phenotypes are pri-
marily caused by inactivating changes in RHD.
In individuals of African ancestry, 66% have a
37-bp insertion in RHD,37 which results in a
premature stop codon. D-negative pheno-
types in people of Asian ancestry result from
mutations in RHD that are most often associ-
ated with a Ce (r) haplotype, although 10% to
30% of people of Asian ancestry who type as D
negative are actually Del.26,27
Testing for D
Monoclonal antibody technology introduced
in the 1980s freed manufacturers from reliance
on human source material to manufacture
anti-D reagents. However, these antibodies are
specific for a single D epitope and do not de-
tect all D-positive red cells. Consequently,
most Food and Drug Administration (FDA)-
approved anti-D reagents combine a mono-
clonal IgM, which causes direct agglutination
at room temperature, with a monoclonal or
polyclonal IgG that is reactive by IAT for the
determination of weak D. Anti-D for column
agglutination testing is an exception and con-
tains only a monoclonal IgM. Three of the five
FDA-licensed reagents contain unique IgM
clones, and these may exhibit different reactiv-
ity with red cells that have weak D, partial D, or
D-like epitopes (Table 13-4).
The Rh System 䡲 327
Typing Donors for D
The goal of D typing of Red Blood Cell (RBC)
donors, including the identification of units
with weak D or partial D types, is to prevent
anti-D immunization of recipients. Therefore,
the AABB Standards for Blood Banks and
Transfusion Services requires donor blood to
be tested using a method that is designed to
detect weak expression of D. There is no re-
quirement that the typing be done using an
IAT. If the test results are positive, the unit
must be labeled as “Rh positive.”38(p31) Most
weak D or partial D antigen units are detected
but infrequently, some very weak D red cells
are not detected, and Del red cells appear non-
reactive with anti-D. Red cells with weak D an-
tigen are less immunogenic than normal D-
positive red cells, and Del donor units can stim-
ulate anti-D in some D-negative recipients.39-43
A unit labeled Rh negative must be confirmed
by testing an integrally attached segment be-
fore transfusion. Weak D testing is not re-
quired. The RhD type of the units labeled Rh
positive do not require confirmatory test-
ing.38(p35)
Typing Patients for D
When the D type of a patient is determined, a
weak D test is not necessary except to assess
the red cells of an infant whose mother is at
risk of D immunization. Today, monoclonal
IgM reagents type many samples as being D
positive in direct testing that would have pre-
viously been detected only by IAT. As a result,
some of the concerns regarding the unneces-
sary use of Rh-negative blood and RhIG have
been addressed.
DVI is the most common partial D found
in people of European ancestry, and anti-D
produced by women with partial DVI has re-
sulted in fatal hemolytic disease.44 Current
FDA-licensed monoclonal IgM reagents are
selected to be nonreactive with RBCs from
partial DVI in direct tests (Table 13-4). There-
fore, performing only the direct test on red
cells from female children and women of
childbearing potential avoids the risk of sensi-
tization by classifying DVI as D negative for
transfusion and RhIG prophylaxis. However,
328 䡲 AABB TECHNICAL MANUAL
because no IAT is performed, the results of
positive rosetting tests (to detect fetomaternal
hemorrhage) must be carefully evaluated; ma-
ternal weak D types that are reactive only in
the IAT phase have a false-positive rosette test
result.
D Typing Discrepancies
D typing discrepancies should always be in-
vestigated and resolved (see “Resolving D Typ-
ing Discrepancies” below). An Rh-negative
blood transfusion is an appropriate option for
patients needing immediate transfusion, but a
thorough clerical and serologic investigation
should be performed. RHD genotyping is also
useful to resolve D typing discrepancies.45
Because donor centers test for weak D, a
donor who is correctly classified as Rh positive
may be classified as Rh negative as a recipient.
This discrepancy should not be considered
problematic but, rather, should be communi-
cated to the patient and health-care staff and
be noted in the patient’s medical record.
Clinical Considerations
The long history of transfusing patients who
have weak D red cells with D-positive RBCs
suggests that weak D Types 1, 2, and 3, which
are present in the majority of people of Euro-
pean ancestry with weak D, are unlikely to
make anti-D. People with these weak D types
can therefore safely receive D-positive blood.
The less common weak D Types 11 and 15
have been reported to make anti-D, suggesting
that they have altered D epitopes.3 Empirical
data are needed to determine the risk of pro-
duction of anti-D in people with other weak D
types.
Unfortunately, serologic reagents cannot
typically be used to distinguish people with
partial D that is reactive only with enhanced
methods and techniques from D-positive indi-
viduals. Many partial D red cells (eg, DIIIa, the
most common partial D type in people of Afri-
can ancestry) type as strongly D positive in di-
rect tests and are recognized only after the pa-
tients produce anti-D.
Policies regarding D typing procedures
and selection of blood components for trans-
fusion should be based on the patient popula-
tion, risk of immunization to D, and limited
supply of D-negative blood components.
These policies should address what should be
done when a partial D type is found before the
patient makes anti-D. Anti-D is a clinically sig-
nificant antibody, and preventing immuniza-
tion in females of childbearing potential is im-
portant to avoid the complications of HDFN.
For other patients, the complications of anti-D
are less serious, and the decision to transfuse
Rh-positive or Rh-negative blood should take
into consideration dependence on D-negative
blood transfusions for future bleeding epi-
sodes and a possible increased risk of multiple
blood group antibodies in addition to anti-D.46
Not all D-negative patients make anti-D
when they are exposed to D-positive RBCs.
The incidence in D-negative hospitalized pa-
tients switched to D-positive blood compo-
nents is much lower, than anticipated.47 AABB
Standards requires that transfusion services
must have policies that address the adminis-
tration of D-positive red cells to D-negative
patients and the use of RhIG, which is a hu-
man blood product that is not entirely without
risk.38(p37)
G Antigen
The G antigen is found on red cells possessing
C or D and maps to the 103Ser residue on RhD,
RhCe, and RhCE (Fig 13-1). Antibodies to G ap-
pear as anti-D plus anti-C and cannot be sepa-
rated. However, the antibody can be adsorbed
by either D–C+ or D+C– red cells. The presence
of anti-G can explain why a D-negative person
who was transfused with D– (C+) blood or a D-
negative woman who delivered a D– (C+) child
and subsequently appeared to have made
anti-D. Anti-D, -C, and -G can be distinguished
by adsorption and elution studies.48 These
analyses are not usually necessary in the pre-
transfusion setting. However, it is important to
provide RhIG prophylaxis to pregnant women
who have anti-G only.
C/c and E/e Antigens
The common RHCE alleles encode the princi-
pal C or c and E or e antigens. However, more
 CHAPTER 13
than 50 different RHCE alleles are known, and
many are associated with altered or weak ex-
pression of the principal antigens and, in some
cases, loss of high-prevalence antigens.37 Par-
tial C and many partial e antigens are well rec-
ognized, and the majority are reported in indi-
viduals of African ethnicity.
Compound Antigens (ce, Ce, cE, and CE)
Compound antigens define epitopes that de-
pend on conformational changes that result
from amino acids associated with both C/c
and E/e. These antigens were previously
referred to as “cis products” to indicate that the
antigens were expressed from the same haplo-
type. However, it is now known that these anti-
gens are expressed on a single protein. These
antigens are shown in Table 13-5 and include
ce or f, Ce or rhi, cE or Rh27, and the uncom-
mon CE or Rh22.
Altered or Variant C and e Antigens
Nucleotide changes in RHCE result in quanti-
tative and qualitative changes in C/c or E/e
antigen expression; altered C and altered e are
encountered most frequently. In persons of
European ancestry, altered C is associated with
amino acid changes on the first extracellular
loop of RhCe and the expression of CW
(Gln41Arg) or CX (Ala36Thr) antigens. Altered
C is also associated with changes that result in
the expression of the novel antigens JAHK
(Ser122Leu) and JAL (Arg114Trp). These red
TABLE 13-5. Compound Rh Antigens on Rh
Proteins
Compound Present on Red
Antigen Cells with These
Designation Rh Protein Haplotypes
ce or f Rhce Dce (R0) or ce (r)
Ce or rhi, RhCe DCe (R1) or Ce (r)
Rh7
cE or Rh27 RhcE DcE (R2) or cE (r)
CE or Rh22 RhCE DCE (Rz) or CE (ry)
The Rh System 䡲 329
cells type as C positive, but these patients can
make apparent anti-C or anti-Ce (rhi) when
they are stimulated.
In individuals of African ancestry, altered
C expression most often results from the in-
heritance of a RHDIIIa-CE(4-7)-D hybrid and
less often of a RHD-CE(4-7)-D hybrid.37 These
hybrids do not encode D antigen, but they do
encode C antigen on a hybrid background that
differs from the normal background (Fig 13-3).
The gene has an incidence of approximately
20% in people of African ancestry. It is inherit-
ed with an RHCE allele designated as
“RHCE*ceS” that encodes altered e antigen and
a V–VS+ phenotype.50 The expressed product
of the hybrid RHDIIIa-CE(4-7)-D linked to
RHCE*ceS is referred to as the “(C)ceS” or “rS”
haplotype. Red cells with the rS haplotype of-
ten type as strongly C-positive with monoclo-
nal reagents, and the presence of the altered C
is undetected.
Patients with these types of red cells fre-
quently make alloantibodies with C-like, and
occasionally e-like, specificities that may
appear to be autoantibodies. The red cells
may also lack the high-prevalence hrB antigen
(hrB–).51,52 Altered or partial e expression is as-
sociated with other RHCE*ce alleles.37,53 These
alleles are found primarily in people of African
ethnicity; some examples are shown in Fig 13-
3. Some people lack the high-prevalence hrS
antigen (hrS–). Anti-hrS is a clinically signifi-
cant antibody and has caused transfusion fa-
talities.53 Not all red cells designated as hrB– or
hrS– by serologic testing are compatible with
antibodies produced by patients with altered
or partial e expression.
An additional complication is that altered
RHCE*ce is often inherited with a partial RHD
(eg, DIII, DAU, or DAR).54 As discussed above,
patients with partial D red cells are at risk of
producing anti-D.
Clinical Considerations
It has long been recognized that alloimmuni-
zation represents a significant problem in pa-
tients with SCD because 25% to 30% or more
of those who are chronically transfused devel-
op red cell antibodies. To address the problem,
330 䡲 AABB TECHNICAL MANUAL
many programs determine the pretransfusion
red cell phenotype in patients with SCD and
transfuse RBCs that are antigen matched for D,
C, E, and K because these antigens are consid-
ered to be the most immunogenic. In addition,
some programs attempt to supply RBCs from
donors of African ancestry whenever possible.
Although there is no consensus on the need to
perform red cell phenotype matching for all
patients with SCD,55 transfusion programs that
transfuse RBCs that are antigen matched for D,
C, E, and K have reduced the incidence of allo-
antibody production.56 Unfortunately, despite
matching for D, C/c, and E/e, some patients
become sensitized because they express Rh
variants.57
R H G E N OT Y PIN G
RH genotyping is a powerful adjunct to sero-
logic testing for the typing of transfused pa-
tients, RHD zygosity determination, noninva-
sive fetal D typing, determination of D status,
and identification of antigen-matched blood
for patients with SCD.
Typing Multitransfused Patients
In patients receiving chronic or massive trans-
fusions, the presence of donor red cells in the
peripheral blood often makes red cell pheno-
typing by agglutination difficult or inaccurate.
Genotyping overcomes this limitation because
blood grouping can be determined with DNA
prepared from a blood sample, even if the
sample was collected after transfusion.58
RHD Zygosity Testing
RHD zygosity can be determined by assaying
RHD dosage or confirming the presence of a
hybrid rhesus box.18,59 In prenatal practice, pa-
ternal RHD zygosity testing is important to
predict fetal D status when the mother has
anti-D. Care must be taken in the interpreta-
tion of testing results using either approach.
Several different genetic events cause an ap-
parent D-negative haplotype, and multiple
targets must be tested to accurately determine
zygosity.59,60
Fetal RHD Typing
To determine the D antigen status of a fetus,
fetal DNA can be isolated from cells obtained
by amniocentesis or chorionic villus sampling.
An alternative, noninvasive approach is to test
the maternal plasma, which contains cell-free,
fetal-derived DNA beyond 5 weeks’ gesta-
tion.61,62 In the future, determination of fetal
RHD status using this noninvasive procedure
could become routine in clinical practice to
eliminate the unnecessary administration of
antepartum RhIG to women who are carrying
a D-negative fetus.62
Confirming D Status
RHD genotyping is useful to distinguish partial
D from weak D or to resolve serologic D typing
discrepancies. Although patients with an un-
certain D status can be treated as D negative
for transfusion and RhIG administration, this
approach may be unsatisfactory for females of
childbearing potential who face unnecessary
RhIG injections and puts a strain on the limit-
ed Rh-negative blood supply. RHD genotyping
allows informed decisions to be made in this
setting (see “Weak D Types” above).
For donors, D typing discrepancies must
be resolved because errors in determining
their D status may be reportable to the FDA
and result in the recall of blood components.
D-negative, first-time donors are screened for
RHD to detect red cells with very weak D in
some European centers.3,63,64
RH Genotyping for Patients with SCD
Currently, extensive RH genotyping is time
consuming and is used to find compatible do-
nors in the American Rare Donor Program for
patients with antibodies to high-prevalence
Rh antigens.52 The future availability of high-
throughput RH genotyping platforms enables
donors to be identified by genotyping. Howev-
er, RH genotype matching patients with SCD
who have rare Rh variant types may not be
possible for chronic prophylactic transfu-
sions.65 These patients may be candidates for
stem cell transplantation.66
 CHAPTER 13
RH NULL SYNDRO ME AND RHAG
B LOO D G RO U P S Y S T EM
Erythrocytes express a third Rh-protein, RhAG.
RhAG shares 38% of its identity with RhD/
RhCE, has the same topology in the mem-
brane, and is encoded by a single gene on
chromosome 6. RhAG associates with the Rh
blood group proteins in the membrane to
form an Rh-core complex. Three red cell anti-
gens resulting from single amino acid substi-
tutions form the RHAG blood group system:
Duclos is RHAG1, Ol(a) is RHAG2, and DSLK is
RHAG3 and RHAG4.67
Red cells lacking all Rh antigens are desig-
nated as “Rhnull.” Although uncommon, the
phenotype most often results from nucleotide
changes in RHAG, known as a “regulator” type
Rhnull, indicating that the RhAG protein plays a
critical role in trafficking RhCE and RhD to the
membrane. Less often, Rhnull individuals have
RHCE nucleotide changes along with the com-
mon deletion of RHD, and these individuals
are called “amorph.”49
Rhnull red cells are stomatocytic and asso-
ciated with mild anemia, suggesting that the
Rh proteins have an important structural role
in the erythrocyte membrane. The Rh complex
is linked to the membrane skeleton through
CD47-protein 4.2 interactions and Rh/RhAG-
ankryin interactions.68,69
RH A NT IBO D IE S
Most Rh antibodies are IgG, although some
sera may have an IgM component. Typically,
Rh antibodies do not activate complement, al-
though rare exceptions have been reported. As
a result, in transfusion reactions involving Rh
antibodies, hemolysis is primarily extravascu-
lar rather than intravascular.
Most Rh antibodies have the potential to
cause clinically significant HDFN and transfu-
sion reactions. Anti-c, which is clinically the
most important Rh antibody after anti-D, may
cause severe HDFN. Anti-C, -E, and -e do not
often cause HDFN, and when they do, it is usu-
ally mild. The reactivity of Rh antibodies is en-
hanced by enzyme treatment of red cells, and
The Rh System 䡲 331
most Rh antibodies are optimally reactive at
37 C.
Concomitant Rh Antibodies
Some Rh antibodies are often found together.
For example, a DCe/DCe (R1R1) patient with
anti-E most certainly has been exposed to the
c antigen as well. Anti-c may be present in ad-
dition to anti-E but the anti-c may be weak
and undetectable at the time of testing. When
seemingly compatible E-negative blood is
transfused, it is most likely to be c positive and
may elicit an immediate or delayed transfu-
sion reaction. Therefore, some experts advo-
cate for avoiding the transfusion of c-positive
blood in this situation. In contrast, testing for
anti-E in serum containing anti-c is not war-
ranted because the patient has probably been
exposed to c without being exposed to E. In
addition, the vast majority of c-negative donor
blood is E negative (see Table 13-3).
Antibodies to High-Prevalence Rh
Antigens
Alloantibodies to high-prevalence Rh antigens
include anti-Rh29, made by some Rhnull indi-
viduals who lack Rh antigens, and others (anti-
hrS, -hrB, -HrB, and -Hr) that are most often en-
countered in transfused patients with SCD.
TE CH N I CA L CO N S I D E R AT I ON S
F O R R H T Y PIN G
High-Protein Reagents and Controls
Some Rh reagents for use in slide, rapid tube,
or microplate tests contain high concentra-
tions of protein (20% to 24%) and other mac-
romolecular additives. These reagents are pre-
pared from pools of human sera and give
reliable results; however, high-protein levels
and macromolecular additives may cause
false-positive reactions (see “Causes of False-
Positive and False-Negative Rh Typing Results”
below). These reagents must be used accord-
ing to the manufacturers’ instructions and with
the appropriate controls. False-positive results
could cause a D-negative patient to receive D-
positive blood and become immunized. If red
332 䡲 AABB TECHNICAL MANUAL
cells exhibit aggregation in the control test, the
results of the test are not valid.
Low-Protein Reagents and Controls
Most Rh reagents in routine use are low-pro-
tein reagents formulated predominantly with
IgM monoclonal antibodies. Spontaneous ag-
glutination causing a false-positive result can
occur, although this happens much less fre-
quently than with high-protein reagents. A
negative result from a test that was performed
concurrently with a similar reagent serves as a
control. For example, for ABO and Rh typing,
the absence of agglutination by anti-A or anti-B
serves as a negative control for spontaneous
aggregation. For red cells that show agglutina-
tion with all reagents (eg, type AB or D+), a
control performed as described by the reagent
manufacturer is required (with the exception
of donor retyping). In most cases, a suitable
control is a suspension of the patient’s red cells
with autologous serum or 6% to 10% albumin.
Indirect antiglobulin testing is not valid for red
cells with a positive direct antiglobulin test
(DAT) result unless a method is used to re-
move the IgG antibody. Positive and negative
controls should be tested, and the positive
control cells should have a single dose of the
antigen or be known to show weak reactivity.
Rh Testing Considerations in HDFN
Red cells from an infant with HDFN are coated
with immunoglobulin, and a low-protein re-
agent is usually necessary to test these cells.
Occasionally, red cells with a strongly positive
DAT result may be so heavily coated that they
are not agglutinated by a reagent with the
same specificity as the bound antibody. This
“blocking” phenomenon probably results
from steric hindrance, and occupied antigen
sites show false-negative results. Heat elution
of the antibody performed at 45 C permits red
cell typing, but elution must be performed
with caution to avoid denaturing the antigen.
Although detection of the antibody in an elu-
ate confirms the presence of the antigen on
the eluted red cells, RHD genotyping can be
used for confirmation of D typing.
Causes of False-Positive and False-
Negative Rh Typing Results
False-positive typing results can be caused by:
1. Immunoglobulin coating of the cells as a
result of warm or cold autoagglutinins.
Wash the red cells several times and retest
them with low-protein reagents by direct
methods. If an IAT is required, IgG coating
on the red cells can be removed by treating
the cells with glycine/EDTA (Method 2-21)
or chloroquine (Method 2-20) and retest-
ing.
2. Induction of rouleaux by serum factors that
can be eliminated by thoroughly washing
the red cells and retesting.
3. Use of the wrong reagent.
4. Contamination with reagent from another
vial.
5. Nonspecific aggregation of the red cells due
to some component of the reagent other
than the antibody (ie, a preservative, antibi-
otic, or dye).
6. Testing of polyagglutinable red cells agglu-
tinated with reagents that contain human
serum.
False-negative typing results can be caused by:
1. Failure to add the reagent. It is good prac-
tice to add typing reagent to all test tubes or
wells before adding the red cells.
2. Use of the wrong reagent.
3. A red cell suspension that is too heavy for a
tube test or too weak for a slide test.
4. Failure to detect a weak D reaction with
direct testing (immediate centrifugation).
5. Nonreactivity of a reagent with a weak or
partial form of the antigen.
6. Aggressive resuspension of the red cell but-
ton dispensing the agglutination.
7. Contamination, improper storage, or out-
dating of the reagent.
8. Red cells with a strongly positive DAT result
and antigen sites blocked because of a large
amount of bound antibody (most common
in severe HDFN caused by anti-D).
 CHAPTER 13 The Rh System 䡲 333

Resolving D Typing Discrepancies to specify the reactivity of their reagents with

To investigate D typing discrepancies, errors in
sample identification or of a clerical nature
should be eliminated by obtaining and testing
a new sample. Beyond clerical errors, multiple
variables contribute to D typing discrepancies.
These variables include the use of different
methods (ie, slide, tube, microplate, gel, and
automated analyzers using enzyme-treated
red cells), phase of testing (direct or IAT), dif-
ferent IgM clones in manufacturers’ reagents,
and large number of RHD gene variations that
affect the level of expression and epitopes of
the D antigen.
It is important to know the characteristics
of the D typing reagent used and to always
consult and follow the manufacturer’s instruc-
tions during D typing. The FDA has drafted
recommendations that require manufacturers
partial DIV, DV, and DVI red cells.70
The IgM anti-D in all of the tube reagents
currently licensed by the FDA is reactive by di-
rect testing (initial spin) with DIV and DV red
cells but has been selected to be nonreactive
with partial DVI red cells in direct testing. Lim-
ited studies have been performed to charac-
terize the reactivity of anti-D reagents with
other partial D and weak D red cells. These
studies have shown that the anti-D reagents
cannot reliably predict whether a D variant is a
weak or partial D antigen.71,72 Table 13-5 shows
the reactivity of D variant red cells that have
predictable patterns among the different anti-
D reagents. In general, an individual with par-
tial D should be considered to be a D-positive
blood donor but a D-negative transfusion re-
cipient.

KEY POINTS

1. The Rh system is highly immunogenic, complex, and polymorphic. More than 50 Rh anti-
gens have been characterized, although the five principal antigens—D, C, c, E, and e—are
responsible for the majority of clinically significant antibodies.
2. “Rh positive” and “Rh negative” refer to the presence or absence, respectively, of the D anti-
gen.
3. Contemporary Rh terminology distinguishes between antigens (such as D and C), genes
(such as RHD and RHCE), alleles (such as RHCE*ce and RHCE*Ce), and proteins (such as
RhD and Rhce).
4. Most D-negative (Rh-negative) phenotypes result from complete deletion of the RHD gene.
Exposure of D-negative individuals to RhD often results in the development of anti-D.
5. RHCE encodes both C/c and E/e antigens on a single protein. C and c differ by four amino
acids, whereas E and e differ by one amino acid.
6. Routine donor and patient Rh typing procedures test only for D. Testing for other common
Rh antigens is used to resolve or confirm antibody identification and, for many SCD trans-
fusion programs, or for other patients receiving chronic transfusions to match patients and
donors for D, C, and E.
7. Weak D phenotypes are defined as having a reduced amount of D antigen and are detected
by IAT. Weak D usually results from amino acid changes that impair the insertion of the pro-
tein in the membrane. Many different mutations cause weak expression of D.
8. RHD genotyping can identify those pregnant females and blood transfusion recipients with
a serologic weak D phenotype who can be managed safely as Rh positive.
9. Most anti-D reagents approved by the FDA combine a monoclonal IgM (that is reactive at
room temperature for routine testing) and a monoclonal or polyclonal IgG (that is reactive
by IAT for the determination of weak D). The exception is anti-D for column agglutination
testing, which contains only IgM. These reagents may show different reactivity with red cells
that have weak D, partial D, or D-like epitopes.
334 䡲 AABB TECHNICAL MANUAL

10. When determining the D type of a patient, an IAT for weak expression of D is not necessary
except when testing the red cells of an infant born to a mother at risk of D immunization.
Rh-negative donors must be tested by a method that detects weak D.
11. Most Rh antibodies are IgG, although some may have an IgM component. With rare excep-
tions, Rh antibodies do not activate complement and, thus, cause primarily extravascular
rather than intravascular hemolysis. Antibodies almost always result from red cell immuni-
zation through pregnancy or transfusion.

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35. Race RR, Sanger R. Blood groups in man. 6th
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37. Reid ME, Lomas-Francis C, Olsson ML. The
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40. Wagner T, Kormoczi GF, Buchta C, et al. Anti-D
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41. Yasuda H, Ohto H, Sakuma S, Ishikawa Y. Sec-
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42. Flegel WA, Khull SR, Wagner FF. Primary anti-
D immunization by weak D type 2 RBCs.
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alloimmunization by weak D type 1 red blood
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44. Lacey PA, Caskey CR, Werner DJ, Moulds JJ.
Fatal hemolytic disease of a newborn due to
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Transfusion 1983;23:91-4.
45. Flegel WA, Denomme GA, Yazer MH. On the
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746-52.
46. Schonewille H, van de Watering LM, Brand A.
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blood transfusions in a nonhematologic allo-
immunized patient cohort: Is it time to take
precautionary measures? Transfusion 2006;
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47. Frohn C, Dumbgen L, Brand J-M, et al. Proba-
bility of anti-D development in D–patients re-
ceiving D+ RBCs. Transfusion 2003;43:893-8.
48. Issitt PD, Anstee DJ. Applied blood group se-
rology. 4th ed. Durham, NC: Montgomery Sci-
entific Publications, 1998.
49. Singleton BK, Green CA, Avent ND, et al. The
presence of an RHD pseudogene containing a
37 base pair duplication and a nonsense mu-
tation in Africans with the Rh D-negative
blood group phenotype. Blood 2000;95:12-18.
50. Daniels GL, Faas BH, Green CA, et al. The VS
and V blood group polymorphisms in Afri-
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Transfusion 1998;38:951-8.
51. Reid ME, Storry JR, Issitt PD, et al. Rh haplo-
types that make e but not hrB usually make VS.
Vox Sang 1997;72:41-4.
52. Vege S, Westhoff CM. Molecular characteriza-
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can Rare Donor Program. Immunohematol
2006;22:143-7.
53. Noizat-Pirenne F, Lee K, Pennec PY, et al. Rare
RHCE phenotypes in black individuals of Afro-
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336 䡲 AABB TECHNICAL MANUAL
54. Westhoff CM, Vege S, Halter-Hipsky C, et al.
DIIIa and DIII Type 5 are encoded by the same
allele and are associated with altered RHCE*ce
alleles: Clinical implications. Transfusion
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55. Ness PM. To match or not to match: The ques-
tion for chronically transfused patients with
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56. Vichinsky EP, Luban NL, Wright E, et al. Pro-
spective RBC phenotype matching in a stroke
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57. Chou ST, Jackson T, Vege S, et al. High preva-
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58. Reid ME, Rios M, Powell VI, et al. DNA from
blood samples can be used to genotype pa-
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59. Pirelli KJ, Pietz BC, Johnson ST, et al. Molecular
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60. Matheson KA, Denomme GA. Novel 3 rhesus
box sequences confound RHD zygosity assign-
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Presence of fetal DNA in maternal plasma and
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Presence of RHD in serologically D–, C/E+
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67. Tilley L, Green C, Poole J, et al. A new blood
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69. Nicolas V, Le Van Kim C, Gane P, et al.
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 C h a p t e r
Other Blood Group Systems
and Antigens
Jill R. Storry, PhD, FIBMS
T H E I N T E R N A T I O N A L S O C I E T Y of
Blood Transfusion (ISBT) currently rec-
ognizes 339 antigen specificities, of which 297
belong to 1 of 34 blood group systems.1-3 Each
system represents either a single gene or two
or three closely linked homologous genes. The
ABO and Rh systems are the best known and
clinically most important systems and are de-
scribed in detail in Chapters 12 and 13. The
antigens of the H, Lewis, I, P1PK, and Globo-
side systems are carbohydrate structures that
are biochemically closely related to ABO anti-
gens and are discussed in Chapter 12. The re-
maining systems are described in this chapter;
some, generally the most important in transfu-
sion medicine, are described in some detail,
others in a few lines. They are listed in ISBT or-
der, as in Table 14-1.
In addition to the 34 systems, some
groups of antigens that are serologically, bio-
chemically, or genetically related but not eligi-
ble to join a system are classified together as
collections. Other antigens that are not eligible
to join a system or collection are of either low
or high prevalence in most major populations
Jill R. Storry, PhD, FIBMS, Associate Professor, Region Skane Office of Medical Services, Department of Clini-
cal Immunology and Transfusion Medicine, Lund, Sweden.
The author has disclosed no conflicts of interest.
1 4
and make up the 700 and 901 series, respec-
tively.1 They are all discussed at the end of this
chapter.
The full ISBT classification can be found
on the ISBT website and in Appendix 6.4 Many
more references to blood group systems and
antigens than can be provided here are avail-
able in various textbooks and reviews.5-7
The most important aspect of blood
group antigens in transfusion medicine is
whether their corresponding antibodies are
hemolytic and therefore have the potential to
cause hemolytic transfusion reactions (HTRs)
and hemolytic disease of the fetus and new-
born (HDFN).5 A guide to the potential clinical
significance of blood group antibodies is pro-
vided in Table 14-1.
T HE M N S S Y S TE M
MNS is a highly complex blood group system
consisting of 46 antigens. As with the Rh sys-
tem, much of its complexity arises from re-
combination between closely linked homolo-
gous genes.
337
TABLE 14-1. Clinical Significance of Antibodies to Blood Group Antigens
338

ISBT System No. of Hemolytic Transfusion Reaction (HTR), Acute (AHTR), or Hemolytic Disease of the Fetus and Newborn
No. Name Antigens Delayed (DHTR) (HDFN)
䡲

001 ABO 4 See Chapter 12. See Chapter 12.

002 MNS 46 Rare examples of anti-M and -N active at 37 C cause AHTRs Anti-S, -s, -U, and some other antibodies cause severe
and DHTRs; anti-S, -s, -U, and some other antibodies may HDFN; anti-M rarely causes severe HDFN.
cause AHTRs and DHTRs.

003 P1PK 3 Only very rare examples active at 37 C cause AHTRs and No.
DHTRs.

004 Rh 55 Rh antibodies can cause severe AHTRs and DHTRs Anti-D can cause severe HDFN (see Chapter 22).
(see Chapter 13).

005 Lutheran 20 Anti-Lua and -Lub have caused mild DHTRs; anti-Lu8 has No.
caused AHTRs.

006 Kell 35 Kell antibodies can cause severe AHTRs and DHTRs. Anti-K can cause severe HDFN.
AABB TECHNICAL MANUAL

007 Lewis 6 Anti-Lea and -Leb are not generally considered to be clinically No.
significant.

008 Duffy 5 Anti-Fya, -Fyb, and -Fy3 cause AHTRs and DHTRs; anti-Fy5 Anti-Fya and -Fyb cause HDFN.
causes DHTRs.

009 Kidd 3 Anti-Jka is a common cause of DHTRs; anti-Jka and -Jk3 also No. Anti-Jka does not usually cause HDFN.
cause AHTRs.

010 Diego 22 One anti-Dia caused DHTR, but there is little evidence; anti-Dib, Anti-Dia, -Dib, -Wra, and -Wrb plus some others have caused
DHTRs, and anti-Wra cause HTRs. severe HDFN.

011 Yt 2 Anti-Yta has very rarely caused HTR. No.

012 Xg 2 No. No.

013 Scianna 7 No. No.

014 Dombrock 8 Anti-Doa and -Dob cause AHTRs and DHTRs. No.

015 Colton 4 Anti-Coa causes AHTRs and DHTRs; anti-Cob and -Co3 have Anti-Coa has caused severe HDFN, and -Co3 has caused mild
caused mild HTRs. HDFN.

016 LW 3 No. No.

017 Ch/Rg 9 No. No.

018 H 1 Anti-H in Bombay phenotype can cause severe intravascular Anti-H in Bombay phenotype has the potential to cause
HTRs; anti-HI in para-Bombay is not usually clinically signifi- severe HDFN (see Chapter 12).
cant (see Chapter 12).

019 Kx 1 Anti-Kx and -Km in McLeod syndrome has caused severe Antibodies found only in males.
HTRs.

020 Gerbich 11 No. Three examples have been reported of anti-Ge3 causing
CHAPTER 14

HDFN.

021 Cromer 22 No. No.

022 Knops 9 No. No.

b
023 Indian 4 There is one example of anti-In causing an HTR. No.

024 Ok 3 Anti-Oka is very rare and no cases of HTR have been reported. No.
Other Blood Groups

025 Raph 1 No. No.

026 JMH 6 One example has been reported of anti-JMH causing AHTR. No.
䡲

(Continued)
339
 340
䡲

TABLE 14-1. Clinical Significance of Antibodies to Blood Group Antigens (Continued)

ISBT System No. of Hemolytic Transfusion Reaction (HTR), Acute (AHTR), or Hemolytic Disease of the Fetus and Newborn
No. Name Antigens Delayed (DHTR) (HDFN)

027 I 1 Anti-I in adult i phenotype has caused increased destruction No.

of I+ red cells.

028 Globoside 1 Globoside has caused intravascular HTRs. No, but anti-PP1Pk is associated with a high rate of spontane-
ous abortion.

029 Gill 1 No. No.

030 RHAG 3 No. No.

031 FORS 1 No. No.

032 JR 1 Mild DHTRs and one case of AHTR have been reported to be Two examples of severe HDFN due to anti-Jra have been
AABB TECHNICAL MANUAL

caused by anti-Jra. reported.

033 Lan 1 Mild to severe HTR due to anti-Lan has been reported. Anti-Lan is generally not a cause of HDFN, although cases of
mild HDFN have been reported.

034* Vel 1 Severe AHTR and mild to severe DHTR due to anti-Vel have Anti-Vel is generally not a cause of HDFN, although cases of
been reported. severe HDFN have been reported.

*Provisionally assigned blood group systems.

ISBT = International Society of Blood Transfusion.
 CHAPTER 14 Other Blood Groups 䡲 341

The MNS Glycoproteins and the Genes TABLE 14-2. Frequencies of Some Phenotypes
that Encode Them of the MNS System

The antigens of the MNS system are located on

one or both of two glycoproteins: glycophorin
A (GPA, CD235A) and glycophorin B (GPB,
CD235B). Each crosses the membrane once
and has an external N-terminal domain and a
C-terminal cytosolic domain. The extracellular
domains of both molecules have many sialic
acid-rich O-glycans; GPA is N-glycosylated at
asparagine-45 (26 in the mature protein),
whereas GPB is not N-glycosylated. The long
cytosolic tail of GPA interacts with the cyto-
skeleton. GPA is abundant, with about 106 cop-
ies per red cell, whereas GPB has only about
200,000 copies per cell. GPA forms an associa-
tion in the membrane with Band 3 (Diego
blood group system), and both GPA and GPB
appear to be part of the Band 3/Rh ankyrin
complex (Fig 14-1).8,9
GYPA and GYPB, the genes encoding GPA
and GPB, comprise seven and five exons, re-
spectively. A region of intron 3 of GYPB is ho-
mologous to exon 3 of GYPA but is not ex-
pressed because of a defective splice site (Fig
14-2).10 Exon 1 of each gene encodes a 19-ami-
no-acid signal peptide that is not present in
the mature protein. A third gene, GYPE, proba-
bly produces a third glycoprotein, glycopho-
rin E, but this plays little or no part in MNS an-
tigen expression.
GPA is restricted to blood cells of ery-
throid origin and is often used as an erythroid
marker. Both GPA and GPB are exploited by
the malarial parasite Plasmodium falciparum
as receptors for binding to red cells and may
be critical to the invasion process.11,12 A GPA-
like molecule has been detected on renal en-
dothelium.
M ( MNS1 ), N (MNS2) , S ( MNS3 ),
AND s (MNS4)
M and N (as detected by most anti-N reagents)
are antithetical antigens and polymorphic in
all populations tested (Table 14-2). M and N
are located at the N-terminus of GPA. M-active
GPA has serine and glycine at the first and fifth
Frequency (%)
Phenotype Whites Blacks
M+ N– 30 25
M+ N+ 49 49
M– N+ 21 26
S+ s– 10 6
S+ s+ 42 24
S– s+ 48 68
S– s– 0 2
positions of the mature protein (positions 20
and 25); N-active GPA has leucine and glutam-
ic acid at those positions.
S and s are another pair of polymorphic
antithetical antigens of the MNS system (Table
14-2). Family studies show tight linkage be-
tween M/N and S/s. The S and s antigens repre-
sent a Met48Thr (29 in mature protein) poly-
morphism in GPB. The amino-terminal 26
amino acids of GPB mature protein are usually
identical to those of the N form of GPA. Conse-
quently, in almost all people of European an-
cestry and most people of other ethnicities,
GPB expresses ‘N.’ However, because GPB is
much less abundant than GPA, most anti-N re-
agents do not detect the ‘N’ antigen on GPB.
The N-terminal region of GPA is cleaved from
intact red cells by trypsin, whereas that of GPB
is not. Consequently, M and N antigens on
GPA are trypsin sensitive, and S, s, and ‘N’ on
GPB are trypsin resistant. In contrast, with -
chymotrypsin treatment of red cells, M and N
activity is only partially reduced, whereas S, s,
and ‘N’ expression is completely destroyed. M,
N, S, s, and ‘N’ are all destroyed by treatment
of the red cells with papain, ficin, bromelin, or
pronase, although this effect with S and s is
variable.
 342
䡲
AABB TECHNICAL MANUAL

FIGURE 14-1. Model of two proposed membrane complexes containing Band 3 and Rh proteins: 1) containing tetramers of Band 3 and heterotrimers of RhD, RhCE, and
RhAG, and linked to the spectrin matrix of the cytoskeleton through Band 3, protein 4.2, and ankyrin; and 2) containing Band 3, RhD, and RhCE, and linked to the
spectrin/actin junction through glycophorin C (GPC), p55, and protein 4.1 and through Band 3 and adducin.
 CHAPTER 14 Other Blood Groups 䡲 343

FIGURE 14-2. GYPA, GYPB, and the hybrid GYPB–A–B gene responsible for GP.Mur, and a representation of the
proteins they encode, showing the regions of proteins encoded by the various exons.
 = pseudoexon not represented in the mRNA or the encoded protein.

S–s–U– Phenotype testing and screening for antibodies, these an-

The red cells of about 2% of Americans of Afri-
can ancestry and a higher proportion of
Africans are S–s– and lack the high-prevalence
antigen U (MNS5). The S–s–U– phenotype of-
ten results from homozygosity for a deletion of
the coding region of GYPB, but other, more
complex molecular phenomena involving hy-
brid genes may also give rise to an S–s– pheno-
type with expression of a variant U antigen. U
is generally resistant to denaturation by prote-
ases—papain, ficin, trypsin, -chymotryp-
sin—although anti-U is not reactive with pa-
pain-treated red cells in rare cases.
M, N, S, s, and U Antibodies and Their
Clinical Significance
Anti-M is a relatively common antibody,
whereas anti-N is quite rare. Most anti-M and
-N are not active at 37 C and are not clinically
significant.13 They can generally be ignored in
transfusion practice. If room-temperature in-
cubation is eliminated from compatibility
tibodies are not detected. When M or N anti-
bodies active at 37 C are encountered, antigen-
negative or red cells that are compatible by an
indirect antiglobulin test (IAT) should be pro-
vided.5 Very occasionally, anti-M and -N have
been implicated as the cause of acute and de-
layed HTRs, and anti-M has very rarely been
responsible for severe HDFN. A few cases of
warm autoimmune hemolytic anemia (AIHA)
caused by autoanti-N have been described,
one of which had a fatal outcome. Autoanti-M
responsible for warm AIHA has not been re-
ported.
Anti-S and -s are usually immune globu-
lin G (IgG) antibodies that are active at 37 C.
They have been implicated in HTRs and have
caused severe and fatal HDFN. Autoanti-S has
caused AIHA. If immunized, individuals with
S–s–U– red cells may produce anti-U. Anti-U
has been responsible for severe and fatal HTRs
and HDFN. Autoanti-U has been implicated in
AIHA.
344 䡲 AABB TECHNICAL MANUAL
Other MNS Antigens and Antibodies
The other MNS antigens are either of high or
low prevalence in most populations. The simi-
larity of sequence between certain regions of
GYPA and GYPB may occasionally lead to
GYPA pairing with GYPB during meiosis. If re-
combination then occurs, either by crossing
over or by a less well-defined mechanism
called “gene conversion,” a hybrid gene can be
formed consisting partly of GYPA and partly of
GYPB. A large variety of these rare hybrid
genes exists, and they give rise to low-preva-
lence antigens and, in the homozygous state,
to phenotypes that lack high-prevalence anti-
gens.10 Red cells of some of the phenotypes re-
sulting from hybrid genes react with an anti-
body called “anti-Mia.” The phenotypes were
grouped together as the Miltenberger series,
but this classification is obsolete because the
hybrids are now well defined.14
One example that is relatively common in
Southeast Asia is the hybrid gene that is re-
sponsible for the GP.Mur (previously Mi.III)
phenotype. The hybrid gene is mostly GYPB,
but a small region of GYPB encompassing the
3 end of the pseudoexon and the 5 end of the
adjacent intron has been replaced by the
equivalent region from GYPA. This means that
the defective splice site in GYPB is now re-
placed by the functional splice site from GYPA,
and the new, composite exon is expressed in
the mRNA and represented in the protein.10
This provides an unusual amino acid se-
quence that is immunogenic and represents
the antigen Mur. The amino acid sequence
that results from the junction of exons B3 and
A3 gives rise to Hil and MINY (Fig 14-2).
Mur antigen is rare in people of European
and African ethnicity but has a prevalence of
about 7% in people of Chinese and 10% in
people of Thai ancestry. Anti-Mur has the po-
tential to cause severe HTRs and HDFN. In
Hong Kong and Taiwan, anti-Mur is the most
common blood group antibody after anti-A
and -B. In Southeast Asia, it is important that
red cells for antibody detection include a Mur+
sample.15
Antibodies to regions of GPA with the ge-
neric name Ena that may be made by very rare
individuals who lack all or part of GPA have
caused severe HTRs and HDFN.
T HE LUT H E R A N SY S TE M
Lutheran is a complex system consisting of 20
antigens, including four antithetical pairs: Lua/
Lub (His77Arg); Lu6/Lu9 (Ser275Phe); Lu8/
Lu14 (Met204Lys); and Aua/Aub (Thr529Ala).16
The other Lutheran antigens are highly preva-
lent in all populations tested. Lua (LU1) has a
prevalence of about 8% in people of European
or African ethnicity but is rare elsewhere; its
antithetical antigen, Lub (LU2), is common
everywhere. In the other Lutheran polymor-
phism, Aua and Aub have a prevalence of
around 80% and 50%, respectively, in people of
European ancestry.
Lutheran antigens are destroyed by treat-
ment of the red cells with trypsin or -chymo-
trypsin, whereas papain and ficin have little ef-
fect. Most Lutheran antibodies are not reactive
with red cells treated with the sulfhydryl re-
agents 2-aminoethylisothiouronium bromide
(AET) or dithiothreitol (DTT), which reduce
the disulfide bonds of the immunoglobulin su-
perfamily (IgSF) domains (Method 3-18).
The Lutheran antigens are located on a
pair of glycoproteins that span the membrane
once, have five extracellular IgSF domains, and
differ by the length of their cytoplasmic do-
mains as a result of alternative RNA splicing.
The isoform with the longer cytoplasmic do-
main interacts with spectrin of the membrane
skeleton.17 The location of the Lutheran anti-
gens on the IgSF domains is shown in Fig 14-3.
The Lutheran glycoproteins are adhesion mol-
ecules that bind isoforms of laminin that con-
tain -5 chains. Laminin is a glycoprotein of
the extracellular matrix, and Lutheran-laminin
interactions may play a role in the migration of
mature erythroid cells from the marrow to the
peripheral blood at the latest stages of erythro-
poiesis. Upregulation of Lutheran glycopro-
teins on red cells of patients with sickle cell
disease could play a part in adhesion of these
cells to the vascular endothelium and the re-
sultant crises of vascular occlusion.17
 CHAPTER 14
FIGURE 14-3. Diagram of the two isoforms of the
Lutheran glycoprotein, showing the five extracellular
immunoglobulin superfamily domains and the
location of the Lutheran antigens on these domains,
the single membrane-spanning domain, and the
cytoplasmic domains.
The extremely rare Lunull phenotype aris-
es from homozygosity for inactive LU gene.18
The red cells lack any expression of Lutheran
antigens, and individuals with this phenotype
Other Blood Groups 䡲 345
may produce anti-Lu3, which is reactive with
all red cells except those from other Lu(a–b–)
individuals. Heterozygosity for inactivating
mutations in the erythroid transcription fac-
tor KLF1 is responsible for In(Lu), a Lumod phe-
notype with extremely weak expression of Lu-
theran antigens that are detectable only by
adsorption/elution techniques. Mutations in
KLF1 also affect other blood group genes and
cause weakened expression of several other
antigens, including P1, Inb, and AnWj.19 The
In(Lu) phenotype has a prevalence of around
0.03%. In one family, hemizygosity for a muta-
tion in the X-linked gene for the major ery-
throid transcription factor GATA-1 resulted in
an Lumod phenotype with an X-linked mode of
inheritance.20
Lutheran antibodies are most often IgG
and demonstrate reactivity best by IAT, but
have generally been implicated only in mild
delayed HTRs and have not caused severe
HDFN. Anti-Lua may be “naturally occurring”
or immune, and it is often IgM but may also be
IgG and IgA. These antibodies are usually reac-
tive by direct agglutination of Lu(a+) red cells
but often also reactive by an IAT and may show
a mixed-field-like agglutination that is charac-
teristic of this and other antibodies to Luther-
an antigens.
TH E K E L L A N D K X S Y S T EMS
The antigen often referred to as “Kell,” but cor-
rectly named “K” or “KEL1,” is the original an-
tigen of the Kell system and the first blood
group antigen to be identified following the
discovery of the antiglobulin test in 1946. Its
antithetical antigen, k or KEL2, was identified
3 years later. The Kell system now consists of
35 antigens numbered from KEL1 to KEL38, of
which three are obsolete.21,22 The Kell system
includes six pairs (K/k, Jsa/Jsb, K11/K17, K14/
K24, VLAN/VONG, and KYO/KYOR) and one
triplet (Kpa/Kpb/Kpc) of Kell antithetical anti-
gens. Initially, most antigens joined the Kell
system through genetic associations observed
in family studies. These associations have now
been confirmed by DNA sequencing of the
KEL gene.
346 䡲 AABB TECHNICAL MANUAL

The Kell Glycoprotein and the KEL TABLE 14-3. Frequencies of Some Kell
Gene Phenotypes

The Kell antigens are located on a red cell

membrane glycoprotein (CD238) with four or Frequency (%)
five N-glycans but no O-glycosylation. Kell is
Phenotype Whites Blacks
unique as a blood group antigen because it is a
Type II membrane glycoprotein; it spans the K– k+ 91.0 98
membrane once and has a short N-terminal
K+ k+ 8.8 2
domain in the cytosol and a large C-terminal
domain outside the membrane (Fig 14-4).23 K+ k– 0.2 Rare
The extracellular domain has 15 cysteine resi-
Kp(a–b+) 97.7 100
dues and is extensively folded by disulfide
bonding, although crystallographic studies are Kp(a+b+) 2.3 Rare
required to determine the molecule’s three-
Kp(a+b–) Rare 0
dimensional structure. Kell system antigens
depend on the conformation of the glycopro- Js(a–b+) 100.0 80
tein and are sensitive to disulfide-bond-reduc-
Js(a+b+) Rare 19
ing agents, such as DTT and AET.
The Kell glycoprotein is linked through a Js(a+b–) 0 1
single disulfide bond to the Xk protein (Fig 14-
K, Kpa, and Jsa are extremely rare in populations of
Asian ancestry.

4), an integral membrane protein that express-

es the Kx blood group antigen (XK1). Absence
of Xk protein from the red cell results in re-
duced expression of the Kell glycoprotein and
weakened Kell antigens (McLeod phenotype,
see below).
The KEL gene is located on chromosome
7q34. It is organized into 19 exons: exon 1 en-
codes the probable translation-initiating me-
thionine; exon 2, the cytosolic domain; exon 3,
the membrane-spanning domain; and exons 4
through 19, the large extracellular domain.

FIGURE 14-4. Diagram of the Kell and Xk proteins
linked by a single disulfide bond. The Xk protein has
cytoplasmic N- and C-terminal domains and 10
membrane-spanning domains. The Kell glycoprotein
has a large, folded, extracellular, C-terminal domain
and an intracellular N-terminal domain.
Kell Antigens
K has a prevalence of about 9% in people of
European ancestry and about 1.5% in people
of African ancestry. It is rare in East Asia (Table
14-3). The k antigen is highly prevalent in all
populations. K and k result from a single nu-
cleotide polymorphism (SNP) in exon 6, which
encodes Met193 in K and Thr193 in k. Asn191
 CHAPTER 14
is N-glycosylated in the product of the k but
not the K allele.
Kpa (KEL3) is found in about 2% of people
of European ancestry and is not present in
people of African or Japanese ancestry (Table
14-3); Kpb (KEL4) has high prevalence in all
populations. Whereas 2.3% of people of Euro-
pean ancestry are Kp(a+) only 1.2% of K+ per-
sons of that same ancestry are Kp(a+). Nine
percent of people of European ancestry are K+,
but only 2.7% of Kp(a+) people from the same
population are K+. This strong allelic associa-
tion has been confirmed by family studies.
Only one example of the KKpa haplotype has
been found.24 Kpc (KEL21), an antigen with
very low prevalence, is the product of another
allele of Kpa and Kpb. KEL alleles encoding the
three Kp antigens differ by single base substi-
tutions within codon 281 (exon 8): Kpa, TGG,
Trp281; Kpb, CGG, Arg281; and Kpc, CAG,
Gln281. The mutation associated with Kpa ex-
pression appears to reduce the quantity of Kell
glycoprotein in the red cell membranes, giving
rise to a slight reduction in expression of Kell
antigens in Kpa/Kpa homozygotes but a more
obvious weakening of Kell antigens in individ-
uals who are heterozygous for Kpa and the null
allele K0.
Jsa (KEL6) is almost completely confined
to people of African ethnicity. The prevalence
of Jsa in African Americans is about 20% (Table
14-3). Jsb (KEL7) is highly prevalent in all popu-
lations, and Js(a+b–) has not been found in
persons of non-African ethnicity. Jsa represents
Pro597; Jsb, Leu597.
K17 (Wka) (Ala302) has a prevalence of
0.3% in English donors; K11 (Val302), its anti-
thetical antigen, has very high prevalence. K14
(Arg180) and K24 (Pro180) are very high- and
very low-prevalence antigens, respectively.
VLAN and VONG are low-prevalence antigens
associated with Arg248Gln and Arg248Trp, re-
spectively.
The presence of the low-prevalence anti-
gens Ula, KYO, and K23 and the absence of the
high-prevalence antigens K12, K13, K18, K19,
K22, TOU, RAZ, KALT, KTIM, KUCI, KANT,
KASH, KELP, KETI, and KHUL result from sin-
gle amino acid substitutions in the Kell glyco-
protein.
Other Blood Groups 䡲 347
Kell antigens are resistant to papain, ficin,
trypsin, and -chymotrypsin but are de-
stroyed by a mixture of trypsin and -chymo-
trypsin. They are also destroyed by DTT and
AET (see above) and by EDTA glycine.
Kell Antibodies and Their Clinical
Significance
Kell antibodies are usually IgG, predominantly
IgG1.13 They should be considered potentially
clinically significant from the perspective of
causing severe HDFN and HTRs. Patients with
Kell antibodies should be transfused with anti-
gen-negative blood whenever possible.
Anti-K is the most common immune red
cell antibody outside the ABO and Rh systems;
one-third of all non-Rh red cell immune anti-
bodies are anti-K. An antiglobulin test is usual-
ly the method of choice for detecting anti-K,
although occasional samples may agglutinate
red cells directly. Most anti-K appears to be in-
duced by blood transfusion. Because anti-K
can cause severe HDFN, it is usual practice in
some countries for girls and women of child-
bearing potential to receive only K– red cells.
Anti-K, -k, -Kpa, -Kpb, -Jsa, -Jsb, -Ku, -Ula, -K11,
-K19, and -K22 are all reported to have caused
severe HDFN. Anti-K, -k, -Kpb, -Jsa, -Jsb, -Ku,
and -K19 have all been implicated in acute or
delayed HTRs.
The pathogenesis of HDFN caused by
anti-K differs from that resulting from anti-D.
Anti-K HDFN is associated with lower concen-
trations of amniotic fluid bilirubin than anti-D
HDFN of comparable severity. Postnatal hy-
perbilirubinemia is not prominent in infants
with anemia caused by anti-K. There is also re-
duced reticulocytosis and erythroblastosis in
the anti-K disease compared with anti-D
HDFN. These symptoms suggest that anti-K
HDFN is associated with a lower degree of he-
molysis and that fetal anemia in anti-K HDFN
results predominantly from a suppression of
erythropoiesis. The Kell glycoprotein appears
on erythroid progenitors at a much earlier
stage of erythropoiesis than do Rh antigens.
Consequently, anti-K probably facilitates
phagocytosis of K+ erythroid progenitors at an
early stage of development by macrophages in
348 䡲 AABB TECHNICAL MANUAL
the fetal liver, before the erythroid cells pro-
duce hemoglobin.25
Antibodies mimicking Kell specificities
have been responsible for severe AIHA. Pres-
ence of the autoantibody is often associated
with apparent depression of all Kell antigens.
Although most examples of anti-K are stimu-
lated by pregnancy or transfusion, a few cases
of apparently non-red-cell immune anti-K
have been described. In some cases, the anti-
bodies were found in untransfused, healthy,
male blood donors; in others, microbial infec-
tion was implicated as an immunizing agent.
Null (K0) and Mod Phenotypes
Like most blood group systems, Kell has a null
phenotype (K0) in which none of the Kell anti-
gens is expressed and the Kell glycoprotein
cannot be detected in the membrane. Immu-
nized K0 individuals may produce anti-Ku
(anti-KEL5), an antibody that is reactive with
all cells except those of the K0 phenotype. Ho-
mozygosity for a variety of nonsense, mis-
sense, and splice-site mutations have been as-
sociated with K0 phenotype.26
Kmod red cells have only very weak expres-
sion of Kell antigens, and individuals with this
phenotype are homozygous (or doubly hetero-
zygous) for missense mutations, resulting in
single-amino-acid substitutions within the
Kell glycoprotein.27 Some Kmod individuals
make an antibody that resembles anti-Ku but
differs in being nonreactive with Kmod red cells.
Other phenotypes in which Kell antigens have
substantially depressed expression result from
Kpa/K0 heterozygosity (see above), absence of
Xk protein (see below), and absence of the
Gerbich antigens Ge2 and Ge3, which are lo-
cated on the glycophorins C and D. The reason
for this phenotypic association between Kell
and Gerbich is not known, although Kell glyco-
proteins, Xk, and glycophorins C and D could
all be located within the same membrane pro-
tein complex (Fig 14-1).
Functional Aspects
The Kell protein has structural and sequence
homology with a family of zinc-dependent en-
dopeptidases that process a variety of peptide
hormones. Although the function of the Kell
glycoprotein is not known, it is enzymatically
active and can cleave the biologically inactive
peptide big-endothelin-3 to create the biologi-
cally active vasoconstrictor endothelin-3. Con-
sequently, Kell might play a role in regulating
vascular tone, but there is no direct evidence
for this.28 No obvious pathogenesis is associat-
ed with the K0 phenotype.
In addition to erythroid cells, Kell anti-
gens may be present on myeloid progenitor
cells, and Kell glycoprotein has been detected
in testis, lymphoid tissues, and with Xk protein
in skeletal muscle.
Kx Antigen (XK1), McLeod Syndrome,
and McLeod Phenotype
Kx is the only antigen of the Kx blood group
system. It is located on a polytopic protein that
spans the red cell membrane 10 times and is
linked to the Kell glycoprotein by a single di-
sulfide bond (Fig 14-4). Xk protein is encoded
by the XK gene on chromosome Xp21.1.
McLeod syndrome is a very rare X-linked
condition that develops almost exclusively in
males and is associated with acanthocytosis
and a variety of late-onset muscular, neurolog-
ic, and psychiatric symptoms. It results from
hemizygosity for inactivating mutations and
deletions of the XK gene.29 McLeod syndrome
is associated with McLeod phenotype, in
which Kell antigens are expressed weakly and
Km (KEL20) as well as Kx are absent. When
transfused, people with McLeod phenotype
without chronic granulomatous disease (CGD)
produce anti-Km only, which is compatible
with both McLeod and K0 phenotype red cells.
The function of the Xk-Kell complex is not
known, but Xk has structural resemblance to a
family of neurotransmitter transporters.
Deletion of part of the X chromosome
that includes XK may also include CYBB, ab-
sence of which is responsible for X-linked
CGD. When transfused, CGD patients with
McLeod syndrome usually produce anti-Kx
plus anti-Km, making it almost impossible to
find compatible donors. It is recommended,
 CHAPTER 14
therefore, that transfusion of males with CGD
and McLeod syndrome be avoided.
THE DUF FY SY STE M
The Duffy system officially includes five anti-
gens that reside on a glycoprotein known as
the Duffy antigen receptor for chemokines
(DARC). The DARC gene consists of two exons,
with exon 1 encoding only the first seven ami-
no acids of the Duffy glycoprotein.21,22 DARC is
on chromosome 1q23.2.
Fya (FY1) and Fyb (FY2)
In people of European and Asian ancestry, the
Duffy polymorphism consists of two antigens,
Fya and Fyb, giving rise to three phenotypes:
Fy(a+b–), Fy(a+b+), and Fy(a–b+) (Table 14-4).
The Fya and Fyb alleles represent an SNP in
exon 2 of DARC that encodes Gly42 and Asp42,
respectively, in the N-terminal extracellular
domain of the glycoprotein (Fig 14-5). Fya and
Fyb are very sensitive to most proteolytic en-
zymes, including bromelin, -chymotrypsin,
ficin, papain, and pronase, but are not de-
stroyed by trypsin. People of African ethnicity
have a third allele, Fy, that is more abundant
than Fya and Fyb. Fy produces no Duffy glyco-
protein on red cells and, hence, neither Fya nor
Fyb. Individuals who are homozygous for Fy
have the red cell phenotype Fy(a–b–), whose
prevalence varies from about 70% in African
Americans to 100% in residents of Gambia (Ta-
ble 14-4).
TABLE 14-4. Duffy Phenotypes and Genotypes in Selected Populations
Genotype
European or
Phenotype Asian Ethnicity African Ethnicity
Fy(a+b–) Fy a/Fy a Fy a/Fy a or Fy a/Fy
Fy(a+b+) Fy a/Fy b Fy a/Fy b
Fy(a–b+) Fy b/Fy b Fy b/Fy b or Fy b/Fy
Fy(a–b–) Fy/Fy Fy/Fy
Other Blood Groups 䡲 349
The coding region for the Fy allele in peo-
ple of African ancestry is identical to that of
the Fyb allele, which encodes Asp42. Fy pro-
duces no Duffy glycoprotein and no Fyb anti-
gen in red cells because of an SNP in the pro-
moter region of DARC. This mutation disrupts
the binding site for the erythroid-specific
GATA-1 transcription factor and prevents ex-
pression of the gene in erythroid tissue.30 Duffy
glycoprotein is present on many cells through-
out the body; thus, Fy(a–b–) people of African
ethnicity lack Duffy glycoprotein on their red
cells but not on cells from other tissues. This
explains why they do not make anti-Fyb and
only very rarely make anti-Fy3 or anti-Fy5 (see
below). Although the GATA-1 binding site mu-
tation in people of African ancestry has been
found only in Duffy genes with the Fyb se-
quence, the mutation has been detected in
silent Fya alleles in Papua New Guinea and
Brazil.
Fyx is a weak form of Fyb that results from
an Fyb allele with a missense mutation encod-
ing an Arg89Cys substitution in a cytosolic do-
main of the glycoprotein (Fig 14-5). Fyb anti-
gen may be undetected in some samples of
anti-Fyb, although the antigen can be detected
by adsorption/elution.
Fy3, Fy4, Fy5, and Fy6
Very rare people of non-African ancestry with
Fy(a–b–) red cells are homozygous for inacti-
vating mutations in their DARC genes. These
individuals have no Duffy glycoprotein on
Frequency (%)
Whites Blacks Japanese
20 10 81
48 3 15
32 20 4
0 67 0
350 䡲 AABB TECHNICAL MANUAL
FIGURE 14-5. Diagram of the Duffy glycoprotein
(DARC), with a glycosylated external N-terminal
domain, seven membrane-spanning domains, and
cytoplasmic C-terminus. The positions of the Fya/Fyb
polymorphism and of the amino acid substitution
responsible for the Fyx phenotype are shown.
their red cells and would not be expected to
have it in any other tissues. All were found
through the presence in their sera of anti-Fy3,
an antibody that is reactive with all red cells
except those of the Fy(a–b–) phenotype. FY6,
like Fya and Fyb, is sensitive to protease treat-
ment, whereas FY3 and FY5 are resistant. Fy5
is also absent from cells of the Fy(a–b–) pheno-
type, but unlike Fy3, Fy5 is also absent from
cells of the Rhnull phenotype. The Duffy glyco-
protein may belong to the junctional mem-
brane protein complex, which also contains
Rh proteins (Fig 14-1).9 Anti-Fy5 has been
found only in multitransfused individuals of
African ethnicity. Anti-Fy6 was the designation
given to a monoclonal antibody that produced
serologic reactions very similar to those of
anti-Fy3. Anti-Fy4 appeared to be reactive with
red cells of individuals with the silent Fy allele.
However, the work could not be repeated, the
antibody is no longer available, and Fy4 is now
obsolete.
Duffy Antibodies and Their Clinical
Significance
Anti-Fya is a relatively common antibody, and
anti-Fyb is about 20 times less common.31 IgG1
usually predominates, and these antibodies
are generally detected by an antiglobulin test.
Naturally occurring examples are very rare.
Anti-Fya and anti-Fyb may cause acute or
delayed HTRs. Although generally mild, some
have proved fatal. These antibodies have also
been responsible for from mild to severe
HDFN. Anti-Fy3 has been responsible for
acute and delayed HTRs and anti-Fy5 for de-
layed HTRs.
The Duffy Glycoprotein, a Receptor for
Chemokines
The Duffy glycoprotein is a red cell receptor for
a variety of chemokines, including interleukin-
8, monocyte chemotactic protein-1, and mela-
noma growth stimulatory activity.32 It traverses
the membrane seven times, and a 63-amino-
acid extracellular N-terminal domain contains
two potential N-glycosylation sites and a cyto-
plasmic C-terminal domain (Fig 14-5). This ar-
rangement is characteristic of the G protein-
coupled superfamily of receptors that includes
chemokine receptors.
The function of the DARC on red cells is
not known. It has been suggested that it might
act as a clearance receptor for inflammatory
mediators and that Duffy-positive red cells
function as a “sink,” or as scavengers, for the
removal of unwanted chemokines.33 If so, this
function must be of limited importance be-
cause Duffy is not present on the red cells of
most individuals of African ancestry. It has
been suggested that DARC on red cells reduces
angiogenesis and, consequently, the progres-
sion of prostate cancer by clearing angiogenic
chemokines from the tumor microenviron-
ment. This potential effect of erythroid DARC
could provide an explanation for the substan-
tially higher levels of prostate cancer in men of
African ancestry compared with those of Euro-
pean ancestry.34
DARC is present in many organs, where it
is expressed on endothelial cells lining post-
capillary venules.35 Duffy glycoprotein on vas-
cular endothelium may be involved in the
inhibition of cancer-cell metastasis and induc-
tion of cellular senescence.34 DARC may also
 CHAPTER 14 Other Blood Groups 䡲 351

facilitate movement of chemokines across the

endothelium.

The Duffy Glycoprotein and Malaria

The Duffy glycoprotein is a receptor for mero-
zoites of Plasmodium vivax, the parasite re-
sponsible for a form of malaria that is widely
distributed in Africa but is less severe than ma-
laria resulting from P. falciparum infection.
Red cells with the Fy(a–b–) phenotype are re-
sistant to invasion by P. vivax merozoites. Con-
sequently, the Fy allele confers a selective ad-
vantage in geographic areas where P. vivax is
endemic; this advantage probably balances
out any potential disadvantage resulting from
the absence of the chemokine receptor on red
cells.33

TH E K I D D S Y S TE M FIGURE 14-6. Diagram of the Kidd glycoprotein, a

The Kidd system consists of three antigens lo-
cated on a glycoprotein with 10 membrane-
spanning domains, cytoplasmic N- and C-ter-
mini, and one extracellular N-glycosylation
site (Fig 14-4).22,36 The Kidd gene (SLC14A1)
contains 11 exons, 4 through 11 encoding the
mature protein, and is located on chromo-
some 18q12.3.
Jka (JK1) and Jkb (JK2)
Jka and Jkb are the products of antithetical al-
leles and represent Asp280 and Asn280 in the
fourth external loop of the Kidd glycoprotein
(Fig 14-6). They have similar prevalence in
populations of European and Asian ancestry,
but Jka is more common than Jkb in people of
African ancestry (Table 14-5). Kidd antigens
are resistant to proteolytic enzymes, such as
papain and ficin.
urea transporter, with cytoplasmic N- and C-terminal
domains, 10 membrane-spanning domains, and an
N-glycan on the third extracellular loop. The position
of the Jka/Jkb polymorphism is shown on the fourth
external loop.
sian null allele contains a splice site mutation
in intron 5 that results in the loss of exon 6
from the mRNA. In people of Finnish ancestry,
where Jk(a–b–) is less rare than in other popu-
lations of European ancestry, the mutation re-
sponsible encodes a Ser291Pro substitution.
Immunized individuals with the Jk(a–b–) phe-
notype may produce anti-Jk3. An extremely
rare form of Jk(a–b–) phenotype found in
TABLE 14-5. Kidd Phenotypes in Three
Populations

Jk(a–b–) and Jk3 Frequency (%)

The null phenotype, Jk(a–b–) Jk:–3, usually re-
Phenotype Whites Blacks Asians
sults from homozygosity for a silent gene at
the JK locus. Although very rare in most popu- Jk(a+b–) 26 52 23
lations, the null phenotype is relatively com-
mon in people of Polynesian ethnicity, with a Jk(a+b+) 50 40 50
prevalence of around 1 in 400, but as high as Jk(a–b+) 24 8 27
1.4% in those of Niuean ancestry. The Polyne-
352 䡲 AABB TECHNICAL MANUAL
people of Japanese ethnicity results from het-
erozygosity for a dominant inhibitor gene,
named In(Jk) in analogy with the In(Lu) domi-
nant inhibitor of Lutheran and other antigens.
Very weak expression of Jka and/or Jkb can be
detected on In(Jk) red cells by adsorption/elu-
tion tests.
Kidd Antibodies and Their Clinical
Significance
Anti-Jka and –Jkb are not common antibodies
and are generally found in antibody mixtures.
They are usually IgG1 and IgG3, but some are
partly IgG2, IgG4, or IgM. About 50% of anti-Jka
and –Jkb bind complement.13
Kidd antibodies are often difficult to de-
tect. Some agglutinate antigen-positive cells
directly, but the reactions are usually weak.
Generally, an antiglobulin test is required, and
use of enzyme-treated cells may be necessary
to detect weaker antibodies.
Kidd antibodies may cause severe acute
HTRs. They are also a very common cause of
delayed HTRs, probably because they are of-
ten not detected in pretransfusion testing due
to their tendency to drop to low or undetect-
able levels in plasma. Anti-Jk3 can also cause
acute or delayed HTRs. Despite their hemolyt-
ic potential, Kidd antibodies only very rarely
cause severe HDFN.
Kidd antibodies have been implicated in
acute renal transplant rejection, suggesting
that Kidd antigens can behave as histocom-
patibility antigens.37
The Kidd Glycoprotein in Urea
Transportation
The Kidd antigens are located on a red cell
urea transporter, also known as “human urea
transporter 11” (HUT11 or UT-B1).38 When red
cells approach the renal medulla, which con-
tains a high concentration of urea, the urea
transporter permits rapid uptake of urea and
prevents the cells from shrinking in the hyper-
tonic environment. As the red cells leave the
renal medulla, urea is transported rapidly out
of the cells, preventing the cells from swelling
and carrying urea away from the kidney.
HUT11 has been detected on endothelial cells
of the vasa recta, the vascular supply of the re-
nal medulla, but it is not present in renal tu-
bules.
Normal red cells are rapidly lysed by 2M
urea because urea transported into the cells
makes them hypertonic and they burst as a re-
sult of the osmotic influx of water. Because of
the absence of the urea transporter, Jk(a–b–)
cells are not hemolyzed by 2M urea, and this
can be used as a method for screening for Jk(a–
b–) donors.39
The Jk(a–b–) phenotype is not associated
with any clinical defect, although two unrelat-
ed Jk(a–b–) individuals had a mild urine-
concentrating defect.40
T HE D IE GO S Y ST EM
Band 3, the Red Cell Anion Exchanger
The 22 antigens of the Diego system are locat-
ed on Band 3, the common name for the red
cell anion exchanger or solute carrier family
4 A1 (SLC4A1).41 Band 3 is a major red cell
membrane glycoprotein with approximately
106 copies per red cell. Band 3 has a transmem-
brane domain that traverses the membrane
about 14 times, with an N-glycan on the fourth
extracellular loop. Band 3 also has a long cyto-
plasmic N-terminal domain that interacts with
the membrane skeleton proteins ankyrin,
4.1R, and protein 4.2 and functions as a bind-
ing site for hemoglobin (Figs 14-1 and 14-7).
The short cytoplasmic C-terminal domain
binds carbonic anhydrase II.
Band 3 in red cells has at least two major
functions: the rapid exchange of HCO3– and Cl–
ions, which are important in CO2 transport,
and attachment of the red cell membrane to
the cytoskeleton.9 Tetramers of Band 3 form
the core of the Band 3/Rh ankyrin macrocom-
plex of red cell membrane proteins, which
could function as a gas channel for O2 and
CO2.8 Band 3 is also a component of the junc-
tional complex that links the red cell mem-
brane to the membrane skeleton via glycopho-
rin C (Fig 14-1).9 The Band 3 gene (SLC4A1)
 CHAPTER 14
FIGURE 14-7. Diagram of Band 3, the Diego
glycoprotein and anion exchanger, with cytoplasmic
N- and C-terminal domains, 14 membrane-spanning
domains, and an N-glycan on the fourth extracellular
loop (although the precise conformation is still
controversial). The locations of 22 antigens of Diego
system on the extracellular loops are shown.
consists of 20 exons of coding sequence and is
on chromosome 17.
Dia (DI1) and Dib (DI2); Anti-Dia and -Dib
Dia, the original Diego antigen, is very rare in
people of European or African ancestry but
has a prevalence of 5% in people of Chinese or
Japanese ancestry and an even higher preva-
lence in the indigenous peoples of North and
South America, reaching 54% in the Kaingan-
ges Indians of Brazil. Dib is a high-prevalence
antigen in almost all populations. Dia and Dib
represent an amino acid substitution in the
seventh extracellular loop of Band 3—Leu854
in Dia and Pro854 in Dib.
Anti-Dia and -Dib are usually IgG1 plus
IgG3 and typically require an antiglobulin test
for detection, although a few directly aggluti-
nating samples have been found. Anti-Dia oc-
casionally binds complement and lyses un-
Other Blood Groups 䡲 353
treated red cells. Anti-Dia, which is present in
3.6% of multitransfused patients in Brazil, can
cause severe HDFN. Anti-Dib has, very rarely,
been responsible for serious HDFN. Apart
from one example of anti-Dia causing a de-
layed reaction, neither anti-Dia nor anti-Dib
has been reported to be responsible for an
HTR.13,21
Antigens of the Diego system are not de-
stroyed by proteolytic enzymes, such as papa-
in, ficin, or trypsin; however, the antigens car-
ried on the third extracellular loop (Rba, Tra,
WARR, Vga, Wda, BOW, NFLD, Wu, DISK, Jna,
KREP, and Bpa) are sensitive to -chymotryp-
sin.
Wra (DI3) and Wrb (DI4); Anti-Wra and -Wrb
The low-prevalence antigen Wra and its anti-
thetical antigen of extremely high prevalence,
Wrb, represent an amino acid substitution in
the fourth loop of Band 3—Lys658 in Wra and
Glu658 in Wrb. Wrb expression, however, also
depends on the presence of GPA. Despite its
homozygosity for the Glu658 codon in the
Band 3 gene, Wrb is not expressed in the rare
phenotypes associated with a complete ab-
sence of the MN glycoprotein GPA or of the
part of GPA that is close to insertion into the
red cell membrane. This provides strong evi-
dence for an interaction between Band 3 and
GPA within the red cell membrane.
Anti-Wra is a relatively common antibody,
usually detected by an antiglobulin test but
sometimes by direct agglutination of red cells.
Wra antibodies are mostly IgG1 but sometimes
IgM or IgM plus IgG. Anti-Wra has been re-
sponsible for severe HDFN and HTRs. Alloan-
ti-Wrb is rare and little is known about its clini-
cal significance, but autoanti-Wrb is a relatively
common autoantibody and may be implicated
in AIHA.
Other Diego Antigens
Since 1996, 17 antigens of very low prevalence
have been shown to represent amino acid sub-
stitutions in Band 3 and have joined the Diego
system: Wda, Rba, WARR, ELO, Wu, Bpa, Moa,
Hga, Vga, Swa, BOW, NFLD, Jna, Krep, Tra, Fra,
354 䡲 AABB TECHNICAL MANUAL
and SW1. Anti-DISK detects a high-prevalence
antigen that is antithetical to Wu. Anti-ELO
and anti-BOW have caused severe HDFN.
TH E YT S Y S T EM
Yta (YT1; His353) and Ytb (YT2; Asn353) are an-
tithetical antigens on acetylcholinesterase, an
enzyme that is important in neurotransmis-
sion but has unknown function on red cells.
Ytb has a prevalence of about 8% in people of
European or African ancestry but has not been
found in people of Japanese ancestry; Yta has
relatively high prevalence in all populations.
Yta is not affected by trypsin but is destroyed by
-chymotrypsin treatment of the red cells; pa-
pain and ficin may also destroy the antigen,
but this ability appears to depend on the anti-
Yta used. Yta and Ytb are sensitive to the disul-
fide-bond-reducing agents AET and DTT.
Yt antibodies are usually IgG and require
an IAT for detection. They are not generally
considered clinically significant, although
anti-Yta may cause accelerated destruction of
Yt(a+)-transfused red cells and has been impli-
cated in acute and delayed HTRs.21,41
TH E X G S Y S T EM
Xga (XG1) is the only polymorphic blood group
antigen encoded by an X-linked gene.41 Xga has
a prevalence of about 66% in males and 89% in
females. Part of the XG gene is within the X
chromosome pseudoautosomal region, a sec-
tion at the tip of the short arm that pairs with
the Y chromosome. CD99 (XG2) is the second
antigen of the Xg system. The CD99 gene is
homologous to XG and is located on both X
and Y chromosomes, with pairing occurring at
meiosis. Both CD99 and Xga expression appear
to be controlled by a common regulator gene,
XGR. Although Xga antibodies occasionally ag-
glutinate red cells directly, they are generally
IgG and are reactive by an IAT. They are not re-
active with red cells treated with proteolytic
enzymes. Anti-Xga is not clinically significant.
CD99 antibodies in common use are mostly
monoclonal and of mouse origin; a few human
alloanti-CD99 occur, although little is known
about their characteristics.
T HE S C I A N NA SY S TE M
The Scianna system consists of seven antigens
on erythrocyte membrane-associated protein,
a member of the IgSF that has one IgSF do-
main.41,42 Sc1 (Gly57) and Sc2 (Arg57) are anti-
thetical antigens of high and low prevalence,
respectively. Rd (SC4) has very low prevalence;
Sc3, STAR, SCER, and SCAN all have very high
prevalence. Anti-Sc3 is produced by individu-
als with the very rare Scianna-null phenotype.
No Scianna antibody has been implicat-
ed in an HTR or severe HDFN, although evi-
dence is limited because of the scarcity of the
antibodies. Although directly agglutinating
SC1 antibodies are known, Scianna antibodies
are generally reactive by an IAT. Treatment of
red cells by proteolytic enzymes has little ef-
fect on their reactivity with Scianna antibod-
ies, but disulfide-bond-reducing agents (AET
and DTT) substantially reduce reactivity.
T HE D OM B RO C K S Y ST E M
The Dombrock system consists of eight anti-
gens: the polymorphic antithetical antigens
Doa (DO1; Asn265) and Dob (DO2; Asp265) and
the high-prevalence antigens Gya, Hy, Joa,
DOYA, DOMR, and DOLG.3,43 Doa and Dob have
a prevalence of 66% and 82%, respectively, in
populations of European ancestry (Table 14-
6). The prevalence of Doa is somewhat lower in
populations of African ancestry and substan-
tially lower in people of East Asian ancestry.
Anti-Gya is the antibody that is characteristi-
cally produced by immunized individuals with
the Dombrock-null [Gy(a–)] phenotype that
results from various inactivating mutations.
Two uncommon phenotypes are present in in-
dividuals of African ethnicity: Hy– Jo(a–)
(Gly108Val) and Hy+w Jo(a–) (Thr117Ile), which
are usually associated with weak expression of
Dob and Doa, respectively (Table 14-6). The
Dombrock glycoprotein (CD297) has a struc-
ture that is characteristic of an adenosine di-
phosphate ribosyltransferase, and the Dom-
brock gene has the designation “ART4.”
Dombrock antigens are resistant to papa-
in and ficin treatment of the red cells but are
sensitive to trypsin, -chymotrypsin, and pro-
 CHAPTER 14 Other Blood Groups 䡲 355

TABLE 14-6. Phenotypes of the Dombrock System and Their Approximate Frequencies

Frequency (%)

Phenotype Doa Dob Gya Hy Joa Whites Blacks

Do(a+b–) + – + + + 18 11
Do(a+b+) + + + + + 49 44
Do(a–b+) – + + + + 33 45
Gy(a–) – – – – – Rare 0
Hy– – + w
+w – – 0 Rare

Jo(a–) +w –/+w + +w – 0 Rare

DOYA– – – +w
+w
+w Rare Rare

DOMR– – + – +w +w Rare Rare

DOLG– + – +w + + Rare Rare

+w = weakened expression of antigen.

nase. They are also sensitive to the disulfide-
bond-reducing agents AET and DTT.
Dombrock antibodies are usually IgG and
reactive by an IAT. They are in short supply and
are often of poor quality, with very weak reac-
tivity. Screening for Dombrock-compatible
donors, therefore, is best performed by molec-
ular genetics (SNP testing).
Anti-Doa and -Dob have been responsible
for acute and delayed HTRs. There is little in-
formation regarding the clinical significance of
other Dombrock antibodies. No Dombrock
antibody has caused HDFN.
TH E CO LTO N S Y S TE M
Coa (CO1; Ala45) is a high-prevalence antigen;
Cob (CO2; Val45), its antithetical antigen, has a
prevalence of about 8% in people of European
ancestry but is less common in other ethnic
groups.41 Anti-Co3 is reactive with all red cells
except those of the extremely rare Co(a–b–)
phenotype that results from various inactivat-
ing mutations. Co4 (Gln47) is a high-preva-
lence antigen whose presence is required for
the expression of Coa due to the proximity of
the polymorphism.44 The Colton antigens are
located on aquaporin-1, a water channel (Fig
14-8).45 Colton antibodies are usually IgG and
reactive by an IAT, although agglutinating IgM
anti-Coa has been found. Colton antibodies
have been implicated in severe HDFN and
HTRs. Colton antigens are resistant to proteo-
lytic enzymes.
T HE L AN D ST E IN E R - W IE N ER
S Y S TE M
LWa (LW5) and LWb (LW7; Gln100Arg) are anti-
thetical antigens of high and low prevalence,
respectively.41 Anti-LWab is reactive with all red
cells except those of the extremely rare LW-
null phenotype and Rhnull cells, which are also
LW(a–b–). LW antigens are expressed more
strongly on D+ than D– red cells and more
strongly on umbilical cord red cells than on
those of adults. LW antigens are unaffected by
treatment of the red cells with papain, ficin,
trypsin, or -chymotrypsin but are destroyed
by pronase. Disulfide-bond-reducing agents
(AET and DTT) either destroy or greatly reduce
LWa or LWab (LW6) on red cells.
The LW glycoprotein is intercellular adhe-
sion molecule-4 (ICAM-4), an IgSF adhesion
molecule. ICAM-4 binds integrins on macro-
phages and erythroblasts, and it is probably
356 䡲 AABB TECHNICAL MANUAL

FIGURE 14-8. A three-dimensional model of aquaporin-1, showing the six membrane-spanning domains as
cylinders. The first extracellular loop is glycosylated and contains the Coa/Cob polymorphism. The third
extracellular loop and first intracellular loop contain alanine (A)-proline (P)-asparagine (N) motifs and form a
channel in the membrane through which water molecules pass.

involved in the stabilization of erythroblastic rary LW-negative phenotypes sometimes oc-

islands in the marrow during the later stages of
erythropoiesis.45 ICAM-4 is also part of the
Band 3/Rh ankyrin macrocomplex (Fig 14-1)
of red cell surface antigens and might main-
tain close contact between the red cell surface
and the vascular endothelium.8 Upregulation
of ICAM-4 on red cells of patients with sickle
cell disease could play a part in the adhesion
of these cells to the vascular endothelium and
the resultant crises of vascular occlusion.17,46
Most LW antibodies are reactive by an IAT.
They are not generally considered to be clini-
cally significant and have not been implicated
in HTRs or HDFN. Acquired and often tempo-
cur with production of anti-LWa or anti-LWab, a
phenomenon that is usually associated with
pregnancy or hematologic malignancy. The
transient antibodies behave like alloantibod-
ies but, strictly speaking, should be considered
autoantibodies.
T HE C H I D O / RO D G E R S S Y ST E M
The nine antigens of the Chido/Rodgers sys-
tem are not true blood group antigens because
they are not produced by erythroid cells. They
are located on a fragment of the fourth com-
ponent of complement (C4d) that attaches to
 CHAPTER 14
the red cells from the plasma. Ch1 to Ch6, Rg1,
and Rg2 each have a prevalence greater than
90%; WH has a prevalence of about 15%. A
complex relationship exists between the nine
determinants and SNPs in C4A and C4B, the
genes encoding the C4 chains. The expres-
sion of Chido/Rodgers on red cells is destroyed
by treatment of the cells with proteolytic en-
zymes, such as papain or ficin.
No Chido/Rodgers antibodies are known
to have caused HTRs or HDFN, and antigen-
negative blood is not required for transfusion.
Chido/Rodgers antibodies are generally IgG.
Detection of these antibodies with native red
cells usually requires an IAT, but they often
directly agglutinate red cells coated artificially
with C4d. Binding of Chido/Rodgers antibod-
ies to red cells is readily inhibited by plasma
from Ch/Rg+ individuals; this is a useful aid to
identification of these antibodies (Method 3-
17).
TH E G ER B I C H S Y S TE M
The Gerbich system consists of six highly prev-
alent antigens—Ge2, Ge3, Ge4, GEPL, GEAT,
and GETI—and five antigens with very low
prevalence—Wb, Lsa, Ana, Dha, and GEIS.
These antigens are located on the sialoglyco-
proteins glycophorin C (GPC), glycophorin D
(GPD), or both. These two glycoproteins are
produced by the same gene, GYPC, by initia-
tion of translation at two different sites on the
mRNA. GPD lacks the N-terminal 21 amino ac-
ids of GPC. GPC and GPD are part of the junc-
tional complex of membrane proteins. Their
C-terminal cytoplasmic domains interact with
the membrane skeleton through 4.1R, p55,
and adducin and serve as an important link
between the membrane and its skeleton.9,45
GPC appears to be exploited as a receptor
by some strains of the malarial parasite P. falci-
parum. There are three types of “Gerbich-neg-
ative” phenotypes (Table 14-7). The first of
these phenotypes, Ge:–2,–3,–4, is the true null
in which both GPC and GPD are absent from
the red cells, and the cells are elliptocytic. In
the other phenotypes, Ge:–2,3,4 and Ge:–2,
–3,4, GPD is absent and an abnormal form of
GPC is present. Ge2, Ge3, and Ge4 are de-
Other Blood Groups 䡲 357
stroyed by trypsin treatment of red cells.
Whereas Ge2 and Ge4 are also sensitive to pa-
pain, Ge3 is resistant to papain treatment.
Consequently, papain-treated red cells can be
used for distinguishing anti-Ge2 from anti-
Ge3 in the absence of the very rare Ge:–2,3,4
phenotype red cells.
Gerbich antibodies may be IgM and di-
rectly agglutinating, but most are IgG and re-
quire an IAT for detection. They are not gener-
ally considered to be clinically significant and
have not caused HTRs. However, anti-Ge3 has
caused HDFN that tends to manifest 2 to 4
weeks after birth. Some autoantibodies with
specificities resembling anti-Ge2 or -Ge3 have
been responsible for AIHA.
T HE CROM ER SY ST E M
The 18 Cromer antigens are located on the
complement-regulatory glycoprotein, decay
accelerating factor (DAF or CD55).3,47 They in-
clude the antithetical antigens Tca (Arg52), Tcb
(Leu52), Tcc (Pro52), WESa (Arg82), and WESb
(Leu82). Tca and WESb have high prevalence,
and Tcb, Tcc, and WESa, have low prevalence,
although both Tcb and WESa are present in ap-
proximately 0.5% of people of African ancestry
and WESa is present in 0.6% of people of Finn-
ish ancestry. Other antigens—Cra, Dra, Es, IFC,
UMC, GUTI, SERF, ZENA, CROV, CRAM, and
CROZ, CRUE, and CRAG—have high preva-
lence.
Anti-IFC is the antibody made by individ-
uals with the very rare Cromer-null phenotype
(Inab phenotype), and it is reactive with all red
cells, apart from those of individuals with the
TABLE 14-7. Phenotypes Lacking High-
Prevalence Gerbich Antigens and the
Antibodies that May Be Produced
Phenotype Antibodies
Ge:–2,3,4 (Yus type) Anti-Ge2
Ge:–2,–3,4 (Ge type) Anti-Ge2 or -Ge3
Ge:–2,–3,–4 (Leach Anti-Ge2, -Ge3, or -Ge4
type)
358 䡲 AABB TECHNICAL MANUAL

Inab phenotype. Cromer antigens are readily TABLE 14-8. Approximate Frequencies of
destroyed by -chymotrypsin treatment of red Knops Antigens in Two Populations
cells but not by papain, ficin, or trypsin treat-
ment. The disulfide-bond-reducing agents Frequency (%)
AET and DTT reduce antigen expression only
Antigen Whites Blacks
slightly.
CD55 helps protect the red cells from lysis Kna KN1 99 100
resulting from autologous complement by in- b
Kn KN2 6 0
hibiting the action of C3-convertases. Inab-
a
phenotype red cells do not undergo undue he- McC KN3 98 94
molysis, however, because of the activity of Sl1 (Sla) KN4 98 60
another complement regulatory glycoprotein,
Yka KN5 92 98
CD59. CD55 and CD59 are both linked to the
b
red cell membrane by a glycosylphosphati- McC KN6 0 45
dylinositol (GPI) anchor. Pathological levels of Sl2 KN7 0 80
hemolysis occur in paroxysmal nocturnal he-
moglobinuria, which is associated with a clon- Sl3 KN8 100 100
al defect in GPI biosynthesis and the absence KCAM KN9 98 20
of both CD55 and CD59 in affected red cells.
Cromer antibodies are not usually con-
sidered to be clinically significant because tigens are generally resistant to papain and fi-
there is no firm evidence that any of them has cin, although this may depend on the antibod-
caused an HTR, and the evidence from func- ies used, and are destroyed by trypsin or -
tional cellular assays is equivocal. No Cromer chymotrypsin treatment. They are also de-
antibodies have been implicated in HDFN, stroyed, or at least weakened, by AET and DTT.
and they are probably sequestered by high lev- CR1 appears to be involved in the roset-
els of CD55 in the placenta. Cromer antibodies ting of red cells that is associated with severe P.
are usually IgG and require an IAT for detec- falciparum malaria. The McCb and Sl2 alleles,
tion. They are inhibited by serum or concen- present almost exclusively in individuals of Af-
trated urine from antigen-positive individuals rican ancestry, may confer a degree of protec-
and are removed from serum by platelet con- tion from the parasite. This might explain the
centrates. very strong difference in the prevalence of
some antigens, especially Sl1, McCb, Sl2, and
TH E K N O P S S Y S TE M KCAM, among populations of European and
African ethnicity (Table 14-8).
The nine antigens of the Knops system are lo- Knops antibodies are not clinically signif-
cated on the complement-regulatory glyco- icant and can be ignored when selecting blood
protein complement receptor 1 (CR1 or for transfusion.5 They are usually difficult to
CD35).48 All are polymorphic, although Kna, work with, often making it difficult to distin-
McCa, Sl1, and Yka have relatively high preva- guish antigen-negative cells from those with
lence (Table 14-8). weak expression. They are generally IgG and
Kna/Knb represent Val 1561Met, McCa/ reactive only by an IAT.
b
McC , Lys1590Glu, Sl1/Sl2, and Arg1601Gly. Sl3
requires the presence of Ser1610 and Arg1601
T HE I N D I A N S Y S TE M
(Sl2) for expression. Absence of KCAM results
from an Ile1615Val substitution. An apparent The low-prevalence antigen Ina (Arg46) and its
null phenotype, the Helgeson phenotype, indi- antithetical antigen Inb (Pro46) plus two other
cates very low levels of red cell CR1 and very high-prevalence antigens, INFI and INJA, are
weak expression of Knops antigens. Knops an- located on CD44, the predominant cell surface
 CHAPTER 14
receptor for the glycosaminoglycan hyaluro-
nan, a component of the extracellular matrix.41
AnWj (901009), an antigen with very high prev-
alence, may also be located on or associated
with CD44, but the evidence is incomplete. In-
dian antigens have reduced expression on red
cells with the In(Lu) phenotype, and AnWj is
virtually undetectable on In(Lu) cells. Ina and
Inb are sensitive to treatment of red cells with
proteolytic enzymes—papain, ficin, trypsin, -
chymotrypsin—and are also destroyed by the
disulfide-bond-reducing agents AET and DTT.
AnWj, however, is resistant to all these en-
zymes but shows variable outcomes with re-
ducing agents.
Anti-Ina and -Inb often agglutinate red
cells directly, but the reaction is usually en-
hanced by an IAT. Indian antibodies are not
generally considered to be clinically signifi-
cant, although there is one report of anti-Inb
causing an HTR. AnWj, however, has caused
severe HTRs, and In(Lu) red cells should be se-
lected for transfusion.5
TH E O K SY ST E M
Oka, OKGV, and OKVM have very high preva-
lence and are located on the IgSF molecule
CD147 or basigin, which has two IgSF do-
mains. Oka is resistant to proteolytic enzymes
and disulfide-bond-reducing agents. Only two
alloanti-Oka antigens and a single example
each of anti-OKGV and -OKVM are known; all
are reactive by an IAT.49 In-vivo survival tests
and cellular functional assays with one anti-
Oka have suggested that it could be clinically
significant, but no clinical information exists.
TH E R A PH S Y S TE M
MER2 (RAPH1), which is located on the tet-
raspanin CD151, was initially defined by
mouse monoclonal antibodies that recognized
a quantitative polymorphism, and about 8% of
the population has undetectable levels of
MER2 on their mature red cells. Alloanti-MER2
was found in three Israeli Jews originating
from India who had a RAPH-null phenotype
resulting from a single nucleotide deletion that
led to a premature stop codon. These three in-
Other Blood Groups 䡲 359
dividuals were CD151 deficient and had end-
stage renal failure, sensorineural deafness, and
pretibial epidermolysis bullosa, suggesting
that CD151 is essential for the proper assem-
bly of basement membranes in kidney, inner
ear, and skin.50 MER2-negative individuals
with anti-MER2 but only single amino acid
substitutions in CD151 do not have these
symptoms.
MER2 antigen is resistant to treatment of
red cells with papain but is destroyed by tryp-
sin, -chymotrypsin, and pronase and by AET
and DTT. MER2 antibodies react in an IAT.
There is no evidence that anti-MER2 is clini-
cally significant.
T HE J O H N M I LTO N HAG E N
S Y S TE M
This system consists of six antigens with very
high prevalence—JMH, JMHK, JMHL, JMHG,
JMHM, and JMHQ—on the semaphorin glyco-
protein CD108 (Sema7A). Anti-JMH is typically
produced by individuals with an acquired loss
of CD108. This most often occurs in elderly pa-
tients and is associated with a weakly positive
direct antiglobulin test result. The absence of
the other JMH antigens results from different
missense mutations in SEMA7A.51 JMH anti-
gens are destroyed by proteolytic enzymes and
disulfide-bond-reducing agents. They are not
detected on cord red cells.
JMH antibodies are usually reactive in an
IAT. They are not generally considered to be
clinically significant, although one example
was implicated in an acute HTR.
T HE G IL L S Y S T EM
GIL antibodies detect a very high-prevalence
antigen, GIL, located on aquaporin 3 (AQP3), a
member of the aquaporin superfamily of wa-
ter and glycerol channels (like the Colton anti-
gen).52 AQP3 enhances the permeability of the
red cell membrane by glycerol and water.
GIL antigen is resistant to proteolytic en-
zymes and disulfide-bond-reducing agents.
GIL antibodies are reactive by an IAT. Anti-GIL
has not been implicated in HTRs or HDFN,
360 䡲 AABB TECHNICAL MANUAL
although monocyte monolayer assays have
suggested a potential to cause accelerated de-
struction of GIL+ red cells.
TH E RH AG SY ST E M
The four antigens of the RHAG system are lo-
cated on the Rh-associated glycoprotein
(RhAG), which is also described in Chapter
13.53 RhAG is closely associated with the Rh
protein in the membrane as part of the Band
3/Rh/ankyrin macrocomplex (Fig 14-1). Ola is
very rare, and homozygosity for the allele en-
coding Ola is associated with an Rhmod pheno-
type. Duclos and DSLK have high prevalence,
and absence of these antigens is associated
with an aberrant U (MNS5) antigen. RHAG4 is
a low-prevalence antigen whose antibody was
associated with a single case of severe HDFN.3
TH E F O R S S Y S T EM
FORS is a new blood group system consisting
of a single antigen, Forssman glycosphingolip-
id antigen (FORS1). The presence of FORS1 on
human erythrocytes is unusual and was shown
to be the result of an enzyme-activating amino
acid substitution arising from a missense mu-
tation in the human Forssman synthase gene
GBGT1. FORS1 was demonstrated biochemi-
cally on the red cells of two blood donors from
different families with the Apae phenotype,
which was first described in 1987.54,55 Apae had
been previously thought to constitute a sub-
group of A in the ABO system but has now
been shown to be based on the presence of
FORS1 antigen on red cells. Forssman syn-
thase adds a terminal 3--N-acetylgalactos-
amine to its globoside acceptor. FORS1 is not
usually present on the red cells of primates but
is highly expressed on the red cells and uroepi-
thelia of lower mammals, such as dogs and
sheep. As with other carbohydrate blood
group antigens, naturally occurring antibodies
to FORS1 are present in all human sera, with
the exception of rare FORS1+ individuals, and
have the potential to cause a positive cross-
match.
T HE JR SY ST E M
The high-prevalence antigen Jra has been pro-
moted to a new blood group system, JR, fol-
lowing the independent findings of two groups
demonstrating that the Jr(a–) phenotype was
due to inactivating nucleotide changes in
ABCG2.56,57 The gene encodes ABCG2, a multi-
pass membrane protein family member of the
adenosine triphosphate (ATP)-binding cas-
sette transporters that is broadly distributed
throughout the body. Jra has long been associ-
ated with drug resistance in cancer and resis-
tance to xenobiotics, and it might be impor-
tant for porphyrin homeostasis.58
The Jr(a–) phenotype is present predomi-
nantly in people of Japanese ancestry. Jra anti-
gen is resistant to proteolytic enzymes and
disulfide-bond-reducing agents. Anti-Jra is re-
active by an IAT and has caused HTR. It is not
usually implicated in HDFN, although one
case has been reported.
T HE L AN S Y ST E M
Lan, another high-prevalence antigen, was
also elevated to a new blood group system fol-
lowing the discovery that it was carried on
ABCB6, another ATP-binding cassette trans-
porter molecule on the erythrocyte mem-
brane.59 Unlike Jra, Lan is not associated with
any single geographic or ethnic group, and this
is mirrored by the diversity of mutant alleles in
the Lan– individuals studied. ABCB6 is associ-
ated with porphyrin transport and was
thought to have an important role in heme
synthesis.60 However, the existence of ABCB6-
deleted individuals indicates that there may be
compensation by other transporters in the ab-
sence of ABCB6.
Lan antigen is expressed variably on red
cells in different individuals but is resistant to
proteolytic enzymes and disulfide-bond-
reducing agents. Anti-Lan is reactive by an IAT
 CHAPTER 14
and has been implicated in HTR but not gen-
erally in HDFN.
TH E VE L SY ST E M
Vel is a high prevalence blood group antigen
that has been shown to depend on the pres-
ence of small integral protein 1 (SMIM1), a
protein of unknown function newly discovered
on the erythrocyte surface.60-62 Absence of Vel
antigen in the vast majority of individuals re-
gardless of ethnic background, is due to a 17-
base pair deletion in SMIM1, which results in
the absence of the protein at the cell mem-
brane.
Vel antigen expression is generally weak
on cord RBCs and differs substantially from
one individual to another. Patterns of expres-
sion are a consequence both of zygosity for the
17-bp deletion and also for a SNP in a GATA-1
transcription factor site in intron 2.62 Serologi-
cal expression is not affected by protease treat-
ment although sensitivity to reducing agents
such as 0.2 M DTT is variable. Anti-Vel are of-
ten a mixture of IgG and IgM, readily activate
complement and have been implicated in
mild to severe HTRs, although HDFN is rare.
ANTIGENS THAT DO NOT
BELO NG TO A BLOOD GROU P
SY S TE M
CD59—A Potential New Blood Group
System
An antibody to a high-prevalence antigen de-
tected in the plasma of a transfused CD59-
deficient child was shown to be specific for
CD59.63 The antibody was readily inhibited
with soluble protein. Sequence analysis of
samples from the family revealed that the par-
ents (first-degree cousins) were heterozygous
and the child homozygous for a silencing mu-
tation in CD59. The antibody was an IgG anti-
body, and although the child’s RBCs had been
weakly DAT-positive following transfusion, in-
compatible blood was well-tolerated. Thus,
CD59 has been proposed as a blood group sys-
tem but is as yet, unconfirmed.
Other Blood Groups 䡲 361
Blood Group Collections
Although many antigens belong to blood
group systems, others have not been shown to
belong to a system. These are mostly antigens
with either very high or very low prevalence.
Some of them are included in blood group col-
lections that contain two or more antigens that
are related serologically, biochemically, or ge-
netically but do not fit the criteria for system
status.1
The high-prevalence antigen ABTI is sero-
logically related to Vel. However, it has been
excluded from SMIM1 by sequencing analysis
and thus remains in a collection. Like Vel, ABTI
expression differs substantially and it is gener-
ally expressed only weakly on cord red cells.
ABTI is resistant to treatment of red cells with
proteolytic enzymes or disulfide-bond-reduc-
ing agents. Anti-ABTI has not caused HDFN
and clinical data are limited.
The Cost collection contains Csa and Csb,
antithetical antigens with relatively high and
low prevalence, respectively. These antigens
are serologically related to those of the Knops
system but do not appear to be located on
CR1. Cost antibodies are not clinically signifi-
cant.
Era and Erb are antithetical antigens with
very high and low prevalence, respectively.
Anti-Er3 is produced by individuals with Er(a–
b–) red cells. There is no evidence that Er anti-
bodies are clinically significant.
Carbohydrate antigens of the Ii and GLOB
collections are described in Chapter 12.
Carbohydrate antigens associated with
MNS antigens that are not encoded by GYPA or
GYPB are included in a seventh collection,
MNS CHO. These antigens have been shown to
be due to altered glycosylation of the O-linked
sugars on GPA and GPB.2
High-Prevalence Antigens (901 Series)
The 901 series of the ISBT classification con-
tains six antigens (Table 14-9): five have a
prevalence well in excess of 99%, whereas Sda
has a prevalence of about 91%. All are inherit-
ed, and none is eligible to join a system.1 All six
antigens are resistant to papain, trypsin, -
chymotrypsin, and AET treatment of the red
362 䡲 AABB TECHNICAL MANUAL

TABLE 14-9. Antigens of the 901 Series of High-Frequency Antigens and Their Clinical Significance

Antigen Number Clinical Significance

Ata 901003 Reported to have caused an HTR and mild HDFN

Emm 901008 No evidence of clinical significance
AnWj 901009 Severe AHTRs
a
Sd 901011 No evidence of clinical significance
PEL 901014 No evidence of clinical significance
MAM 901016 Severe HDFN
HTR = hemolytic transfusion reaction; HDFN = hemolytic disease of the fetus and newborn; AHTR = acute HTR.

cells, and all except AnWj and Sda are ex-
pressed strongly on cord cells.
Sda represents carbohydrate structures on
red cells, and the product of the Sda gene is
probably a (1,4)N-acetylgalactosaminyltrans-
ferase. The strength of Sda on red cells is highly
variable, and Sda is not detected on cord red
cells. Agglutination of Sd(a+) red cells has a
characteristic “mixed-field” appearance of ag-
glutinates and free red cells; when viewed mi-
croscopically, the agglutinates are refractile.
Anti-Sda is inhibited by urine from Sd(a+)
individuals (Method 3-19), and by guinea pig
urine.
HLA-B7; Bgb, HLA-B17 (B57 or B58); and Bgc,
HLA-A28 (A68 or A69, which cross-reacts with
HLA-A2). Many individuals, however, do not
express Bg antigens on their red cells, despite
having the corresponding HLA antigens on
their lymphocytes.
There are a few reports of Bg antibodies
causing HTRs.64 These antibodies are some-
times present as contaminants in reagents.
HLA antigens on red cells are not destroyed by
papain, ficin, pronase, trypsin, -chymotryp-
sin, AET, or DTT. They can be stripped from
red cells with chloroquine (Method 2-20) or
EDTA/glycine-HCl (Method 2-21).

Low-Prevalence Antigens (700 Series) ERY TH ROI D PHE N OT Y PES

Eighteen antigens of very low prevalence in all
of the populations tested constitute the 700 se-
ries of the ISBT classification: By, Chra, Bi, Bxa,
Toa, Pta, Rea, Jea, Lia, Milne, RASM, JFV, Kg,
JONES, HJK, HOFM, SARA, and REIT. All are
inherited and do not fit any criteria for joining
or forming a system.1
Antibodies to low-prevalence antigens do
not present transfusion problems because
compatible blood is readily available. These
antibodies remain undetected if a serologic
crossmatch is not employed. Anti-JFV, -Kg,
-JONES, -HJK, and -REIT have all caused
HDFN.
HLA Antigens on Red Cells
“Bg” is the name given to HLA Class I antigens
expressed on mature red cells. Bga represents
CAUS ED BY MUTATI ONS IN
TRANSCR IP TION FACTOR
GE NES
Mutations in genes encoding erythroid tran-
scription factors are emerging as important
modifiers of blood-group-antigen expression.
As described in the “Lutheran System” section
above, heterozygosity for different mutations
in KLF1 has been identified in individuals with
the In(Lu) phenotype. In these individuals, ex-
pression of antigens carried on CD44 (Ina/Inb)
and the AnWj and P1 antigens is weak.19 How-
ever, KLF1 mutations have also been shown to
affect other genes, notably the -globin gene,
resulting in the hereditary persistence of fetal
hemoglobin syndrome.65 Affected individuals
have elevated HbF levels, some 30%, and
demonstrate an In(Lu) phenotype. Further-
 CHAPTER 14 Other Blood Groups 䡲 363

more, discrete mutations in KLF1 appear to
give rise to different phenotypes; for example,
the change of Glu325Lys does not result in the
In(Lu) phenotype but is associated with severe
congenital dyserythropoietic anemia. These
red cells demonstrate weakened expression of
antigens in the Colton (AQP1), Cromer (DAF),
and LW (ICAM-4) blood group systems.66
Although mentioned above in the “Lu-
theran System” section, it is worth repeating
that a mutation in GATA-1 resulted in the X-
linked Lu(a–b–) phenotype in one family.20 It is
likely that additional mutations in these and
other erythroid-specific transcription factors
will be identified as the causes of altered blood
group antigen expression.

KEY POINTS

1. Of 339 recognized antigen specificities, 297 belong to 1 of 34 blood group systems repre-
senting either a single gene or two or three closely linked homologous genes. Some groups
of antigens that are not eligible to join a system are classified together as collections. Anti-
gens not classified in a system or collection have either low or high prevalence and make up
the 700 and 901 series, respectively.
2. M and N are antithetical, polymorphic antigens. M, N, S, s, and ‘N’ are all destroyed by treat-
ment of the red cells with papain, ficin, bromelin, or pronase, although this effect with S and
s is variable. M and N—but not S, s, or ‘N’—are destroyed by trypsin treatment.
3. Anti-M is relatively common, and anti-N is quite rare. Most anti-M and -N are not clinically
significant. When M or N antibodies active at 37 C are encountered, antigen-negative or
compatible red cells should be provided. Anti-S, -s, and -U are generally IgG antibodies that
are active at 37C. They have been implicated in HTRs and severe and fatal HDFN.
4. The antigen often referred to as “Kell” is correctly named “K” or “KEL1”; its antithetical anti-
gen is k or KEL2.
5. Because Kell antibodies can cause severe HDFN and HTRs, patients with Kell antibodies
should be transfused with antigen-negative blood whenever possible. Anti-K is the most
common immune red cell antibody not in the ABO and Rh systems.
6. In people of European or Asian ancestry, the Duffy polymorphism consists of two antigens,
Fya and Fyb, and three phenotypes, Fy(a+b–), Fy(a+b+), and Fy(a–b+). Fya and Fyb are very
sensitive to most proteolytic enzymes. In people of African ancestry, a third allele, Fy, may
result in neither Fya nor Fyb. Individuals who are homozygous for Fy have the red cell pheno-
type Fy(a–b–).
7. Anti-Fya (common) and anti-Fyb (rare) are generally detected by an IAT and may cause acute
or delayed HTRs that are usually mild, although some have been fatal.
8. Kidd antigens are resistant to proteolytic enzymes, such as papain and ficin.
9. Kidd antibodies anti-Jka and -Jkb are not common, are generally present in antibody mix-
tures, and are often difficult to detect. An IAT is usually required, and use of enzyme-treated
cells may be necessary to detect weaker antibodies. Kidd antibodies may cause severe acute
HTRs and are a common cause of delayed HTRs.
10. The 22 antigens of the Diego system are located on Band 3, the red cell anion exchanger.
Anti-Dia and -Wra can cause severe HDFN. Anti-Wra can also cause HTRs.

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62. Cvejic A, Haer-Wigman L, Stephens JC, et al.
SMIM1 underlies the Vel blood group and in-
fluences red blood cell traits. Nat Genet 2013;
45:542-5.
63. Anliker M, von Zabern I, Höchsmann B, et al. A
new blood group antigen is defined by anti-
CD59, detected in a CD59-deficient patient.
Transfusion 2014 (in press).
64. Nance ST. Do HLA antibodies cause hemolytic
transfusion reactions or decreased RBC sur-
vival? Transfusion 2003;43:687-90.
65. Borg J, Papadopoulos G, Georgitsi M, et al.
Haploinsufficiency for the erythroid transcrip-
tion factor KLF1 causes hereditary persistence
of fetal hemoglobin. Nat Genet 2010;42:801-5.
66. Arnaud L, Saison C, Helias V, et al. A dominant
mutation in the gene encoding the erythroid
transcription factor KLF1 causes a congenital
dyserythropoietic anemia. Am J Hum Genet
2010;87:721-7.
 C h a p t e r 1 6

Identification of Antibodies to
Red Cell Antigens

Phyllis S. Walker, MS, MT(ASCP)SBB, and

Janis R. Hamilton, MS, MT(ASCP)SBB

NATURALLY OCCURRING ANTI-A Immunization to red cell antigens may

and anti-B are the only red cell antibod-
ies that are commonly found in human serum
or plasma. All other antibodies are called “un-
expected red cell antibodies.” This chapter dis-
cusses methods for detecting and identifying
unexpected red cell antibodies.
There are two types of unexpected red cell
antibodies: alloantibodies and autoantibodies.
When someone produces an antibody to an
antigen that he or she lacks, the antibody is
called an “alloantibody.” When someone pro-
duces an antibody to an antigen that he or she
possesses, the antibody is called an “autoanti-
body.” Therefore, by definition, alloantibodies
react only with allogeneic red cells that express
the corresponding antigens—not with the an-
tibody producer’s red cells. Conversely, auto-
antibodies are reactive with the red cells of the
antibody producer. In fact, autoantibodies
usually are reactive with most reagent red cells
as well as with autologous red cells.
result from pregnancy, transfusion, transplan-
tation, needle sharing, or injections of immu-
nogenic material. The frequency of alloimmu-
nization is extremely variable depending on
the patients or donors being studied. Healthy
blood donors have very low immunization
rates. In chronically transfused patient popu-
lations, such as those with sickle cell anemia
or thalassemia, as many as 14% to 50% of indi-
viduals are reported to be alloimmunized.1-3
In some instances, no specific immuniz-
ing event can be identified. In such cases,
naturally occurring antibodies have presum-
ably resulted from exposure to environmen-
tal, bacterial, or viral antigens that are similar
to blood group antigens. Also, antibodies de-
tected in serologic tests may be passively ac-
quired from injected immunoglobulin, do-
nor plasma, passenger lymphocytes in
transplanted organs, or hematopoietic pro-
genitor cells (HPCs).

Phyllis S. Walker, MS, MT(ASCP)SBB, retired from Reference Laboratory, Blood Centers of the Pacific-Irwin
Center, San Francisco, California, and Janis R. Hamilton, MS, MT(ASCP)SBB, Manager, Reference Laboratory,
American Red Cross Blood Services, Detroit, Michigan
P. Walker has disclosed no conflict of interest. J. Hamilton has disclosed a financial relationship with Bio-Rad
Laboratories, Inc.

391
392 䡲 AABB TECHNICAL MANUAL
SI G NI F I C A N C E O F
A L LOA N T I B OD I E S
Alloantibodies to red cell antigens may be de-
tected initially with any test that uses serum or
plasma (eg, an ABO test, antibody detection
test, or compatibility test) or in an eluate pre-
pared from red cells coated with alloantibody.
Serum and plasma are interchangeable for an-
tibody testing unless complement is required
for antibody detection. In such rare cases, only
serum provides complement. Throughout this
chapter, serum can be considered to be inter-
changeable with plasma unless the text indi-
cates otherwise.
After an antibody has been detected, its
specificity should be determined and its
clinical significance assessed. A clinically
significant red cell antibody is defined as an
antibody that is frequently associated with he-
molytic disease of the fetus and newborn (HD-
FN), hemolytic transfusion reactions, or a no-
table decrease in transfused red cell survival.
The degree of clinical significance varies
among antibodies with the same specificity;
some cause destruction of incompatible red
cells within hours or even minutes, whereas
others decrease red cell survival by only a few
days, and still others do not shorten red cell
survival discernibly. Some antibodies are
known to cause HDFN, whereas others may
cause a positive direct antiglobulin test (DAT)
result in the fetus without clinical evidence of
HDFN.
P R E A N A LY T I C A L
CO NS I DE RAT IO N S
Before antibody identification testing is start-
ed, the patient’s medical history should be
considered if such information can be ob-
tained. Exposure to foreign red cells through
blood transfusion or pregnancy is the usual
cause of red cell immunization. It is uncom-
mon for patients who have never been trans-
fused or pregnant to produce clinically
significant alloantibodies, although “naturally
occurring” antibodies may be present. If the
patient has been transfused, it is critical to
know when the most recent transfusion was
given. If the patient was transfused during the
past 3 months, primary immunization to red
cell antigens may be a risk. In addition, the
presence of circulating donor red cells may
cause mixed-field results in antigen-typing
tests, and autologous adsorption techniques
should not be used because alloantibodies
could be adsorbed onto transfused donor red
cells. In general, women who may have been
sensitized by pregnancy are more likely to
have alloantibodies than men. Infants who are
younger than 6 months usually do not pro-
duce alloantibodies, but newborns may have
passive antibody of maternal origin.
Certain diseases have been associated
with red cell antibodies; depending on the
methods used, such antibodies may be detect-
able in antibody detection and identification
tests. Cold agglutinin syndrome, Raynaud
phenomenon, and infections with Mycoplas-
ma pneumoniae are often associated with
anti-I. Infectious mononucleosis is sometimes
associated with anti-i. Patients with paroxys-
mal cold hemoglobinuria, which is associated
with syphilis in adults and viral infections in
children, may demonstrate autoantibodies
with anti-P specificity. Warm autoantibodies
often accompany diagnoses such as warm au-
toimmune hemolytic anemia, systemic lupus
erythematosus, multiple myeloma, chronic
lymphocytic leukemia, or lymphoma. Patients
who have received solid-organ or HPC trans-
plants may demonstrate passive antibodies
that originate from donor passenger lympho-
cytes.
Drugs are known to cause antibody iden-
tification problems. (See Chapter 17 for a dis-
cussion of drug-related mechanisms and
drugs that are associated with serologic prob-
lems.) Administration of intravenous immune
globulin (IVIG) and Rh Immune Globulin
(RhIG) can interfere with antibody-screening
tests. Some lots of IVIG have been reported to
contain unexpected antibodies, including
anti-A and anti-B. Intravenous RhIG, which is
sometimes used to treat thrombocytopenia,
could explain the presence of anti-D in an Rh-
positive patient.
Finally, when a patient is suspected of
having an antibody to a high-prevalence anti-
 CHAPTER 16 Identification of Antibodies to Red Cell Antigens
gen, the patient’s ethnic origin may provide
clues to the specificity of the antibody. Table
16-1 lists some rare blood types that are more
commonly associated with certain ethnic
groups.4
A N A LY T I C A L PHA SE O F
A N TI B O DY ID E N T I F I C ATI O N
Specimen Requirements
Either serum or plasma may be used for anti-
body detection and identification; however,
plasma may not be suitable for detecting com-
plement-activating antibodies. Depending on
the test methods used, a 5-mL to 10-mL ali-
quot of whole blood usually contains enough
serum or plasma for identifying simple anti-
body specificities; more whole blood may be
required for complex studies. When autolo-
gous red cells are studied, the use of samples
anticoagulated with EDTA avoids problems as-
sociated with the in-vitro uptake of comple-
ment components by red cells, which may oc-
cur when clotted samples are used.
Reagents and Test Methods
Antibody Detection Red Cells
Group O red cells suitable for pretransfusion
antibody screening are commercially available
and offered as sets of either two or three re-
agent single-donor red cells. Pooled red cells
for antibody detection are usually obtained
from two different donors and may be used
only when donor serum is tested. Each labora-
tory should decide whether to use two or three
reagent donor red cells for antibody-detection
testing. All reagent red cells licensed by the
Food and Drug Administration (FDA) for this
purpose must express the following antigens:
D, C, E, c, e, M, N, S, s, P1, Lea, Leb, K, k, Fya, Fyb,
Jka, and Jkb. Three-cell antibody detection sets
usually offer red cells from presumed homozy-
gous donors with double-dose expression for
the following common antigens: D, C, E, c, e,
M, N, S, s, Fya, Fyb, Jka, and Jkb. Some weakly re-
active antibodies are reactive only with red
cells from donors who are homozygous for the
genes encoding the antigens; this serologic
䡲 393
phenomenon is known as “dosage.” Antibod-
ies in the Rh, MNS, Duffy, and Kidd systems
most commonly demonstrate dosage. Reagent
red cells should be refrigerated when not in
use, and they should not be used for antibody
detection after their expiration date.
Antibody Identification Panels
Identification of an antibody to red cell anti-
gen(s) requires testing the serum against a
panel of selected red cell samples (typically 8-
14 samples) with known antigenic composi-
tion for the major blood groups. Usually, the
red cell samples are obtained from commer-
cial suppliers, but institutions may assemble
their own panels using red cells from local
sources. Except in special circumstances, pan-
el cells are group O, thereby allowing the se-
rum of any ABO group to be tested.
Each reagent red cell of the panel is from a
different donor. The reagent red cells are se-
lected so that if one takes all of the examples of
red cells into account, a distinctive pattern of
positive and negative reactions exists for each
of many antigens. To be functional, a reagent
red cell panel must make it possible to identify
with confidence those clinically significant al-
loantibodies that are most commonly encoun-
tered, such as anti-D, -E, -K, and -Fya. The phe-
notypes of the reagent red cells should be
distributed so that single specificities of the
common alloantibodies can be clearly identi-
fied and most others can be excluded. Ideally,
patterns of reactivity for most examples of sin-
gle alloantibodies should not overlap with any
other (eg, all of the K+ samples should not be
the only ones that are also E+). Also, it is im-
portant to include reagent red cells with dou-
ble-dose antigen expression to detect com-
mon antibodies that frequently show dosage.
Commercial panels are accompanied by a
sheet that lists the phenotypes of the reagent
red cells. The combination of reagent red-cell
samples varies from lot to lot, so it is essential
to use the correct phenotype sheet when inter-
preting panel results. Commercial reagent red
cells for tube testing are diluted to a 2% to 5%
suspension in a preservative solution that can
be used directly from the bottle. Washing the
394 䡲 AABB TECHNICAL MANUAL

TABLE 16-1. High-Prevalence Antigens Absent In Certain Ethnic Populations

Phenotype Population

AnWj– Transient>Israeli Arabs (inherited)

At(a–) Blacks

Cr(a–) Blacks

Di(b–) South Americans>Native Americans>Japanese

DISK– Dutch>Europeans>any

Dr(a–) Jews from Bukhara>Japanese

En(a–) Finns>Canadians>English>Japanese
Es(a–) Mexicans, South Americans, Blacks

Fy(a–b–) Blacks>Arabs/Jews>others of Mediterranean ancestry>Whites

Ge:-2,-3 (Gerbich phenotype) Papua New Guineans>Melanesians>Whites>any

Ge:-2,3 (Yus phenotype) Mexicans>Israelis>Mediterraneans>any

Ge:-2,-3,-4 (Leach phenotype) Any

GUTI– Chileans

Gy(a–) Eastern Europeans (Romany)>Japanese

B
hr – Blacks

hrs– Blacks

Hy– Blacks

IFC (Crnull, Inab) Japanese>any

In(b–) Indians>Iranians>Arabs

Jk(a–b–) Polynesians>Finns>Japanese>any

Jo(a–) Blacks

Jr(a–) Japanese>Asians>Europeans>Bedouin Arabs>any

Js(b–) Blacks

k– Whites>any

Ko (Knull) Reunion Islanders>Finns>Japanese>any

K12– Whites

K14– French-Cajuns

K22– Israelis

KCAM– Blacks>any

Kn(a–) Whites>Blacks>any
 CHAPTER 16 Identification of Antibodies to Red Cell Antigens 䡲 395

TABLE 16-1. High-Prevalence Antigens Absent In Certain Ethnic Populations (Continued)

Phenotype Population

Kp(b–) Whites>Japanese

KUCI– Native Americans

Lan– Whites>Japanese>Blacks>any

Lu(a–b–) Any

Lu20– Israelis

Lu21– Israelis

LW(a–b–) Transient in any>inherited in Canadians

LW(a–) Those of Baltic Sea region

MAM– Arabs>any

MAR– Finns>any

McC(a–) Blacks>Whites>any

MER2– Indian Jews, Turks, Portuguese

k k
MM Swiss>Japanese

Oh (Bombay) Indians>Japanese>any

Ok(a–) Japanese

P– Japanese>Finns>Israelis>any

Para-Bombay Reunion Islanders>Indians>any

PEL– French-Canadians
k
PP1P – Swedes>Amish>Israelis>Japanese>any

Sl(a–) Blacks>Whites>any

Tc(a–b+c-) Blacks

Tc(a–b–c+) Whites

SERF– Thais

U– and S–s–U+var Blacks

UMC– Japanese

Vel– Swedes>any

WES(b–) Finns>Blacks>any

Yk(a–) Whites>Blacks>any

Yt(a–) Arabs>Jews>any
Used with permission from Reid, Lomas-Francis, and Olsson.4
Any = may be found in any population; > = more prevalent than.
396 䡲 AABB TECHNICAL MANUAL
red cells before use is usually unnecessary un-
less the preservative solution is suspected of
interfering with antibody identification.
Panel cells should not be used after the
expiration date; however, this restriction is not
always practical. Most serologists use in-date
reagent cells for initial antibody identification
panels and, if necessary, use expired reagent
red cells to exclude or confirm specificities.
Each laboratory should establish a policy for
using expired reagent red cells and validate
any procedures associated with this prac-
tice.5(p21)
Antiglobulin Reagents
Most antibody detection and identification
studies include an indirect antiglobulin test
(IAT) phase. Antihuman globulin (AHG) can
be specific only for human immunoglobulin G
(IgG), or a polyspecific reagent that contains
anti-IgG and anti-complement may be used. A
polyspecific reagent may detect—or may de-
tect more readily—antibodies that bind com-
plement. To detect complement binding, se-
rum rather than plasma must be used because
the anticoagulant binds calcium, making it
unavailable for complement activation. Al-
though complement binding may be advanta-
geous in some instances, such as the detection
of certain JK system antibodies, many serolo-
gists prefer the routine use of IgG-specific
AHG reagents to avoid unwanted reactivity re-
sulting from in-vitro complement binding by
cold-reactive antibodies.6
Enhancement Media
Although the test system may consist solely of
serum and red cells (either reagent red cells as
provided by the manufacturer or saline-sus-
pended red cells), most serologists use some
type of enhancement medium. Several differ-
ent enhancement media are available, includ-
ing low-ionic strength saline (LISS), polyethyl-
ene glycol (PEG), and 22% bovine albumin.
Additional enhancement techniques may be
used for complex studies. Enhancement tech-
niques are discussed in more detail later in this
chapter.
Test Methods
Hemagglutination testing performed in tubes
has been the gold standard of immunohema-
tology testing for decades, but testing by gel-
column agglutination and solid-phase tech-
nology are commonplace. These methods
offer stable and possibly less subjective end-
points, work-flow standardization, and the
ability to be incorporated into semiautomated
or automated systems. They provide a sensi-
tive detection system for most blood-group
antibodies. Both gel and solid-phase methods
have also been shown to enhance serologic re-
activity that may not be clinically significant in
the selection of units for transfusion, including
the reactivity of warm autoantibodies. Reactiv-
ity that is dependent on the diluent in com-
mercially prepared reagent cells can also be
seen in gel-column tests. Published studies
have compared the various methods for detec-
tion of wanted and unwanted red cell alloanti-
bodies.7-9
Recent reports have illustrated the poten-
tial effect of using red cell membranes vs intact
red cells to coat the microwells in solid-phase
tests. 10,11 In selected samples, reactivity was
found with the disrupted red cell membranes,
but the samples were not reactive when the
same intact red cells were used. Laboratories
using these techniques for routine testing and
initial problem solving should be familiar with
the unique reactivity characteristics of their
chosen method. Testing algorithms that in-
volve both nontube and tube techniques are
frequently developed to address anomalous
reactivity in either gel-column or solid-phase
testing if specificity cannot be assigned. When
careful review of initial and extended testing
using these methods does not allow identifica-
tion of alloantibody specificity(ies), a tube test
performed with LISS or PEG enhancement
media has been used to further evaluate the
sample for clinically significant alloantibodies.
Basic Antibody Identification
Identification Panel
For initial antibody identification panels, it is
common to use the same methods and test
 CHAPTER 16 Identification of Antibodies to Red Cell Antigens
phases as in the antibody-detection test or
crossmatch. The gel and solid-phase methods
involve a single reading of the test at the indi-
rect antiglobulin phase. Tube-testing proto-
cols have greater flexibility for readings at dif-
ferent test phases but many serologists also
utilize this single test reading because the IAT
detects the overwhelming majority of clinical-
ly significant alloantibodies.
Some serologists using tube methods may
choose to include an immediate centrifuga-
tion reading, a room-temperature incubation
that is read before an enhancement medium is
added, or both. Such an approach may en-
hance the detection of certain antibodies (eg,
anti-M, -N, -P1, -I, -Lea, or –Leb) and may help
explain reactions detected in other phases.
These steps are frequently omitted in initial
antibody-identification studies because most
antibodies that are reactive only at lower tem-
peratures have little or no clinical signifi-
cance.
Test observation after 37 C incubation in
tube testing is influenced by the enhancement
media used. Tests employing PEG enhance-
ment cannot be centrifuged and read because
the reagent causes nonspecific aggregation of
all red cells. LISS, albumin, and saline (no en-
hancement) tests do not have this restriction.
A 37 C reading can detect some antibodies (eg,
potent anti-D, -E, or -K) that may cause direct
agglutination of red cells. Other antibodies (eg,
anti-Lea or -Jka) may occasionally be detected
by the lysis of antigen-positive red cells during
the 37 C incubation if serum is tested. Omit-
ting centrifugation and the reading at 37 C
should lessen the detection of unwanted posi-
tive reactions caused by clinically insignifi-
cant cold-reactive autoantibodies and alloan-
ti bo di es. However, in som e i ns ta nces,
potentially clinically significant antibodies are
detected only by their 37 C reactivity. Exam-
ples of 103 such antibodies (63-E, 27-K, 5-Jka ,
4-D, 3-cE, and 1-C) were identified in 87,480
samples. 12 If the 37 C reading is desired in a
specific antibody study, an alternative strategy
to avoid the uptake of cold antibodies is to set
up duplicate tests. One test is read after the 37
C incubation, and the other test is read only at
the AHG phase.
䡲 397
Selected Cell Panel
If a patient has antibodies that were identified
previously, the known antibodies should be
considered when selecting panel cells to test.
For example, if the patient has known anti-e, it
will not be helpful to test the patient’s serum
with a routine panel, in which 9 of 10 cells are
e-positive. Testing a selected panel of e-nega-
tive red cells is a better approach to find any
newly formed antibodies. It is not necessary to
test e-positive cells to reconfirm the previously
identified anti-e.
If the patient’s red cell phenotype is
known, reagent red cells selected to detect
only those alloantibodies that are potentially
formed by the patient may be tested. For
example, if the patient’s Rh phenotype is C–
E+c+e–, cells selected to exclude anti-E and
anti-c should not be necessary or can be limit-
ed to a single selected cell because the patient
is not expected to form alloantibodies to these
antigens. Exceptions include patients with
weak or altered (partial) Rh antigens, which
are usually found in minority ethnic groups,
and patients whose Rh phenotype was deter-
mined by deoxyribonucleic acid (DNA) testing
rather than serology and who could be carry-
ing a silenced allele. This approach can mini-
mize the amount of testing required.
INTERPRETING RE SULTS. Antibody-detec-
tion results are interpreted as positive or nega-
tive according to the presence or absence of
reactivity (ie, agglutination or hemolysis), re-
spectively. Interpretation of panel results can
be a more complex process combining tech-
nique, knowledge, and intuitive skills. Panel
results generally include both positive and
negative results, sometimes at different phases
of testing; each positive result should be ex-
plained by the final conclusion. The patient’s
phenotype and the probability of the antibody
specificity are also taken into account in the
final interpretation.
POSITIVE AND NEGATIVE RE ACTIONS.
Both positive and negative reactions are im-
portant in antibody identification. The phase
and strength of positive reactions can suggest
certain specificities. (See Method 1-9 for
398 䡲 AABB TECHNICAL MANUAL
grading agglutination.) Positive reactions are
compared to the antigen patterns of the panel
cells to help assign specificity. A single alloan-
tibody usually produces a clear pattern with
antigen-positive and antigen-negative re-
agent red cells. For example, if a serum sample
is reactive only with red cell samples 3 and 5 of
the reagent red cell panel shown in Table 16-2,
anti-E is very likely present. Both reactive red
cells express the antigen, and all nonreactive
cells lack it. When there is no discernible pat-
tern to explain the reactivity, possible explana-
tions include multiple antibodies, dosage, and
variations in antigen expression. These factors
are discussed in more detail later in this chap-
ter. Negative reactions are important in anti-
body identification because they allow tenta-
tive exclusion of antibodies to antigens
expressed on the nonreactive red cells. Exclu-
sion of antibodies is an important step in the
interpretation process and must be performed
to ensure proper identification of all of the an-
tibodies present.
EXCLUSION, “ RULE OUT,” OR “ CROSS
OUT.” A widely used first approach to the in-
terpretation of panel results is to exclude spec-
ificities on the basis of nonreactivity of the pa-
tient’s serum with red cells that express the
antigen. Such a system is sometimes referred
to as a “cross-out” or “rule-out” method. Once
results have been recorded on the work sheet,
the antigen profile of the first nonreactive red
cell is examined. If an antigen is present on the
red cell sample and the serum was not reactive
with it, the presence of the corresponding an-
tibody may tentatively be excluded. Many
technologists actually cross out such antigens
from the list at the top of the panel sheet to fa-
cilitate the process. After all of the antigens on
the list for that red cell have been crossed out,
the same process is performed with the other
nonreactive red cells; additional specificities
are then excluded. In most cases, this process
leaves a group of antibodies that have not
been excluded.
Next, the red cells that are reactive with
the serum are evaluated. The pattern of reac-
tivity for each specificity that was not excluded
is compared to the pattern of reactivity ob-
tained with the test serum. If there is an anti-
gen pattern that matches the test serum pat-
tern exactly, this pattern most likely identifies
the specificity of the antibody in the serum. If
there are remaining specificities that were not
excluded, additional testing is needed to elimi-
nate the possibilities and confirm the suspect-
ed specificity. This process requires testing of
the serum with additional selected red cells.
Although the exclusion (rule-out) ap-
proach often identifies simple antibodies, it
should be considered only as a provisional
step, particularly if the rule out was based on
the lack of reactivity with red cells that have
weaker expression of the antigen (eg, cells
from heterozygous donors).
SELECTED CELLS. Selected cells are red cells
that have been chosen because they express
some specific antigens and lack others. Select-
ed red cells with different antigen combina-
tions can be used to confirm or rule out the
presence of antibodies. For example, if a pat-
tern of reactive red cells fits anti-Jk a exactly,
but anti-K and anti-S were not excluded, the
serum should be tested with selected red cells.
Ideally, red cells with the following phenotypes
should be chosen: Jk(a–), K+, S–; Jk(a–), K–, S+;
and Jk(a+), K–, S–. The reaction pattern with
these red cells should both confirm the pres-
ence of anti-Jka and include or exclude anti-K
and anti-S. Whenever possible, selected red
cells should have a strong expression of the
antigen being tested (ie, from homozygous do-
nors or red cells with double-dose expression).
Such red cells help ensure that nonreactivity
with the selected red cell indicates the absence
of the antibody and not that the antibody was
too weak to be reactive with a selected red cell
that had a weak expression of the antigen.
PROBABILIT Y. To ensure that an observed
pattern of reactions is not the result of chance
alone, conclusive antibody identification re-
quires the serum to be tested against a suffi-
cient number of reagent red cell samples that
lack—and express—the antigen that corre-
sponds with the antibody’s apparent specifici-
ty. A standard approach (which is based on
Fisher’s exact method) has been to require that
TABLE 16-2. Example of a Reagent Red Cell Panel for Antibody Identification

Cell Rh-hr MNS Kell P Lewis Duffy Kidd Others Cell Results
w a a a b a b a b
D C E c e f C V M N S s K k Kp Js P1 Le Le Fy Fy Jk Jk 37 C AHG
CHAPTER 16

1 + + 0 0 + 0 0 0 + 0 0 + 0 + 0 0 + 0 + + + 0 + Bg(a+) 1

2 + + 0 0 + 0 + 0 + + + 0 0 + 0 0 + 0 0 0 0 + 0 2

3 + 0 + + 0 0 0 0 0 + 0 + 0 + 0 0 0 + 0 0 + + + 3

4 0 + 0 + + + 0 0 + 0 + + 0 + 0 0 + 0 + + 0 + 0 4

5 0 0 + + + + 0 0 0 + + + 0 + 0 0 + 0 + 0 + 0 + 5

6 0 0 0 + + + 0 0 + 0 + 0 + + 0 0 + 0 + + 0 0 + 6

7 0 0 0 + + + 0 0 + + + + 0 + 0 0 + 0 + 0 + + 0 7

8 + 0 0 + + + 0 + 0 + 0 0 0 + 0 0 + 0 0 0 0 0 + 8

9 0 0 0 + + + 0 0 + + + + + 0 0 0 0 + 0 + 0 + + 9

10 0 0 0 + + + 0 0 + 0 0 + + + + 0 + 0 0 0 + + + Yt(b+) 10
Identification of Antibodies to Red Cell Antigens

11 + + 0 0 + 0 0 0 + + 0 + 0 + 0 0 + 0 + 0 + 0 + 11

AC AC
+ indicates presence of antigen, 0 indicates absence of antigen, AC = autocontrol, AHG = antihuman globulin.
䡲
399
400 䡲 AABB TECHNICAL MANUAL

three antigen-positive red cell samples are re-
active and that three antigen-negative red cell
samples are not reactive for each specificity
identified.13 In some cases, the use of two reac-
tive and two nonreactive red cell samples is
also an acceptable approach for antibody con-
firmation.5,14 When that approach is not possi-
ble, a more liberal approach (which is derived
from calculations by Harris and Hochman15)
allows the minimum requirement for a proba-
bility (p) value of 0.05 to be met with two re-
active and three nonreactive red cell samples
or with one reactive and seven nonreactive red
cell samples (or the reciprocal of either combi-
nation). Comparative p-value calculations are
shown in Table 16-3. Additional details on cal-
culating probability may be found in the sug-
gested readings list at the end of this chapter.
The possibility of false-negative results with
antigen-positive red cells must be considered
as well as that of unexpected positive results
(ie, caused by the presence of an additional
antibody or an error in the presumptive anti-
body identification).
Autologous Control
The autologous control (autocontrol), in
which serum and autologous red cells are test-
ed under the same conditions as serum and
reagent red cells, is an important part of anti-
body identification. The autocontrol is not the
same as or equivalent to a DAT (Method 3-14).
Incubation and the presence of enhancement
reagents may cause reactivity in the autocon-
trol that is only an in-vitro phenomenon. If the
autocontrol is positive in the antiglobulin
phase, a DAT should be performed. If the DAT
result is negative, antibodies to an enhance-
ment medium constituent or autoantibodies
that are reactive only in the enhancement me-
dium should be considered. Warm autoanti-
bodies and cold autoantibodies, such as anti-I,
-IH, or -Pr, may be reactive in an IAT when cer-
tain enhancement media are used; therefore,
testing should be repeated in another medi-
um. If the DAT result is positive, it must be in-
terpreted with careful attention to the transfu-
sion history. Autoantibodies or drugs could
explain a positive DAT result; however, if the
patient has an alloantibody and was recently
transfused with blood that expressed the
corresponding antigen, the circulating donor
red cells could be coated with alloantibody,
resulting in a positive DAT result associated
with a clinically significant delayed transfu-
sion reaction.

TABLE 16-3. Probability Values for Selected Antibody Identification Approaches

No. No. No.

Tested Positive Negative Fisher13 Harris-Hochman15

5 3 2 0.100 0.035
6 4 2 0.067 0.022
6 3 3 0.050 0.016
7 5 2 0.048 0.015
7 4 3 0.029 0.008
8 7 1 0.125 0.049
8 6 2 0.036 0.011
8 5 3 0.018 0.005
8 4 4 0.014 0.004
 CHAPTER 16 Identification of Antibodies to Red Cell Antigens
Phenotyping Autologous Red Cells
Determining the phenotype of the autologous
red cells is an important part of antibody iden-
tification. When an antibody is tentatively
identified in the serum, the corresponding an-
tigen is expected to be absent from the autolo-
gous red cells. For example, if serum from an
untransfused individual appears to contain
anti-Fya but the autologous red cells have a
negative DAT result and type as Fy(a+), the re-
sults are clearly in conflict and further testing
is indicated. Thus, knowing the patient’s phe-
notype can help guide exclusion testing.
It may be difficult to determine the pa-
tient’s phenotype if he or she was transfused in
the past 3 months. A pretransfusion speci-
men, if available, should be used to determine
the phenotype. If a pretransfusion sample is
not available, the patient’s newly formed
autologous red cells can be separated from the
transfused red cells and then typed (Method 2-
22). Separation of young red cells by centrifu-
gation is based on the difference in the densi-
ties of new and mature red cells. Centrifuga-
tion is most successful when 3 or more days
have elapsed since the last transfusion, which
will provide time for new autologous red cell
production. New autologous red cells must be
isolated from the sample while it is fresh. The
technique is ineffective if the sample is too old
(>24 hours), the patient is not producing new
red cells, or the patient has sickle cell anemia.
Sickle cells are quite dense, and centrifu-
gation is not an effective technique for sepa-
rating the autologous red cells from the trans-
fused donor red cells in a patient with sickle
cell disease. However, autologous sickle cells
may be separated from donor red cells using
washes with hypotonic saline (Method 2-23).
Sickle cells containing hemoglobin SS are re-
sistant to lysis by hypotonic saline, whereas
donor red cells containing hemoglobin AA are
lysed.
The use of potent blood-typing reagents,
appropriate controls, and observation for
mixed-field reactions can allow a specimen
contaminated with donor red cells to be phe-
notyped. Phenotyping results on posttransfu-
sion samples can be misleading, however, and
䡲 401
should be interpreted with caution. 16 If the
specificity of the antibody(ies) in the patient’s
plasma is clear, extensive efforts to separate
and type the patient’s own red cells are not
necessary. A compatible AHG crossmatch us-
ing antigen-negative donor units provides ad-
ditional confirmation of the antibody’s speci-
ficity.17(p37) Definitive typing can be performed
on the patient’s red cells 3 months after the last
transfusion if the patient is not transfusion de-
pendent or experiencing marrow aplasia or
failure.
Cold and warm autoantibodies may also
complicate red cell typing. When red cells are
coated with cold autoantibodies, it may be
possible to remove the autoantibodies with
warm (37 C) saline washes (Method 2-17). If
the cold autoantibodies are very potent, it may
be necessary to treat the red cells with dithio-
threitol (DTT) to dissociate IgM molecules
that cause spontaneous agglutination (Meth-
od 2-18). When red cells are coated with IgG
autoantibodies, it is not possible to perform
typing that requires an IAT without first re-
moving the bound IgG. However, it is often
possible to type antibody-coated red cells with
direct-agglutinating antisera, such as IgM
monoclonal reagents. With rare exceptions,
most direct-agglutinating monoclonal re-
agents give valid phenotyping results despite a
positive DAT result.18 For antisera that require
an IAT (eg, anti-Fya and anti-Fyb), it is neces-
sary to remove the IgG from the test red cells
before typing. Common techniques for remov-
ing IgG antibodies include gentle heat elution
(Method 2-19), treatment with chloroquine di-
phosphate (Method 2-20), and treatment with
acid glycine/EDTA (Method 2-21).
Molecular DNA genotyping offers an al-
ternative to serologic typing and is especially
useful when the patient has been recently
transfused or the patient’s red cells are heavily
coated with IgG. Molecular testing relies on
the extraction of DNA from white cells. Be-
cause of the short life-span of white cells in
vivo and the assay design, the presence of
transfused white cells from donors is not a lim-
iting factor in determining the patient’s geno-
type.
402 䡲 AABB TECHNICAL MANUAL
There are situations, however, where the
genotype of a person may not predict the red
cell phenotype. Mutations that inactivate gene
expression or rare new alleles may not be iden-
tified by the specific assay performed. The
genotype obtained from DNA isolated from
leukocytes and other hematopoietic cells may
differ from that of other tissues in people with
a history of transplantation.19
When DNA testing is used as a tool in an-
tibody identification, the predicted red cell
phenotype should be used judiciously. If a
sample appears to contain an antibody speci-
ficity when the predicted phenotype of the pa-
tient is antigen positive, the apparent antibody
should be further investigated. This discrepan-
cy may indicate that the patient’s red cells do
not actually express the antigen due to a gene
mutation not detected by the assay. Alterna-
tively, the patient might have an altered or par-
tial antigen due to an additional gene poly-
morphism. It is important to remember that
the antibody in the sample could be, in fact, an
alloantibody.
Complex Antibody Problems
Not all antibody identifications are simple.
The exclusion procedure does not always lead
directly to an answer, and additional testing
may be required. When an antibody screen or
incompatible crossmatch detects an unex-
pected antibody, the next step may be to deter-
mine whether the antibody is an autoantibody
or alloantibody. An autocontrol, which may
not have been performed in the initial testing,
can start the identification process. Figure 16-
1 shows some approaches to identifying anti-
bodies in a variety of situations when the auto-
control is negative, and Fig 16-2 shows some
approaches to identifying antibodies when the
autocontrol is positive.
Selected Serologic Procedures
Many techniques and methods are used in
complex antibody identification. Some of the
methods described in this chapter are used
routinely by many laboratories; others are
used selectively and may apply only in special
circumstances. It is important to remember
that no single method is optimal for detecting
all antibodies. Any laboratory that performs
antibody detection or identification should
use routine methods and have access to some
alternative approaches.
When a pattern of weak reactions fails to
indicate specificity or the presence of an anti-
body is suspected but cannot be confirmed,
the use of enhancement techniques or testing
of panel cells treated with enzymes or chemi-
cals may be helpful. An autocontrol should al-
ways be included with each technique.
LISS and PEG
LISS and PEG techniques (Methods 3-4 and 3-
5) are used to enhance reactivity and reduce
incubation time. LISS may be used to suspend
test red cells for use in tube or column aggluti-
nation tests or as an additive medium for tube
or solid-phase tests. Care should be taken to
follow the instructions in the manufacturer’s
product insert closely to ensure that the ap-
propriate proportion of serum to LISS is
achieved. Commercially prepared LISS addi-
tives or PEG additives may contain additional
enhancing agents. Because LISS and PEG en-
hance autoantibodies, their use may compli-
cate alloantibody identification in samples
that also contain autoantibodies.20,21
Enzymes
Ficin and papain are the most frequently used
enzymes for complex antibody identification.
They destroy or weaken antigens, such as M,
N, Fya, Fyb, Xga, JMH, Ch, and Rg (Table 16-4).
Antibodies to these antigens are nonreactive
with treated red cells. Conversely, ficin-treated
and papain-treated red cells show enhanced
reactivity with other antibodies (eg, Rh, P, I,
Kidd, and Lewis). Additional enzymes that are
commonly used in immunohematology labo-
ratories include trypsin, -chymotrypsin, and
pronase. Depending on the enzyme and meth-
od used, other antigens may be altered or de-
stroyed. Antigens that are inactivated by one
proteolytic enzyme may not be inactivated
by other enzymes. The clinical significance
of antibodies that are reactive only with
enzyme-treated cells is questionable; such
 CHAPTER 16
Identification of Antibodies to Red Cell Antigens
䡲

FIGURE 16-1. Antibody identification with negative autocontrol.

DTT = dithiothreitol.
403
 404
䡲
AABB TECHNICAL MANUAL

FIGURE 16-2. Antibody identification with positive autocontrol.

HPCs = hematopoietic progenitor cells; IVIG = intravenous immune globulin.
 CHAPTER 16 Identification of Antibodies to Red Cell Antigens 䡲 405

TABLE 16-4. Alterations of Antigens by Various Agents

Agent Antigens Usually Denatured or Altered*

†
Proteolytic enzymes M, N, S, Fya, Fyb, Yta, Ch, Rg, Pr, Tn, Mg, Mia/Vw, Cla, Jea, Nya, JMH,
some Ge, and Inb
Dithiothreitol (DTT) or Yta, JMH, Kna, McCa, Yka, LWa, LWb, all Kell, Lutheran, Dombrock, and
2-aminoethylisothiouronium Cromer blood group antigens
bromide (AET)
*Some antigens listed may be weakened rather than completely denatured. Appropriate controls should be used with modi-
fied red cells.
†
Different proteolytic enzymes may have different effects on certain antigens.

“enzyme-only” antibodies may not have clini-
cal significance.22
In addition to enhancing the reactivity of
certain antibodies, enzyme techniques may be
used to separate mixtures of antibodies. For
example, if a serum sample contains anti-Fya
and anti-Jka, many of the red cell samples on
the initial panel would be reactive. Then, if a
panel of enzyme-treated red cells were tested,
the anti-Jk a reactivity would be enhanced,
whereas the anti-Fy a reactivity would be de-
stroyed. Procedures for the preparation and
use of proteolytic enzymes are given in
Methods 3-8 to 3-13.
Temperature Reduction
Some antibodies (eg, anti-M, -N, -P1, -Le a,
-Leb, and -A1) react better at room temper-
ature or below, and their specificity may be ap-
parent only at a temperature below 22 C. An
autocontrol is especially important for tests at
low temperatures because many sera also
contain anti-I or other cold-reactive autoanti-
bodies.
Increased Serum-to-Cell Ratio
Increasing the volume of serum incubated
with a standard volume of red cells may en-
hance the reactivity of antibodies that are
present in low concentrations. One acceptable
procedure involves mixing four volumes
(drops) of serum with one volume of a 2% to
5% saline suspension of red cells and incubat-
ing the mixture for 60 minutes at 37 C. Periodic
mixing during the incubation promotes con-
tact between the red cells and the antibodies.
It is helpful to remove the serum before wash-
ing the cells for an IAT because the standard
three to four washes may be insufficient to re-
move all of the unbound immunoglobulin if
increased amounts of serum are used. More
than four washes are not recommended be-
cause bound antibody molecules may dissoci-
ate. Increasing the serum-to-red-cell ratio is
not appropriate for tests using LISS or com-
mercial PEG, which may contain LISS. Tests
performed in a low-ionic-strength medium
require specific proportions of serum and
additive.
Increased Incubation Time
For some antibodies, a 15-minute incubation
period may not be sufficient to achieve equi-
librium; therefore, the reactions may be nega-
tive or weak, particularly in saline or albumin
media. Extending the incubation time to be-
tween 30 and 60 minutes for albumin or saline
tests often improves the reactivity and helps
clarify the pattern of reactions. Extended incu-
bation may be contraindicated when LISS or
PEG is used. If the incubation period exceeds
the recommended times for these methods,
the reactivity may be diminished or lost. Care
must be taken to use all reagents according to
the manufacturer’s directions.
406 䡲 AABB TECHNICAL MANUAL
Alteration in pH
Altering the pH of the test system can change
the reactivity of certain antibodies, enhancing
the reactivity of some and decreasing that of
others.
Some examples of anti-M are enhanced
when the pH of the test system is lowered to
6.5.23 If anti-M is suspected because the only
reactive red cells are M+N–, a definitive pat-
tern (ie, reactivity with M+N+ red cells also)
may be seen if the serum is acidified. The addi-
tion of one volume of 0.1 N HCl to nine vol-
umes of serum lowers the pH to approximately
6.5. The acidified serum should be tested with
known M-negative cells to control for nonspe-
cific agglutination.
Lowering the pH, however, significantly
decreases the reactivity of other antibodies.24 If
unbuffered saline with a pH <6.0 is used to
prepare red cell suspensions or for washing in
an IAT, antibodies in the Rh, Duffy, Kidd, and
MNS blood groups may lose reactivity. Phos-
phate-buffered saline (Method 1-8) can be
used to control the pH and enhance the detec-
tion of antibodies that are poorly reactive at a
lower pH.25
Inhibition Techniques
Soluble forms of some blood group antigens
exist in body fluids, such as saliva, urine, and
plasma. These substances are also present in
other natural sources, and they can be pre-
pared synthetically. Soluble substance can be
used to inhibit the reactivity of the corre-
sponding antibody that could mask the pres-
ence of underlying nonneutralizable antibod-
ies. Also, when an antibody is suspected,
inhibition of the reactivity by a soluble sub-
stance can help with the identification of the
antibody. For example, if a suspected anti-P1
does not produce a definitive pattern of agglu-
tination, the loss of reactivity after the addition
of soluble P1 substance strongly suggests that
the specificity is anti-P1 if a parallel dilution
control with saline. Inhibition results can be
interpreted only when the test is nonreactive
and the dilution control that substitutes an
equal volume of saline for the soluble sub-
stance is reactive.
The most commonly used substances for
inhibition include the following:
1. Lewis substances. Lea substances, Leb sub-
stances, or both are present in the saliva of
individuals who possess the Lewis gene
(FUT3). Lea substance is present in the
saliva of Le (a+b–) individuals, and both Lea
and Leb substances are present in the saliva
of Le(a–b+) individuals (Method 2-8). Com-
mercially prepared Lewis substance is
available.
2. P1 substance. Soluble P1 substance is pres-
ent in hydatid cyst fluid and the ovalbumin
of pigeon eggs. Commercially prepared P1
substance is available.
3. Sd a substance. Soluble Sda blood group
substance is present in various body fluids,
but urine has the highest concentration of
Sda.26 To confirm the presence of anti-Sda in
a serum sample, urine from a known Sd(a+)
individual (or a pool of urine specimens)
can be used to inhibit the antibody reactiv-
ity (Method 3-19).
4. Chido and Rodgers substances. Ch and Rg
antigens are epitopes on the fourth compo-
nent of human complement (C4).27,28 Most
normal red cells have a trace amount of C4
on their surface. Anti-Ch and anti-Rg are
reactive with this C4 in an IAT. If red cells
are coated in vitro with excess C4, these
antibodies may cause direct agglutina-
tion.29 A useful test to identify anti-Ch and
anti-Rg is inhibition of the antibodies with
plasma from Ch+, Rg+ individuals (Method
3-17).
Inactivation of Blood Group Antigens
Certain blood group antigens can be destroyed
or weakened by chemical treatment of the
cells (Table 16-4). Modified red cells can be
useful for both confirming the presence of sus-
pected antibodies and detecting additional
antibodies. The use of modified red cells can
be especially helpful if a sample contains an
antibody to a high-prevalence antigen because
antigen-negative red cells are rare. Proteolytic
enzymes, described above, are commonly
used to alter red cell antigens. Sulfhydryl re-
 CHAPTER 16 Identification of Antibodies to Red Cell Antigens
agents, such as 2-aminoethylisothiouronium
bromide (AET), 2-mercaptoethanol (2-ME), or
DTT, can be used to weaken or destroy anti-
gens in the Kell system and some other anti-
gens (Method 3-18).30,31 ZZAP reagent, which
contains proteolytic enzyme and DTT, dena-
tures antigens that are sensitive to DTT (eg, all
Kell system antigens) as well as antigens that
are sensitive to enzymes (Method 4-8).32 Gly-
cine-HCl/EDTA treatment of red cells destroys
Bg and Kell system antigens as well as the Era
antigen (Methods 2-21 and 4-2).33 Chloroquine
diphosphate can be used to weaken the ex-
pression of Class I HLA antigens (Bg antigens)
on red cells.34 Chloroquine treatment also
weakens some other antigens, including Rh
antigens (Method 2-20).
Sulfhydryl Reagents
Sulfhydryl reagents, such as DTT and 2-ME,
can be used to cleave the disulfide bonds that
join the monomeric subunits of the IgM pen-
tamer. Intact 19S IgM molecules are cleaved
into 7S Ig subunits, which have altered sero-
logic reactivity.35 The interchain bonds of 7S Ig
monomers are relatively resistant to such
cleavage. Sulfhydryl reagents (DTT and 2-ME
as well as AET) can also be used to cleave di-
sulfide bonds that are responsible for the con-
formation of certain blood group antigens
and, therefore, are used to destroy certain red
cell antigens.
Uses of sulfhydryl reagents include the
following:
1. Determining the Ig class of an antibody
(Method 3-16).
2. Identifying antibodies in a mixture of IgM
and IgG antibodies, particularly when an
agglutinating IgM antibody masks the pres-
ence of IgG antibodies.
3. Determining the relative amounts of IgG
and IgM components of a given specificity
(eg, anti-A or -B).
4. Dissociating red cell agglutinates caused by
IgM autoantibodies (Method 2-18).
5. Dissociating IgG antibodies from red cells
using a mixture of DTT and proteolytic
enzyme (ZZAP reagent) (Method 4-8).
䡲 407
6. Destroying selected red cell antigens for use
in antibody investigations (eg, antigens in
the Kell, Dombrock, Cartwright, LW, and
Knops systems) (Method 3-18).
Adsorption
Antibody can be removed from a serum sam-
ple by adsorption onto red cells that express
the corresponding antigen. After the antibody
attaches to the membrane-bound antigens
and the serum and cells are separated, the spe-
cific antibody remains attached to the red
cells. It may be possible to harvest the bound
antibody by elution or examine the adsorbed
serum for antibody(ies) remaining after the
adsorption process.
Adsorption techniques are useful for the
following purposes:
1. Separating multiple antibodies present in a
single serum.
2. Removing autoantibody to permit the
detection or identification of underlying
alloantibodies.
3. Removing unwanted antibody (often anti-
A, anti-B, or both) from serum that contains
an antibody that is suitable for reagent use.
4. Confirming the presence of specific anti-
gens on red cells by their ability to remove
antibody of corresponding specificity from
previously characterized serum.
5. Confirming the specificity of an antibody
by showing that it can be adsorbed onto red
cells of only a particular blood group phe-
notype.
Adsorption serves different purposes in
different situations; no single procedure is sat-
isfactory for all purposes (Methods 4-5, 4-8,
4-9, and 4-10). A basic procedure for antibody
adsorption can be found in Method 3-20. The
usual serum-to-cell ratio is one volume of se-
rum to an equal volume of washed, packed red
cells. To enhance antibody removal, a larger
volume of red cells increases the proportion of
antigen. The incubation temperature should
be that at which the antibody is optimally re-
active. Pretreating red cells with a proteolytic
enzyme may enhance antibody uptake and
408 䡲 AABB TECHNICAL MANUAL
reduce the number of adsorptions required to
remove an antibody completely. Because en-
zymes destroy some antigens, antibodies di-
rected against those antigens are not removed
by enzyme-treated red cells. To ensure that an
adsorption process is complete (ie, that no un-
adsorbed antibody remains), it is essential to
confirm that the adsorbed serum is nonreac-
tive with a reserved sample of the adsorbing
red cells that was not used for adsorption. Ad-
sorption requires a substantial volume of red
cells, and vials of reagent red cells are usually
not sufficient. Blood samples from donor units
or staff members are the most convenient
sources.
When separating mixtures of antibodies,
the selection of red cells of the appropriate
phenotype is extremely important. If one or
more antibodies have been previously identi-
fied, red cells that express the corresponding
antigens can be used to remove the known an-
tibodies and leave the unknown antibody(ies)
in the adsorbed serum. For example, if a per-
son who types K+k–, Fy(a–b+) has produced
anti-k, it may be necessary to adsorb the anti-k
onto K–k+, Fy(a–b+) reagent red cells to re-
move the anti-k. Then, the adsorbed serum
can be tested with common K–k+, Fy(a+b–)
red cells to detect possible anti-Fy a.
Elution
Elution dissociates antibodies from sensitized
red cells. Bound antibody may be released by
changing the thermodynamics of antigen-
antibody reactions, neutralizing or reversing
forces of attraction that hold antigen-antibody
complexes together, or disturbing the struc-
ture of the antigen-antibody binding site. The
usual objective is to recover bound antibody in
a usable form.
Various elution methods have been de-
scribed in the literature. Selected procedures
are given in Methods 4-1 through 4-4. No sin-
gle method is best for all situations. Heat or
freeze-thaw elution techniques are usually re-
stricted to the investigation of HDFN caused
by ABO incompatibility because these elution
procedures rarely work well for other antibod-
ies. Acid or organic solvent methods are used
for eluting warm-reactive auto- and alloanti-
bodies. Commercial kits are also available for
performing elution. (See Table 17-2 for a list of
elution methods and their uses, advantages,
and disadvantages.)
Elution techniques are useful for the fol-
lowing:
1. Investigation of a positive DAT result
(Chapter 17).
2. Concentration and purification of antibod-
ies, detection of weakly expressed antigens,
and identification of multiple antibody
specificities. Such studies are used in con-
junction with an appropriate adsorption
technique, as described below and in
Method 2-7.
3. Preparation of antibody-free red cells for
phenotyping or autologous adsorption
studies. Procedures used to remove cold-
and warm-reactive autoantibodies from red
cells are discussed in Methods 4-5 and 4-8.
Technical factors that influence the out-
come of elution procedures include the follow-
ing:
1. Incomplete washing. Sensitized red cells
should be thoroughly washed before an
elution to prevent contamination of the
eluate with unbound residual antibody. Six
washes with saline are usually adequate,
but more washes may be needed if the
serum contains a high-titer antibody (the
considerations in Item 3 below should be
kept in mind). To confirm the efficacy of the
washing process, supernatant fluid from
the final wash should be tested for antibody
activity and found to be nonreactive.
2. Binding of protein to glass surfaces. If an
eluate is prepared in the test tube that was
used during the sensitization or washing
phases, antibody that nonspecifically binds
to the test tube surface may dissociate dur-
ing the elution. Similar binding can also
occur from a whole blood sample when a
patient has a positive DAT result and has
free antibody in the serum. To avoid such
contamination, red cells used to prepare an
 CHAPTER 16 Identification of Antibodies to Red Cell Antigens
eluate should be transferred to a clean test
tube before washing and then to another
clean tube before the elution procedure is
initiated.
3. Dissociation of antibody before elution. IgM
antibodies, such as anti-A or anti-M, or
low-affinity IgG may spontaneously disso-
ciate from the red cells during the wash
phase. To minimize the loss of bound anti-
body, cold (4 C) saline or wash solution pro-
vided by the manufacturer should be used
for washing.
4. Incorrect technique. Such factors as incom-
plete removal of organic solvents or failure
to correct the tonicity or pH of an eluate
may cause the reagent red cells used to test
the eluate to hemolyze or appear “sticky.”
The presence of stromal debris may inter-
fere with the reading of test results. Careful
technique and strict adherence to proce-
dures should eliminate such problems.
5. Instability of eluates. Dilute protein solu-
tions, such as those obtained by elution
into saline, are unstable. Eluates should be
tested as soon after preparation as possible.
Alternatively, bovine albumin may be
added to a final concentration of 6% w/v,
and the preparation may be frozen during
storage. Eluates can also be prepared in
antibody-free plasma, 6% albumin, or a
similar protein medium. When commercial
elution kits are used, the manufacturer’s
instructions for preparation and storage
should be followed.
Combined Adsorption-Elution
Combined adsorption-elution tests can be
used to separate a mixture of antibodies in a
single serum sample, detect weakly expressed
antigens on red cells, or help identify weakly
reactive antibodies. The process consists of
first incubating serum with selected red cells
and then eluting antibody from the adsorbing
red cells.
Care must be taken when selecting the
adsorbing cells to separate a mixture of anti-
bodies. The cells should express only one of
the antigens corresponding to an antibody in
the mixture so that the eluate from the cells
䡲 409
will contain only that antibody. Both the eluate
and adsorbed serum can be used for further
testing. Unmodified red cells are generally
used for adsorptions when subsequent elu-
tions are being prepared.
Titration
The titer of an antibody is usually determined
by testing serial twofold dilutions of the serum
with selected red cells. Results are expressed as
the reciprocal of the highest serum dilution
that shows macroscopic agglutination. Titra-
tion values can provide information about the
relative amount of antibody present in a se-
rum sample or the relative strength of antigen
expression on red cells.
Titration studies are useful for the follow-
ing purposes:
1. Prenatal studies. When the antibody has a
specificity that is known to cause HDFN or
the antibody’s clinical significance is
unknown, the results of titration studies
may contribute to the decision about per-
forming additional procedures (eg, Doppler
sonography or amniocentesis). (See Chap-
ter 22 and Method 5-3.)
2. Antibody identification. Some antibodies
that agglutinate virtually all reagent red cells
may give an indication of specificity by dem-
onstrating reactivities of different strengths
with different red cell samples in titration
studies. For example, potent undiluted auto-
anti-I may be reactive with both adult and
umbilical cord blood red cells, but titration
studies may reveal reactivity with adult I+
cells at a higher dilution than with cord
blood I– red cells. The reactivity of most
antibodies weakens progressively with serial
dilutions (ie, a 2+ reaction becomes 1+ in the
next dilution), and weak antibodies (<1+)
may lose their reactivity when diluted. How-
ever, some antibodies that have weak reac-
tions when undiluted continue to react at
dilutions as high as 1 in 2048. Such anti-
bodies include anti-Ch, -Rg, -Csa, -Yka, -Kna,
-McCa, and -JMH. When weak reactions are
observed in an IAT, titration studies may
be performed to determine whether the
410 䡲 AABB TECHNICAL MANUAL
reactivity is consistent with the antibodies in
this group; however, not all examples of
these antibodies demonstrate such high
titer, low-avidity characteristics. Thus, the
serologic characteristics may suggest certain
specificities, but failure to do so does not
eliminate these possibilities. The antibodies
listed above are not expected to cause short-
ened red cell survival, although there are
examples of other antibodies (eg, anti-
Lu b, -Hy, and –Yta) that may mimic these
serologic characteristics and cause short-
ened red cell survival. Details about titration
are given in Method 3-15.
3. Separating multiple antibodies. Titration
results may suggest that one antibody is
reactive at higher dilutions than another
antibody. That information can allow the
serum to be diluted before it is tested with a
red cell panel, which effectively removes
one antibody and allows the other to be
identified. For example, if a serum contains
anti-c that is reactive to a titer of 2 and anti-
Jka that is reactive to a titer of 16, it may be
possible to eliminate the anti-c reactivity by
diluting the serum to a titer of 8.
Other Methods
Methods other than traditional tube, gel, or
solid-phase techniques may be used for anti-
body identification. Some methods are espe-
cially useful for testing small volumes of serum
or reagents. Such methods include testing in
capillary tubes, microplates, or enzyme-linked
immunosorbent assays. Other methods that
are useful in laboratories with specialized
equipment include radioimmunoassay, im-
munofluorescence, flow cytometry, and im-
munoblotting.
Factors Affecting Antibody
Identification
Variation in Antigen Expression
For a variety of reasons, antibodies are not al-
ways reactive with all red cells that express the
corresponding antigen. Basic interpretation by
exclusion, as described previously, may result
in an antibody being excluded because the se-
rum is nonreactive with an antigen-positive
red cell sample, despite the presence of the an-
tibody. Technical error, weak antibody reactiv-
ity, and variant or weak antigenic expression
are all possible causes. Therefore, whenever
possible, antibody exclusions should be based
only on red cells that strongly express the anti-
gen. Enhancement techniques often help re-
solve problems associated with variations in
antigen expression (Methods 3-3 through 3-
13).
ZYGOSIT Y. Reaction strength of some anti-
bodies may vary because of dosage, in which
antibodies are more strongly reactive (or only)
with red cells that possess a “double-dose” ex-
pression of the antigen. Double-dose antigen
expression occurs when an individual is ho-
mozygous for the gene that encodes the anti-
gen. Red cells from individuals who are het-
erozygous for the gene may express fewer
antigens and, therefore, may be weakly reac-
tive or nonreactive with a weak example of the
corresponding antibody. Alloantibodies vary
in their tendency to demonstrate dosage.
Many antibodies to antigens in the Rh, Duffy,
MNS, and Kidd blood groups demonstrate
dosage.
VARIATION IN ADULTS AND INFANTS.
Some antigens (eg, I, P1, Lea, and Sda) show
variable expression on red cells from different
adults. The antigenic differences can be dem-
onstrated serologically; however, the variabili-
ty is unrelated to zygosity. Certain antibodies
(eg, anti-I or –Lub) demonstrate weaker reac-
tivity with cord red cells than with red cells
from adults (Table 16-5).
CHANGES WITH STORAGE . Blood group an-
tibodies may be more weakly reactive with
stored red cells than with fresh red cells. Some
antigens (eg, Fya, Fyb, M, P1, Kna/McCa, and
Bg) deteriorate more rapidly than others dur-
ing storage, and the rate varies among red cells
from different individuals.36 Because red cells
from donors are often fresher than commer-
cial reagent red cells, some antibodies have
stronger reactions with donor red cells than
with reagent red cells. Similarly, storage of red
cells in a freezer may cause antigens to deteri-
 CHAPTER 16 Identification of Antibodies to Red Cell Antigens
TABLE 16-5. Antigen Expression on Cord Red Blood Cells*
Expression Antigens
Negative Lea, Leb, Sda, Ch, Rg, and AnWj
Weak I, H, P1, Lua, Lub, Yta, Vel, Bg, KN and DO antigens, Yka, Csa, and Fy3
Strong i, LWa, and LWb
*Modified with permission from Reid, Lomas-Francis and Olsson.4
orate, thus producing misleading antibody
identification results.
The pH or other characteristics of the
storage medium can affect the rate of antigen
deterioration.36,37 For example, Fya and Fyb an-
tigens may weaken when the red cells are
stored in a medium with low pH and low ionic
strength. Thus, certain antibodies may dem-
onstrate differences in reactivity with red cells
from different manufacturers if the suspend-
ing media are different.
The age and nature of the specimen must
be considered when red cells are typed. Anti-
gens on red cells from clotted samples tend to
deteriorate more quickly than antigens on red
cells from donor units that are collected in ci-
trate anticoagulants, such as acid-citrate-dex-
trose or citrate-phosphate-dextrose. Red cells
in donor units collected in approved anticoag-
ulants usually retain their antigens throughout
the standard shelf life of the blood component.
EDTA samples up to 14 days old are suitable
for antigen typing.38 However, the manufactur-
er’s instructions should be consulted when
commercial typing reagents are used.
No Discernible Specificity
Factors other than variation in antigen expres-
sion may contribute to difficulty in interpret-
ing results of antibody identification tests. If
the reactivity with the serum is very weak, the
pattern of reactivity and cross-out process
have excluded all likely specificities, or both,
alternative approaches to interpretation should
be used.
PRESENCE OF ANTIGENS IN COMMON. In-
stead of excluding antibodies to antigens on
nonreactive red cells, it might be helpful to ob-
䡲 411
serve which antigens the reactive red cells
have in common. For example, if all of the red
cells reactive at room temperature are P1+ but
the anti-P1 pattern is not complete, the anti-
body could be anti-P1 that is not reactive with
red cells with a weaker expression of the anti-
gen. (Sometimes, such red cells are designated
on the panel sheet as “+w.”) In this case, it
might be helpful to use a method that enhanc-
es anti-P1, such as testing at a colder tempera-
ture.
If all of the reactive red cells are Jk(b+) but
not all Jk(b+) red cells are reactive, the reactive
red cells might be Jk(a–b+) with a double-dose
expression of the antigen. In this case, en-
hancement techniques, such as enzymes,
LISS, or PEG, might help demonstrate reactivi-
ty with all of the Jk(b+) red cells. Typing the pa-
tient’s red cells to confirm that they lack the
corresponding antigen is also very helpful.
Finally, the presence of some antigens
in common may suppress the expression of
certain antigens. This suppression can cause
weak antibodies to be missed or certain cells
to be unexpectedly nonreactive when a sus-
pected antibody fails to show reactivity with all
antigen-positive cells. For example, In(Lu) is
known to suppress the expression of Lutheran
antigens, P1, In b, and AnWj. Similarly, Kp a is
known to weaken the expression of Kell anti-
gens. (See Chapter 14 for a more detailed dis-
cussion.)
INHERENT VARI ABILIT Y. Neb u lo u s re a c -
tion patterns that do not appear to fit any par-
ticular specificity are characteristic of certain
antibodies, such as anti-Bga, -Kna, -McCa, -Sla,
-Yka, -Csa, and -JMH. Antigens corresponding
to these antibodies vary markedly in their
412 䡲 AABB TECHNICAL MANUAL
expression on red cells from different individ-
uals. For example, the expression of Knops
blood group antigens shows marked differenc-
es between individuals of either African or Eu-
ropean ethnicity, and this difference in expres-
sion is caused by variations in the CR1 copy
numbers on the red cells.39 Rarely, a pattern of
reactive and nonreactive red cells cannot be
interpreted because the typing result for a re-
agent red cell is incorrect or the reagent red
cell has a positive DAT result. If the red cell
sample is from a commercial source, the man-
ufacturer should be notified immediately of
the discrepancy.
UNLISTED ANTIGENS. Sometimes, a serum
reacts with an antigen that is not routinely list-
ed on the antigen profile supplied by the re-
agent manufacturer—Ytb is an example. Even
though serum studies yield clearly reactive
and nonreactive test results, anti-Ytb may not
be suspected. In such circumstances, it is use-
ful to ask the manufacturer for additional phe-
notype information. If only one cell is unex-
pectedly reactive, this reaction is most likely
caused by an antibody to a low-prevalence an-
tigen. These antibodies are discussed in more
detail later in this chapter.
ABO T YPE OF RED CELLS TESTED. A serum
sample may be reactive with many or all of the
group O reagent red cells but not with red cells
of the same ABO group as the autologous red
cells. Such a reaction occurs most frequently
with anti-H, anti-IH, or anti-LebH. Group O and
A2 red cells have more H antigen than A1 and
A1B red cells, which express very little H. (See
Chapter 12 for more information.) Thus, sera
containing anti-H or anti-IH are strongly reac-
tive with group O reagent red cells, whereas
autologous A 1 or A 1 B red cells or donor red
cells used for crossmatching may be weakly re-
active or nonreactive. Anti-LebH is strongly re-
active with group O, Le(b+) red cells but weak-
ly reactive or nonreactive with Le(b+) red cells
from A1 or A1B individuals.
Multiple Antibodies
When a serum sample contains two or more
alloantibodies, it may be difficult to interpret
the results of testing performed with a single
panel of reagent red cells. Perhaps the easiest
way to identify multiple antibodies is to deter-
mine the phenotype of the patient’s pretrans-
fusion autologous red cells and then use a se-
lected cell panel to identify or exclude all
common antibodies to the red cell antigens
that the patient lacks. (See the discussion on
selected cells earlier in this chapter.) The
presence of multiple antibodies may be sug-
gested by a variety of test results, such as the
following:
1. The observed pattern of reactive and nonre-
active red cells does not fit a single antibody.
When the exclusion approach fails to indi-
cate a specific pattern, it is helpful to deter-
mine whether the pattern matches two
combined specificities. For example, if the
reactive red cells on the panel in Table 16-2
are numbers 3, 5, 6, 9, and 10, none of the
specificities remaining after crossing out
fits a pattern exactly. However, if both E and
K are considered together, a pattern is dis-
cerned, with cells 3 and 5 showing reactivity
because of anti-E, and cells 6, 9, and 10
because of anti-K. If the reaction pattern
does not fit two combined specificities, the
possibility that more than two antibodies
are present must be considered. The more
antibodies a serum sample contains, the
more complex identification and exclusion
become, but the basic process remains the
same.
2. Reactivity occurs at different test phases.
When reactivity occurs at several phases,
each phase should be analyzed separately.
The pattern at room temperature may indi-
cate a different specificity from the pattern
at the AHG phase. It is also helpful to look
for variations in the strength of the reac-
tions at each phase of testing. Table 16-6
provides information about the character-
istic reactivity of several antibodies.
3. Unexpected reactions occur when attempts
are made to confirm the specificity of a sus-
pected single antibody. If a serum suspected
to contain anti-e is reactive with some e-
negative cells, another antibody may be
present or the suspected antibody may not
 CHAPTER 16 Identification of Antibodies to Red Cell Antigens 䡲 413

TABLE 16-6. Serologic Reactivity of Some Common Blood Group Antibodies

Immuno- Reactivity Associated with

globulin Papain/ DTT
Antibody Class 4C 22 C 37 C AHG Ficin (200 mM) HDFN HTR

Anti-M IgG > IgM Most Most Rare Sensitive Resistant Rare Rare
Anti-N IgM > IgG Most Most Rare Sensitive Resistant No Rare
Anti-S IgG > IgM Most Most Variable Resistant Yes Yes
Anti-s IgG > IgM Most Variable Resistant Yes Yes
Anti-U IgG Most Resistant Resistant Yes Yes
Anti-P1 IgM Most Most Resistant Resistant No Rare
(IgG rare)
Anti-D IgG > IgM Some Some Most Resistant Resistant Yes Yes
(IgA rare)
Anti-C IgG > IgM Some Some Most Resistant Resistant Yes Yes
Anti-E IgG > IgM Some Some Most Resistant Resistant Yes Yes
Anti-c IgG > IgM Some Some Most Resistant Resistant Yes Yes
Anti-e IgG > IgM Some Some Most Resistant Resistant Yes Yes
a
Anti-Lu IgM > IgG Most Most Resistant or Variable Rare No
weakened
Anti-Lub IgG > IgM Some Most Resistant or Variable Mild Yes
weakened
Anti-K IgG > IgM Some Most Resistant Sensitive Yes Yes
Anti-k IgG > IgM Most Resistant Sensitive Yes Yes
a
Anti-Kp IgG Most Resistant Sensitive Yes Yes
Anti-Kpb IgG > IgM Most Resistant Sensitive Yes Yes
Anti-Jsa IgG > IgM Most Resistant Sensitive Yes Yes
b
Anti-Js IgG Most Resistant Sensitive Yes Yes
a
Anti-Le IgM > IgG Most Most Most Most Resistant Resistant No Rare
Anti-Leb IgM > IgG Most Most Most Most Resistant Resistant No No
a
Anti-Fy IgG > IgM Most Sensitive Resistant Yes Yes
b
Anti-Fy IgG > IgM Most Sensitive Resistant Yes Yes
a
Anti-Jk IgG > IgM Most Resistant Resistant Yes Yes
Anti-Jkb IgG > IgM Most Resistant Resistant Yes Yes
a
Anti-Di IgG Most Resistant Resistant Yes Yes
b
Anti-Di IgG Most Resistant Resistant Yes Yes
414 䡲 AABB TECHNICAL MANUAL

TABLE 16-6. Serologic Reactivity of Some Common Blood Group Antibodies (Continued)

Immuno- Reactivity Associated with

globulin Papain/ DTT
Antibody Class 4C 22 C 37 C AHG Ficin (200 mM) HDFN HTR

Anti-Yta IgG Most Variable Sensitive No Yes

or weak-
ened
Anti-Ytb IgG Most Variable Sensitive No No
or weak-
ened
Anti-Xga IgG > IgM Some Most Sensitive Resistant No No

Anti-Sc1 IgG Most Resistant Variable No No

Anti-Sc2 IgG Most Resistant Variable No No

Anti-Doa IgG Most Resistant Variable No Yes

Anti-Dob IgG Most Resistant Variable No Yes

Anti-Coa IgG > IgM Most Resistant Resistant Yes Yes

Anti-Cob IgG Most Resistant Resistant Yes Yes

AHG = antiglobulin reagent; DTT = dithiothreitol; HDFN = hemolytic disease of the fetus and newborn; HTR = hemolytic
transfusion reaction; Ig = immunoglobulin.

be anti-e. Testing a panel of selected e-neg-
ative red cells may help identify an addi-
tional specificity.
4. No discernible pattern emerges. When vari-
able reaction strengths are observed and
dosage or other variations in antigen
strength do not provide an explanation,
additional approaches and methods of test-
ing are needed. Some helpful steps include
the following:
a. If strongly positive results are
obtained, use the exclusion method
with nonreactive cells to eliminate
some specificities from initial consid-
eration.
b. If weak or questionable positive results
are obtained, test the serum against
cells with a strong expression of the
antigen that corresponds with any sus-
pected specificity, and combine this
approach with methods that enhance
the reactivity.
c. Type the patient’s red cells for com-
mon red cell antigens, and eliminate
from consideration specificities that
correspond to antigens on the patient’s
autologous red cells. This step may not
be possible if the patient has been
transfused recently or has had a posi-
tive DAT result.
d. Use a method to inactivate certain
antigens on the reagent cells. Enzyme
treatment renders cells negative for
such antigens as Fya and Fyb (see Table
16-4).
e. Use adsorption or elution methods to
separate antibodies (Methods 3-20, 4-1,
and 4-2).
f. Enhance antibody reactivity by using a
more sensitive method (eg, PEG,
enzymes, increased incubation time,
or increased serum-to-cell ratio; see
Methods 3-5 and 3-8 through 3-13).
 CHAPTER 16 Identification of Antibodies to Red Cell Antigens
Antibodies to High-Prevalence Antigens
If all reagent red cells are reactive but the auto-
control is nonreactive, an alloantibody to a
high-prevalence antigen should be consid-
ered, especially if the strength and test phase
of reactions are uniform for all of the red cells
tested. Antibodies to high-prevalence antigens
can be identified by testing selected red cells of
rare phenotypes and by typing the patient’s
autologous red cells with antisera to high-
prevalence antigens. Knowing the race or eth-
nic origin of the antibody producer can be
helpful when selecting additional tests to per-
form (Table 16-1). Red cells that lack all of the
antigens in a blood group system (eg, Rhnull or
Ko) or chemically modified red cells (eg, DTT-
treated red cells) can help limit possible speci-
ficities (Table 16-4).
If red cells negative for a particular high-
prevalence antigen are not available, red cells
that are positive for the lower-prevalence anti-
thetical antigen can sometimes be helpful. For
example, if a serum contains anti-Coa, which
reacts with a high-prevalence antigen, weaker
reactions may be observed with Co(a+b+) red
cells than with Co(a+b–) red cells because of a
dosage effect.
Antibodies to high-prevalence antigens
may be accompanied by antibodies to com-
mon antigens, which can make identification
much more difficult. In such cases, it may be
necessary to determine the patient’s pheno-
type for common antigens, choose a pheno-
typically similar red cell sample (ie, one that
lacks the patient’s common antigens) that is
incompatible with the patient’s plasma, and
adsorb the antibody to the high-prevalence
antigen onto that red cell sample. This ap-
proach leaves antibodies to common red cell
antigens in the adsorbed plasma, where they
can be identified with a routine selected red
cell panel. Because the identification of anti-
bodies to high-prevalence antigens is compli-
cated, it may be necessary to refer such speci-
mens to a reference laboratory.
SE ROLOGIC CLUES. Knowing the serologic
characteristics of particular antibodies to
high-prevalence antigens and/or the preva-
䡲 415
lence of such antigens in different popula-
tions may help with identification.
1. Reactivity in tests at room temperature
suggests anti-H, -I, -IH, -P, -PP1P k (-Tj a),
-Ena, -LW (some), -Ge (some), -Sda, or -Vel.
2. Lysis of reagent red cells during testing with
fresh serum is characteristic of anti-Vel, -P,
-PP1Pk (-Tja), -Jk3, and some examples of
anti-H and -I.
3. Reduced or absent reactivity with enzyme-
treated red cells occurs with anti-Ch,
-Rg, -Ena, -Inb, -JMH, -Ge2, and some
examples of anti-Yta. Weak nebulous reac-
tions in the AHG phase are often associated
with anti-Kna, -McCa, -Yka, and -Csa.
4. Complement-binding autoantibodies, such
as anti-I and -IH, or alloantibodies, such as
anti-Lub, -Vel, and -Yta, may give similar
results when polyspecific AHG reagent is
used.
ETHNIC CLUES. Antibodies, such as anti-U,
-McCa, -Sla, -Jsb, -Hy, -Joa, -Tca, -Cra, and –Ata,
should be considered if the serum is from an
individual of African ethnicity because the
antigen-negative phenotypes occur almost ex-
clusively in persons of African ethnicity. Indi-
viduals with anti-Kpb are almost always of Eu-
ropean ethnicity. Anti-Di b is usually found
among populations of Asian, South American
Indian, and Native American ethnicity (Table
16-1).
INTERPRETING A POSITIVE DAT RESULT.
When a patient produces an antibody to a
high-prevalence antigen after transfusion, the
patient’s posttransfusion red cells may have a
positive DAT result, and both serum and elu-
ate may be reactive with all red cells tested. Be-
cause this pattern of reactivity is identical to
that of many warm-reacting autoantibodies
that appear after transfusion, the two scenari-
os can be very difficult to differentiate. A post-
transfusion alloantibody to a high-prevalence
antigen would be expected to produce a DAT
result of mixed-field appearance (ie, some red
cells agglutinated among many unagglutinated
red cells) because only the transfused red cells
would be coated with antibody. In practice,
416 䡲 AABB TECHNICAL MANUAL
however, weak sensitization and mixed-field
agglutination can be difficult to differentiate. If
a pretransfusion specimen is not available, it
may be helpful to use red cell separation pro-
cedures to isolate autologous red cells for test-
ing or perform a DNA-based genotype, as de-
scribed earlier in this chapter. Performing a
DAT on autologous red cells, testing the post-
transfusion serum with DAT-negative autolo-
gous red cells, or both may help distinguish an
autoantibody from an alloantibody. If a DAT
result from autologous red cells is negative, the
reactivity is consistent with an alloantibody. If
the posttransfusion serum is reactive with
DAT-negative autologous red cells, the reactiv-
ity is consistent with an autoantibody (Chap-
ter 17 and Fig 16-2).
Antibodies to Low-Prevalence Antigens
If a serum sample is reactive only with a single
donor or reagent red cell sample, an antibody
to a low-prevalence antigen should be sus-
pected. To identify such an antibody, one can
test the serum with a panel of reagent red cells
that express low-prevalence antigens. Alterna-
tively, the one reactive red cell sample can be
tested with known antibodies to low-preva-
lence antigens. Unfortunately, sera that con-
tain antibodies to low-prevalence antigens
often contain multiple antibodies to low-prev-
alence antigens. Although low-prevalence an-
tigens are rare by definition, antibodies that
recognize some of them are much less rare.
Many antibodies to low-prevalence antigens
are reactive only at temperatures below 37 C
and therefore have doubtful clinical signifi-
cance. To confirm the suspected specificities,
one may need the expertise and resources of a
reference laboratory.
If an antibody to a low-prevalence anti-
gen is suspected, transfusion should not be
delayed while identification studies are per-
formed. Because antisera to type donor units
for low-prevalence antigens are rarely avail-
able, it is usually necessary to rely on the cross-
match to avoid transfusion of antigen-positive
units. When the serum is reactive with only
one donor unit or reagent red cell, the most
likely cause is an antibody to a low-prevalence
antigen; however, other possible explanations
include that the red cells may be ABO incom-
patible, have a positive DAT result, or be poly-
agglutinable.
If an antibody in the serum of a pregnant
woman is suspected of reacting with a low-
prevalence antigen, testing the father’s red
cells with maternal plasma (if the plasma is
ABO compatible) can predict the possibility
that the fetal red cells also carry the paternal
antigen and are incompatible with the mater-
nal antibody, even if the antibody specificity is
unknown. If a newborn has a positive DAT re-
sult, testing the mother’s serum or an eluate
from the infant’s red cells against the father’s
red cells can implicate an antibody to a low-
prevalence antigen as the probable cause.
That test can be performed only if the mother’s
plasma is ABO compatible with the father’s red
cells or if the eluate from the infant’s red cells
does not contain anti-A or -B that would be re-
active with the father’s red cells. Some refer-
ence laboratories do not attempt to identify
antibodies to low-prevalence antigens be-
cause the antibodies are not clinically mean-
ingful. Antibodies may be identified when
time permits and suitable reagents are avail-
able.
Antibodies to Reagent Components and
Other Anomalous Serologic Reactions
Antibodies to a variety of drugs and additives
can cause positive results in antibody detec-
tion and identification tests. The mechanisms
are probably similar to those discussed in
Chapter 17. Most of these anomalous reac-
tions are in-vitro phenomena and have no
clinical significance in transfusion therapy,
other than causing laboratory problems that
delay transfusions. The reactions rarely cause
erroneous interpretations of ABO typing that
could endanger the patient. For a more de-
tailed discussion, see the suggested reading by
Garratty.
ROULEAUX. Rouleaux are aggregates of red
cells that often look like a stack of coins when
viewed microscopically. Rouleaux formation is
an in-vitro phenomenon produced by abnor-
 CHAPTER 16 Identification of Antibodies to Red Cell Antigens
mal serum protein concentrations. It may be
difficult to detect antibodies by direct aggluti-
nation in a test serum that contains rouleaux-
producing proteins; however, it is not a prob-
lem in an IAT, where the serum is washed
away. The saline replacement technique can
be used to detect direct-agglutinating antibod-
ies in the presence of rouleaux (Method 3-7).
PRESERVATIVE SOLUTIONS. A n t i b o d i e s
that are reactive with an ingredient in the solu-
tion used to preserve reagent red cells (eg,
chloramphenicol, neomycin, tetracycline, hy-
drocortisone, EDTA, sodium caprylate, or vari-
ous sugars) may agglutinate red cells suspend-
ed in that solution. The autologous control is
often nonreactive unless a suspension of au-
tologous red cells is prepared with the manu-
facturer’s red cell diluent or a similar preserva-
tive. Such reactions can often be avoided by
washing the reagent red cells with saline be-
fore testing. Adding the medium to the autolo-
gous control and converting a nonreactive test
to a reactive test can often confirm the role of
the preservative. In some cases, however,
washing the reagent red cells does not circum-
vent the reactivity, and the resolution may be
more complex.
ENHA NCEME NT MEDIA . Antibodies that are
reactive with ingredients in other reagents,
such as commercially prepared LISS additives
or albumin, can cause agglutination in tests
that use reagent red cells, donor red cells, au-
tologous red cells, or all three. Ingredients that
have been implicated include parabens (in
some LISS additives), sodium caprylate (in
some albumins), and thimerosal (in some
LISS/saline preparations). Antibody to ingre-
dients in enhancement media may be suspect-
ed if the autologous control is positive but the
DAT result is negative. Omitting the enhance-
ment medium usually circumvents the reac-
tivity.
In some cases, antibodies to reagent in-
gredients show blood group specificity (eg,
paraben-dependent anti-Jka, paraben-depen-
dent antibody to Rh protein, or caprylate-de-
pendent autoanti-e).40-42 The autocontrol may
be reactive if the patient’s own red cells express
䡲 417
the antigen, but the DAT result should be neg-
ative.
RE D-CELL-REL ATE D ANOMA LIE S. The age
of red cells can cause anomalous serologic re-
actions. Antibodies exist that are reactive only
with stored red cells. Such antibodies can
cause agglutination of reagent red cells by all
techniques, and the reactivity may be en-
hanced with enzyme-treated red cells. Such re-
activity is not affected by washing the red cells,
and the autocontrol is usually nonreactive. No
reactivity is seen when freshly collected red
cells (ie, from freshly drawn donor or autolo-
gous blood samples) are tested.
Patients with a Positive Autocontrol
Reactivity of a patient’s serum with the autolo-
gous cells may indicate the presence of warm or
cold autoantibodies, antibodies to certain
drugs, alloantibodies to transfused red cells if
the patient was recently transfused, or antibod-
ies to a constituent of the test medium. When
the autocontrol is positive, a DAT should be
performed (Chapter 17 and Fig 16-2).
NO RECENT TRANSFUSIONS. If the reactivi-
ty in the serum occurs at room temperature or
below, the cause is often anti-I or another cold
autoagglutinin. If the reactivity occurs in the
AHG phase, the reactivity is usually associated
with a positive DAT result and possible auto-
antibodies. If, in addition, the serum is reac-
tive with all cells tested, autoadsorption or
other special procedures may be necessary to
determine whether there are underlying allo-
antibodies that are masked by the autoanti-
bodies. If the serum is nonreactive or shows
only weak reactivity, an eluate may demon-
strate more potent autoantibody reactivity.
(See “Cold Autoantibodies” and “Warm Auto-
antibodies” sections below and Chapter 17 for
a more detailed discussion.)
RE CENT TRANSFUSIONS. If the autocontrol
is positive in the AHG phase, there may be an-
tibody-coated donor red cells in the patient’s
circulation, resulting in a positive DAT result
that may show mixed-field reactivity. An elu-
tion should be performed, especially when
418 䡲 AABB TECHNICAL MANUAL
tests on serum are inconclusive. For example,
a recently transfused patient may have a posi-
tive autocontrol and serum that is weakly reac-
tive with most but not all Fy(a+) red cells. It
may be possible to confirm anti-Fya specificity
in an eluate because more antibody is often
bound to donor red cells than is free in the
plasma. Furthermore the preparation of an el-
uate concentrates the antibody. It is rare for
transfused red cells to make the autocontrol
positive at other test phases, but this can oc-
cur, especially with a newly developing or
cold-reacting alloantibody.
Some patients may form warm autoanti-
bodies following red cell transfusion; there-
fore, if the positive DAT test does not have a
mixed-field appearance—and especially if the
serum is reactive with all red cells—then the
possibility of an autoantibody should be con-
sidered. Detection of alloantibodies in sam-
ples with autoantibody may require allogeneic
adsorptions (Method 3-20). If the patient’s
phenotype is known for the common red cell
antigens, an allogeneic adsorption may re-
quire only one adsorption using a reagent cell
that is matched for the antigens that the pa-
tient lacks. If the patient’s phenotype is un-
known, it is necessary to perform multiple al-
logeneic adsorptions using a combination of
reagent cells, each of which lacks some of the
common red cell antigens. Cells that are ho-
mozygous for the common red cell antigens,
such as R1 (DCe), R2 (DcE), and r (ce), are fre-
quently used. The adsorbed serum is tested
with reagent cells to rule out the common an-
tibodies that correspond to the antigens that
are missing from the adsorbing cell. For exam-
ple, if the adsorbing cell types R1 (DCe), K–,
Fy(a+b–), Jk(a–b+), and S–s+, the adsorbed
plasma could be tested for anti-c, anti-E, anti-
K, anti-Fy b, anti-Jk a , and anti-S. It is never
possible, however, to rule out antibodies to
high-prevalence antigens when allogeneic ad-
sorption techniques are used.
If the DAT result does have a mixed-field
appearance and the serum is reactive with all
cells tested, a transfusion reaction caused by
an alloantibody to a high-prevalence antigen
should be considered (Fig 16-2).
COLD AUTOANTIBODIES. Potent cold auto-
antibodies that are reactive with all red cells,
including the patient’s own, can create special
problems—especially when the reactivity per-
sists at temperatures above room temperature.
Cold autoantibodies may be benign or patho-
logic. (See Chapter 17 for a more detailed dis-
cussion.)
There are different approaches to testing
sera with potent cold agglutinins. To detect
underlying and potentially clinically signifi-
cant antibodies, methods that circumvent the
cold autoantibody can be used. Procedures for
the detection of alloantibodies in the presence
of cold-reactive autoantibodies include the
following:
1. Prewarming techniques in which red cells
and serum are prewarmed to 37 C sepa-
rately before they are combined (Method 3-
6).
2. The use of anti-IgG rather than polyspecific
AHG reagent.
3. Cold autoadsorption of the patient’s serum
with autologous red cells to remove autoan-
tibodies but not alloantibodies.
4. Adsorption with rabbit erythrocytes or rab-
bit erythrocyte stroma.
WARM AUTOANTIBODIES. Pa t i e n t s w i t h
warm-reactive autoantibodies in their sera
create a special challenge because the anti-
body is reactive with virtually all red cells test-
ed. The majority of warm autoantibodies are
IgG, although some warm autoantibodies are
IgM. IgM warm autoantibodies are unusual,
but they have caused severe (often fatal) auto-
immune hemolytic anemia.43 If a patient with
warm autoantibodies requires a transfusion, it
is important to detect any underlying clinically
significant alloantibodies that the autoanti-
bodies may mask. (For more information, see
Chapter 17 and Methods 4-8 through 4-10.)
The reactivity of most warm-reactive autoanti-
bodies is greatly enhanced by certain methods
such as PEG and enzymes and, to a lesser ex-
tent, by LISS. It is often helpful to omit the en-
hancement media when testing sera that con-
tain warm autoantibodies. If such tests are
nonreactive, the same procedure can be used
 CHAPTER 16 Identification of Antibodies to Red Cell Antigens
for compatibility testing without the need for
adsorptions.
Frequency of Antibody Testing
After a clinically significant antibody has been
identified, antigen-negative Red Blood Cell
(RBC) units must be selected for all future
transfusions, even if the antibodies are no lon-
ger detectable.17(p37) In addition, an AHG cross-
match must be performed. It is rarely neces-
sary to repeat the identification of known
antibodies. AABB Standards for Blood Banks
and Transfusion Services states that in patients
with previously identified antibodies, testing
methods should be used that identify addi-
tional clinically significant antibodies.17(p36)
Each laboratory should define and validate
methods for the detection of additional anti-
bodies in these patients.
Immunohematology Reference
Laboratories
When antibody problems cannot be resolved
or rare blood is needed, a reference laboratory
can provide consultation and assistance
through its access to the American Rare Donor
Program (ARDP). (See Method 3-21.)
P O S TA N A LY TI C A L
CO NS I D E R AT IO N S : SE L E C T I NG
BLOOD FOR TRANSFUSION
After an antibody has been identified, it is im-
portant to determine its clinical significance.
Antibodies that are reactive at 37 C, in an IAT,
or both are potentially clinically significant.
Antibodies that are reactive at room tempera-
ture and below are usually not clinically signif-
icant; however, there are many exceptions. For
example, anti-Vel, -P, and -PP1P k (-Tja) may be
reactive only at cold temperatures yet may
cause red cell destruction in vivo. Anti-Ch,
anti-Rg, and many of the Knops and Cost anti-
bodies have little or no clinical significance
despite their reactivity in an IAT. Reported
experience with examples of antibodies with
the same specificity can be used in assessing the
䡲 419
clinical significance. Table 16-6 summarizes
the expected reactivity and clinical signifi-
cance of commonly encountered alloantibod-
ies. For some antibodies, little or no data exist,
and the decision about clinical significance
must be based on the premise that clinically
significant antibodies are those that are active
at 37 C, in an IAT, or both.
Certain laboratory tests have been used to
predict the clinical significance of antibodies.
The monocyte monolayer assay, which quanti-
fies phagocytosis, adherence of antibody-coat-
ed red cells, or both, can be used to predict the
in-vivo clinical significance of some antibod-
ies. The test for antibody-dependent cellular
cytotoxicity, which measures lysis of antibody-
coated red cells, and the chemiluminescence
assay, which measures the respiratory release
of oxygen radicals after phagocytosis of anti-
body-coated red cells, have been helpful in
predicting in-vivo antibody reactivity—partic-
ularly for predicting the severity of HDFN. For
cold-reactive antibodies, in-vitro thermal am-
plitude studies may predict the likelihood of
in-vivo hemolysis.44
In-vivo tests may also be used to evaluate
the significance of an antibody. The most
common technique is a red cell survival study
in which radiolabeled, antigen-positive red
cells (usually labeled with 51Cr) are infused into
the patient. After a specified period has
elapsed, a sample of blood from the patient is
tested for radioactivity. With this technique, it
is possible to measure the survival of 1 mL or
less of infused cells. Another in-vivo tech-
nique, flow cytometry, can also be used to
measure the survival of infused red cells, but a
larger aliquot of red cells (about 10 mL) is usu-
ally required. Interpretation of in-vivo survival
test results is complicated by the fact that
small aliquots of incompatible red cells may
have a faster rate of destruction than an entire
transfused RBC unit. Comparison with docu-
mented cases in the literature and consulta-
tion with a reference laboratory should pro-
vide guidance about previous examples of
similar specificities.
420 䡲 AABB TECHNICAL MANUAL
Phenotyping Donor Units
Whenever possible, RBC units selected for
transfusion to a patient with potentially clini-
cally significant antibodies should be tested
and found negative for the corresponding an-
tigen(s). Even if the antibodies are no longer
detectable, all subsequent RBC transfusions to
that patient should lack the antigen to prevent
a secondary immune response. The transfu-
sion service must maintain records of all pa-
tients in whom clinically significant antibodies
have been previously identified, and an AHG
crossmatch procedure is required if the serum
contains—or has previously contained—a
clinically significant antibody.17(pp37-38,75)
A potent example of the antibody should
be used to identify antigen-negative blood. Of-
ten, the antibody is a commercial antiserum,
but to save expensive or rare reagents, units
can be tested first for compatibility with the
patient’s serum. Then, the absence of the anti-
gen in compatible units can be confirmed with
commercial reagents. If the antibody is unusu-
al and commercial antiserum is not available,
a stored sample from the sensitized patient
can be used to select units for transfusion at a
later time, especially if the patient’s later sam-
ples lose reactivity. If a patient’s serum is used
as a typing reagent, the antibody should be
well characterized and retain its reactivity after
storage. Appropriate negative and weak-posi-
tive controls (eg, from heterozygous donors)
should be used at the time of the testing. The
following criteria, established by the FDA for
licensing some reagents, should be used as
guidelines for human-source reagents used in
lieu of commercial reagents45,46:
1. Anti-K, -k, -Jka, -Fya, and –Cw: dilution of 1:8
must produce at least a 1+ reaction.
2. Anti-S, -s, -P1, -M, -I, -c (saline), -e (saline),
and -A1: dilution of 1:4 must produce at
least a 1+ reaction.
3. Most other specificities: undiluted must
produce at least a 2+ reaction.
When selecting units for patients with
clinically significant antibodies, some serolo-
gists recommend typing the units with anti-
bodies from two different sources, but others
consider this step unnecessary—especially
when potent reagents are available and an
AHG crossmatch will be performed. Different
lots of antibody from the same manufacturer
and even different reagents from different
manufacturers may have been prepared from
the same source material because manufac-
turers often share these resources.
If a donor unit is tested for selected anti-
gens and labeled by the blood center, the use
of licensed (commercial) reagents, if available,
is required. If no licensed reagent is available,
the unit may be labeled with appropriate
wording (eg, “Tested and found negative for
XX antigen using unlicensed typing re-
agents”).47 Except for results of ABO and D typ-
ing, there is no requirement that the hospital
repeat testing of donor minor antigen typing if
the results are attached to the units on the la-
bel or by a tag. Minor antigen typing results on
packing slips or not physically attached to the
donor unit should be confirmed by the hospi-
tal if the unit is used for clinical care.
Antigen-Negative Blood vs Crossmatch
for Compatibility
For certain antibodies, typing the donor units
may not be necessary, and the patient’s serum
can be used to select serologically compatible
RBC units. This is especially true for antibod-
ies that characteristically are reactive below
37 C (eg, anti-M, -N, -P1, -Lea, -Leb, and -A1)
and that do not ordinarily produce a second-
ary immune response following the transfu-
sion of antigen-positive RBC units.
It can sometimes be a best practice to
provide phenotypically matched, antigen-neg-
ative RBC units as a prophylactic measure
when the patient has no detectable antibody.
When a patient of the R1R1 phenotype produc-
es anti-E, some serologists suggest that RBC
units should be negative for both the E and c
antigens. This recommendation is based on
the assumption that the stimulus to produce
anti-E may also have stimulated anti-c or anti-
cE that remains undetected by routine tests.48
Similarly, for an R2R2 patient with demonstra-
 CHAPTER 16 Identification of Antibodies to Red Cell Antigens 䡲 421

ble anti-C, the use of e-negative donor blood
may be considered.
When a patient has potent warm autoan-
tibody and compatibility cannot be demon-
strated by routine testing or when an antibody
has not been specifically demonstrated but
cannot be conclusively excluded, it may be
prudent to select RBC units that are phenotyp-
ically matched for clinically significant anti-
gens.
When Rare Blood Is Needed
Rare blood includes units that are negative for
high-prevalence antigens or are negative for a
combination of common antigens. When a pa-
tient has multiple antibodies, it can be helpful
to determine the prevalence of compatible do-
nors. To calculate this prevalence, one must
multiply the prevalence of donors who are
negative for one antigen by the prevalence of
donors who are negative for each of the other
antigens. For example, if a serum contains
anti-c, anti-Fya, and anti-S and if the preva-
lence of antigen-negatives are c-negative =
18%, Fy(a–) = 34%, and S-negative = 45%, then
the prevalence of compatible units is 0.18 ×
0.34 × 0.45 = 0.028, or 2.8%. If the patient is
group O, the prevalence of group O donors
(45%) is factored into the calculation as fol-
lows: 0.028 × 0.45 = 0.013, or 1.3%.
If any of these antibodies is present alone,
finding compatible blood is not very difficult;
but the combination requires a large number
of units to provide one compatible unit. The
calculation above uses the prevalence in pop-
ulations of European ethnicity, and prevalence
may be different in populations of non-
European ethnicity. In calculating the proba-
bility of compatible donors, one should use
the prevalence that corresponds with the eth-
nic composition of the donor population, if
available.
When units of rare (<1 in 5000) or uncom-
mon (<1 in 1000) phenotypes are needed, the
ARDP can be very helpful. This program,
which is accessible only to personnel from an
accredited reference laboratory, can identify
blood suppliers that are known to have poten-
tially compatible units (usually frozen RBC
units) and donors who may be eligible to do-
nate (Method 3-21).
Family members are another potential
source of rare blood donors. The absence of
high-prevalence antigens is usually associated
with the inheritance of the same rare recessive
blood group gene from each heterozygous
parent. Children from the same parents have
one chance in four of inheriting the same two
rare genetic mutations, making siblings much
more likely than the general population to
have the rare blood type. In most cases, blood
from the patient’s parents, children, and half of
the patient’s siblings express only one rare
gene. If transfusion is essential and there is no
alternative to transfusing incompatible blood,
these heterozygous (single-dose) donors are
preferable to random donors. For infants with
HDFN resulting from multiple antibodies or
an antibody to a high-prevalence antigen, the
mother (if she is ABO compatible) is often the
logical donor.
If the clinical situation allows, autologous
RBC transfusions should be considered for pa-
tients with rare phenotypes who are expected to
need rare blood in the future. For some patients
with multiple antibodies who are not able to
donate autologous units, it may be necessary to
determine whether any of the antibodies is less
likely to cause red cell destruction and, in a crit-
ical situation, to give blood that is incompatible
for that particular antigen.

KEY POINTS

1. It is important to consider the patient’s medical history (transfusions, pregnancies,

transplantations, diagnoses, drugs, and ethnic origin) before starting antibody identifica-
tion testing.
2. An antibody may be tentatively excluded or ruled out if an antigen is present on a reagent
cell and the patient’s serum does is not reactive with it.
422 䡲 AABB TECHNICAL MANUAL

3. Common clinically significant alloantibodies that should be excluded during antibody

identification testing are anti-D, -C, -E, -c, -e, -K, -Fya, -Fyb, -Jka, -Jkb, -S, and -s.
4. Based on probability, the use of two reactive and two nonreactive red cell samples is an ac-
ceptable approach for antibody confirmation.
5. The autologous control, in which serum and autologous red cells are tested under the same
conditions as the serum and reagent red cells, is an important part of antibody identifica-
tion. The autologous control is not the same as a DAT.
6. The phenotype of the autologous red cells is an important part of antibody identification.
When an antibody has been tentatively identified in the serum, the corresponding antigen
is expected to be absent from the autologous red cells, although exceptions can occur.
7. An antibody can be removed from a serum sample by adsorption onto red cells that express
the corresponding antigen.
8. Elution dissociates antibodies from sensitized red cells. Bound antibody may be released by
changing the thermodynamics of an antigen-antibody reaction, neutralizing or reversing
forces of attraction that hold antigen-antibody complexes together, or disturbing the struc-
tures of the antigen-antibody binding site.
9. A clinically significant red cell antibody is an antibody that is frequently associated with
HDFN, hemolytic transfusion reactions, or a notable decrease in the survival of transfused
red cells.
10. Whenever possible, RBC units selected for transfusion to a patient with a potentially clin-
ically significant antibody should be tested and found negative for the corresponding anti-
gen(s). Even if the antibody is no longer detectable, all subsequent RBC transfusions to the
patient should lack the antigen to prevent a secondary immune response.

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 C h a p t e r 1 7

The Positive Direct Antiglobulin

Test and Immune-Mediated
Hemolysis

Regina M. Leger, MSQA, MT(ASCP)SBB, CMQ/OE(ASQ)

test
I fit to performing a DAT (or autocontrol) as part
THE DIRECT ANTIGLOBULIN
(DAT) is a simple test used to determine
if red cells have been coated in vivo with im-
munoglobulin (Ig), complement, or both. The
DAT is used primarily for the investigation of
hemolytic transfusion reactions, hemolytic
disease of the fetus and newborn (HDFN), au-
toimmune hemolytic anemia (AIHA), and
drug-induced immune hemolysis. A positive
DAT result may or may not be associated with
immune-mediated hemolysis. As shown in Ta-
ble 17-1, there are many causes of a positive
DAT result.
TH E DAT
The DAT should be performed on every pa-
tient in whom the presence of hemolysis has
been established to distinguish immune from
nonimmune hemolytic anemia. The DAT
should also be performed when a positive au-
tocontrol is found in antibody identification
studies (see Chapter 16), but there is no bene-
of routine pretransfusion testing. The predic-
tive value of a positive DAT result is 83% in a
patient with hemolytic anemia, but only 1.4%
in a patient without, hemolytic anemia.1
Small amounts of IgG and complement
that are lower than the detection limit of rou-
tine testing techniques appear to be present
on all red cells. Using sensitive testing tech-
niques, 5 to 90 IgG molecules/red cell2 and 5 to
40 C3d molecules/red cell3 have been detected
in healthy individuals. Depending on the tech-
nique and reagents used, the DAT can detect
100 to 500 molecules of IgG/red cell and 400 to
1100 molecules of C3d/red cell. Positive DAT
results are reported in 1:1000 to 1:14,000 blood
donors and 1% to 15% of hospital patients.4
These large differences in incidence are proba-
bly related to the different DAT techniques
used.
Most blood donors with a positive DAT re-
sult appear to be perfectly healthy, and most
patients with positive DAT results have no

Regina M. Leger, MSQA, MT(ASCP)SBB, CMQ/OE(ASQ), Research Associate, American Red Cross Blood Ser-
vices, Southern California Region, Pomona, California
The author has disclosed no conflicts of interest.

425
426 䡲 AABB TECHNICAL MANUAL
TABLE 17-1. Some Causes of a Positive DAT Result
Autoantibodies to intrinsic red cell antigens
Hemolytic transfusion reactions
Hemolytic disease of the fetus and newborn
Drug-induced antibodies
Passively acquired alloantibodies (eg, from donor plasma, derivatives, or immunoglobulin)
Nonspecifically adsorbed proteins (eg, hypergammaglobulinemia, high-dose intravenous immune globulin, or
modification of red cell membrane by some drugs)
Complement activation due to bacterial infection, autoantibodies, or alloantibodies
Antibodies produced by passenger lymphocytes (eg, in transplanted organs or hematopoietic components)
DAT = direct antiglobulin test
obvious signs of hemolytic anemia. However, a
careful evaluation may show evidence of in-
creased red cell destruction. A recent study
suggests that a positive DAT result in a healthy
blood donor may be a marker of risk of future
development of malignancy.5,6
A positive DAT result in a patient with he-
molytic anemia indicates that the most likely
diagnosis is one of the immune hemolytic ane-
mias. However, the DAT result can be positive,
coincidentally, in patients with hemolytic ane-
mia that is not immune mediated. Conversely,
some patients with immune hemolytic anemia
have a negative DAT result (see “DAT-Negative
AIHA” section below).
The DAT can also be positive for IgG or
complement without a clear correlation with
anemia in patients with sickle cell disease,
beta-thalassemia, renal disease, multiple my-
eloma, autoimmune disorders, AIDS, or other
diseases associated with elevated serum glob-
ulin or blood urea nitrogen levels.7-9 The inter-
pretation of a positive DAT result should take
into consideration the patient’s history, clini-
cal data, and results of other laboratory tests.
Initial transfusion reaction investigations
include a DAT on a posttransfusion specimen.
In the presence of immune-mediated hemoly-
sis, the DAT result may be positive if sensitized
red cells have not been destroyed or negative if
hemolysis and rapid clearance have occurred.
Preparation and testing of an eluate from DAT-
positive posttransfusion-reaction red cells is
indicated. Even if the DAT result is only weakly
positive or negative, testing of an eluate may
be informative. If the DAT result is positive on
the postreaction specimen, a DAT should also
be performed on the pretransfusion specimen
for comparison and appropriate interpreta-
tion.
The Principles of the DAT
The DAT is based on the test developed by
Coombs, Mourant, and Race10 for the detec-
tion of antibodies attached to red cells that do
not produce direct agglutination. This test, an
indirect antiglobulin test, was initially used to
demonstrate antibody in serum, but it was lat-
er applied to demonstrate the in-vivo coating
of red cells with antibody or complement
components (the DAT).
Most of the antiglobulin reactivity is di-
rected at the heavy chains (eg, Fc portion of
the sensitizing antibody) or the complement
component, thus bridging the gap between
adjacent red cells to produce visible agglutina-
tion. The strength of the observed agglutina-
tion is usually proportional to the amount of
bound protein.
The DAT is performed by testing freshly
washed red cells directly with antiglobulin re-
agents containing anti-IgG and anti-C3d. In
the United States, only polyspecific anti-IgG,
-C3d and monospecific anti-IgG, anti-C3d,
 CHAPTER 17
and anti-C3b,-C3d reagents are currently li-
censed. The red cells need to be washed to re-
move free plasma globulins and complement;
otherwise, the antiglobulin reagent can be
neutralized, leading to a false-negative result.
The saline used for washing the red cells
should be at room temperature; washing red
cells with warm (eg, 37 C) saline can result in
the loss of red-cell-bound, low-affinity IgG.
The red cells should be tested immediately af-
ter washing to prevent false-negative results
due to the elution of IgG.
Although any red cells may be tested,
EDTA-anticoagulated blood samples are pre-
ferred. The EDTA prevents in-vitro fixation of
complement by chelating the calcium that is
needed for C1 activation. If red cells from a
clotted blood sample have a positive DAT re-
sult due to complement, the results should be
confirmed on red cells from freshly collected
blood kept at 37 C or an EDTA-anticoagulated
specimen if these results are to be used for di-
agnostic purposes.
The DAT can be initially performed with a
polyspecific antihuman globulin (AHG) reagent
that is capable of detecting both IgG and C3d
(see Method 3-14). If the results are positive,
tests with monospecific reagents (anti-IgG and
anticomplement) need to be performed to ap-
propriately characterize the immune process
involved and determine the diagnosis. Be-
cause polyspecific reagents are usually blend-
ed and testing conditions for optimally detect-
ing IgG and C3d on red cells may differ, some
laboratories perform the DAT initially with
anti-IgG and anti-C3d reagents separately. If
the polyspecific reagent is polyclonal, pro-
teins other than IgG or C3d (eg, IgM, IgA, or
other complement components) can occa-
sionally be detected; however, specific re-
agents to distinguish these other proteins by
serologic techniques are not readily available.
If umbilical cord blood samples are to be test-
ed, it is appropriate to use anti-IgG only be-
cause HDFN results from fetal red cell sensiti-
zation with maternally derived IgG antibody
and complement activation rarely occurs.4
It is important to follow the reagent man-
ufacturer’s instructions and recognize any
product limitations. False-negative or weaker
DAT/Immune Hemolysis 䡲 427
results can be obtained if the washed red cells
are allowed to sit before they are tested with
anti-IgG or if the reading of the results is de-
layed. Some anticomplement reagents, in con-
trast, demonstrate stronger reactivity if cen-
trifugation is delayed for a short time after the
reagent has been added. When the DAT result
is positive with both anti-IgG and anti-C3, the
red cells should be tested with an inert control
reagent (eg, 6% albumin or saline). Lack of ag-
glutination of the red cells in the control re-
agent provides some assurance that the test
results are accurately interpreted. If the con-
trol is reactive, the DAT result is invalid [see
sections below on warm AIHA (WAIHA) and
cold agglutinin disease (CAD)]. Reactivity with
this control reagent can indicate spontaneous
agglutination caused by heavy coating of IgG
or rare warm-reactive IgM, or it can indicate
IgM cold autoagglutinins that were not disso-
ciated during routine washing.
Evaluation of a Positive DAT Result
A positive DAT result alone is not diagnostic of
hemolytic anemia. Understanding the signifi-
cance of this positive result requires knowl-
edge of the patient’s diagnosis; recent drug,
pregnancy, and transfusion history; and the
presence of acquired or unexplained hemolyt-
ic anemia. Dialogue with the attending physi-
cian is important. Clinical considerations to-
gether with laboratory data should dictate the
extent to which a positive DAT result is evalu-
ated.
Patient History
The following situations may warrant further
investigation of a positive DAT result.
1. Evidence of in-vivo hemolysis (ie, red cell
destruction). If a patient with anemia who
has a positive DAT result shows evidence of
hemolysis, testing to evaluate a possible
immune etiology is appropriate. Reticulocy-
tosis; spherocytes observed on the peripheral
blood film; hemoglobinemia; hemoglobin-
uria; decreased serum haptoglobin; and
elevated levels of serum unconjugated
(indirect) bilirubin or lactate dehydroge-
428 䡲 AABB TECHNICAL MANUAL
nase (LDH), especially LDH1, may be asso-
ciated with increased red cell destruction.
These factors are indicative of hemolytic
anemia but not specifically immune hemo-
lytic anemia. If there is no evidence of
hemolytic anemia, no further studies are
necessary unless the patient requires a red
cell transfusion and the serum contains
incompletely identified antibodies to red
cell antigens. Testing an eluate may be
helpful for antibody identification (see
“Elution” section below and Chapter 16).
2. Recent transfusion. When a patient has
recently been transfused, a positive DAT
result may be the first indication of a devel-
oping immune response. The developing
antibody sensitizes the transfused red cells
that have the corresponding antigen, and
the DAT result becomes positive. The anti-
body may not be present in sufficient quan-
tity to be detected in the serum. Antibody
may appear as early as 7 to 10 days after
transfusion in a primary immunization or
as early as 1 to 2 days in a secondary
response.4,11 These alloantibodies could
shorten the survival of red cells that have
already been transfused or administered in
subsequent transfusions. A mixed-field
appearance in the posttransfusion DAT
result (ie, agglutination of donor red cells
and no agglutination of the patient’s red
cells) may or may not be observed.
3. Administration of drugs associated with
immune-mediated hemolysis. Many drugs
have been reported to cause a positive DAT
result and/or immune-mediated hemoly-
sis, but this occurrence is not common.12
(See “Drug-Induced Immune Hemolytic
Anemia” section below.)
4. History of hematopoietic progenitor cell or
organ transplantation. Passenger lympho-
cytes of donor origin produce antibodies
directed against ABO or other blood group
antigens on the recipient’s red cells, causing
a positive DAT result.4
5. Administration of intravenous Ig (IVIG) or
IV anti-D. IVIG may contain ABO antibod-
ies, anti-D, or, sometimes, other antibod-
ies.13 Intravenous anti-D used to treat
immune thrombocytopenia (previously
known as “immune thrombocytopenic
purpura”) causes Rh-positive patients to
develop a positive DAT result. IV anti-D
may also contain other antibodies, includ-
ing ABO antibodies.14
Serologic Investigation
Three investigative approaches are helpful in
the evaluation of a positive DAT result.
1. Test the DAT-positive red cells with anti-IgG
and anti-C3d reagents to characterize the
type of protein(s) coating the red cells. This
will help classify an immune-mediated
hemolytic anemia.
2. Test the serum/plasma to detect and iden-
tify clinically significant antibodies to red
cell antigens. Additional tests that are use-
ful in classifying the immune hemolytic
anemias and procedures for detecting allo-
antibodies in the presence of autoantibod-
ies are described later in this chapter.
3. Test an eluate prepared from the DAT-posi-
tive red cells with reagent red cells to deter-
mine whether the coating protein has red
cell antibody specificity. When the only
coating protein is complement, the eluate
is likely to be nonreactive. However, an elu-
ate from the patient’s red cells coated only
with complement should be tested if there
is clinical evidence of antibody-mediated
hemolysis, for example, after transfusion.
The eluate preparation can concentrate
small amounts of IgG that may not be
detectable in routine testing of the patient’s
plasma.
Results of these tests combined with the
patient’s history and clinical data should assist
in classification of the problem involved.
Elution
Elution can be informative in the following sit-
uations:
䡲 Clinical signs and symptoms of immune
hemolysis are present.
 CHAPTER 17
䡲 Serum test results are negative or inconclu-
sive for a patient who has been recently
transfused.
䡲 HDFN is suspected but no alloantibodies
were detected in the maternal plasma.
Performing an elution routinely on the red
cells of all patients who have a positive DAT re-
sult is not recommended. The majority of pre-
transfusion patients with a positive DAT result
have a nonreactive eluate that is often associat-
ed with an elevated serum globulin level.7-9
Elution frees antibody from sensitized red
cells and recovers antibody in a usable form.
Multiple elution methods have been described
and reviewed.15 Many laboratories use com-
mercial acid elution kits, primarily for ease of
use and decreased exposure to potentially
harmful chemicals; these kits are suitable to
recover antibody in most cases. False-positive
eluate results associated with high-titer anti-
bodies have been reported when the low ionic
wash solution supplied with the commercial
acid eluates was used.16 Because no single elu-
tion method is ideal in all situations, an alter-
native elution method (eg, an organic solvent)
may be used in some high-complexity refer-
ence laboratories when a nonreactive acid elu-
ate result is not in agreement with clinical da-
ta.17
Table 17-2 lists the uses of some common
elution methods. Typically, eluates are tested
TABLE 17-2. Antibody Elution Methods
Method Use
Lui freeze-thaw ABO HDFN
Heat (56 C) ABO HDFN, IgM agglutinating
antibodies
Acid elution kits (commercial) Warm auto- and alloantibodies
Chemical/organic solvent Warm auto- and alloantibodies
HDFN = hemolytic disease of the fetus and newborn; IgM = immunoglobulin M.
DAT/Immune Hemolysis 䡲 429
only at the antiglobulin phase. If an IgM anti-
body is being investigated or suspected, how-
ever, centrifugation and reading after the 37 C
incubation should be performed. Technical
considerations for elution are discussed in
Chapter 16.
In cases of hemolytic transfusion reac-
tions or HDFN, specific antibody (or antibod-
ies) is usually detected in the eluate that may
or may not be detectable in the serum. For
transfusion reactions, newly developed anti-
bodies that are initially detectable only in the
eluate are usually detectable in the serum after
about 14 to 21 days.18 If the eluate is nonreac-
tive and a non-group-O patient has received
plasma containing anti-A or anti-B (as a result
of the transfusion of group O platelets, for ex-
ample) and the recipient appears to have im-
mune hemolysis, the eluate should be tested
against A1 and/or B cells. It may be appropriate
to test the eluate against red cells from
recently transfused donor units, which could
have caused immunization to a rare antigen.
For cases of HDFN when no maternal anti-
body has been detected and paternal red cells
are ABO incompatible with maternal plasma,
testing an eluate prepared from the infant’s red
cells with the paternal red cells may detect a
maternally derived antibody to a low-preva-
lence antigen,
When the eluate reacts with all cells test-
ed, autoantibody is the most likely explana-
Comments
Quick, small volume of red cells
needed, poor recovery of other
antibodies
Easy, poor recovery of IgG allo-
and autoantibodies
Easy, possible false-positive elu-
ate results when high-titer anti-
body is present16
Chemical hazards, eg, flammabil-
ity, toxicity, or carcinogenicity
430 䡲 AABB TECHNICAL MANUAL
tion, especially if the patient has not been
transfused recently. However, if the patient has
been recently transfused, an antibody to a
high-prevalence antigen should be consid-
ered. When no unexpected antibodies are
present in the serum and the patient has not
been transfused recently, no further serologic
testing of an autoantibody detected only in the
eluate is necessary.
The patient’s complete history, including
the presence of potential passive antibodies,
needs to be reviewed when the serologic test
results are evaluated. If both the serum and el-
uate are nonreactive, there is evidence of im-
mune hemolysis, and the patient has received
a drug reported to have caused immune-me-
diated hemolysis, testing to demonstrate drug-
related antibodies should be considered (see
“Laboratory Investigation of Drug-Induced
Immune Hemolysis” section below).
AUTO I M M U N E H E M O LY T I C
ANEMIA
Immune-mediated hemolysis is the shorten-
ing of red cell survival as a result of an immune
response. If the marrow is able to adequately
compensate, the reduced red cell survival may
not result in anemia. Immune-mediated he-
molysis is only one cause of hemolytic anemia,
and many causes of hemolysis are unrelated to
immune reactions.
The serologic investigations carried out in
the blood bank do not determine whether a
patient has a “hemolytic” anemia. The diagno-
sis of hemolytic anemia rests on clinical find-
ings and laboratory data, such as hemoglobin
or hematocrit values; reticulocyte count; red
cell morphology; bilirubin, haptoglobin, and
LDH levels; and, sometimes, red cell survival
studies. The serologic findings help determine
whether the hemolysis has an immune basis
and, if so, what type of immune hemolytic
anemia is present. This is important because
the treatment for each type is different.
In some cases, the destruction of red cells
takes place in the intravascular space with the
release of free hemoglobin into the plasma.
The red cells are ruptured following activation
of the classical complement cascade. The
characteristic features of this rare type of he-
molysis are hemoglobinemia, and, when the
plasma hemoglobin level exceeds the renal
threshold, hemoglobinuria. Conversely and
more commonly, extravascular hemolysis re-
sults when macrophages in the spleen and liv-
er phagocytose red cells completely or partial-
ly (producing spherocytes) or destroy red cells
by cytotoxic events, resulting in an increase in
serum bilirubin. This distinction is a simplifi-
cation, however, because hemoglobin can also
be released into the plasma following extravas-
cular destruction if hemolysis is brisk.
Immune hemolytic anemias can be clas-
sified in various ways. One classification sys-
tem is shown in Table 17-3. The AIHAs are sub-
divided into the major types: WAIHA, CAD,
mixed- or combined-type AIHA, and paroxys-
mal cold hemoglobinuria (PCH). Not all cases
fit neatly into these categories. Table 17-4
shows the typical serologic characteristics of
the AIHAs. Drugs (discussed in the “Drug-In-
duced Immune Hemolytic Anemia” section
below) may also induce immune hemolysis;
the effects of drug-induced autoantibodies are
serologically indistinguishable from WAIHA.
Warm Autoimmune Hemolytic
Anemia
The majority of AIHA cases are caused by
warm-reactive autoantibodies that are opti-
TABLE 17-3. Classification of Immune
Hemolytic Anemias
Autoimmune hemolytic anemia (AIHA)
Warm AIHA
Cold agglutinin disease
Mixed-type AIHA
Paroxysmal cold hemoglobinuria
Alloimmune hemolytic anemia
Hemolytic transfusion reaction
Hemolytic disease of the fetus and newborn
Drug-induced immune hemolytic anemia
 CHAPTER 17 DAT/Immune Hemolysis 䡲 431

TABLE 17-4. Typical Serologic Findings in AIHA

WAIHA CAD Mixed-Type AIHA PCH

DAT IgG C3 only IgG + C3 C3 only

(routine) IgG + C3 C3
C3

Ig type IgG IgM IgG, IgM IgG

Eluate IgG antibody Nonreactive IgG antibody Nonreactive

Serum IAT, 35% aggluti- IgM agglutinating IgG IAT-reactive Negative routine
nate untreated red antibody, titer antibody plus IgM IAT result, IgG
cells at 20 C 1000 (60%) at agglutinating anti- biphasic hemolysin
4 C, reactive at body reactive at in Donath-Land-
30 C 30 C steiner test

Specificity Broadly reactive, Usually anti-I Usually unclear Anti-P

multiple specifici-
ties reported

AIHA = autoimmune hemolytic anemia; WAIHA = warm AIHA; CAD = cold agglutinin disease; PCH = paroxysmal cold
hemoglobinuria; DAT = direct antiglobulin test; IgG = immunoglobulin G; IAT = indirect antiglobulin test.

mally reactive with red cells at 37 C. The auto-
antibody is usually IgG, but it can be IgM or
IgA.
Serologic Characteristics
The DAT result may be positive due to IgG plus
complement (67% of cases), IgG without com-
plement (20%), or complement without IgG
(13%).4 Performing an elution at initial diagno-
sis and/or during pretransfusion testing is use-
ful to demonstrate that the IgG coating the pa-
tient’s red cells is autoantibody.
Typically in WAIHA, the eluate is reactive
with virtually all red cells tested, and reactivity
is enhanced in tests against enzyme-treated
red cells, with polyethylene glycol (PEG) en-
hancement, or in column agglutination and
solid-phase tests. The eluate usually has no se-
rologic activity if the only protein coating the
red cells is complement.
If the autoantibody has been adsorbed by
the patient’s red cells in vivo, the serum may
not contain detectable free antibody. The se-
rum contains free antibody when the amount
of autoantibody exceeds the available binding
sites on the patient’s red cells. The DAT result
in such cases is usually strongly positive.
Autoantibody in the serum typically is re-
active against all cells by an IAT. Approximately
60% of patients with WAIHA have serum anti-
bodies that react with untreated saline-
suspended red cells. When tested with PEG,
enzyme-treated red cells, column agglutina-
tion, or solid-phase methods, more than 90%
of these sera can be shown to contain autoan-
tibody. Agglutination at room temperature is
present in about one-third of patients with
WAIHA, but these cold agglutinins have nor-
mal titers at 4 C and are nonreactive at 30 C
and 37 C. Thus, these cold agglutinins are non-
pathogenic and the patient does not have CAD
in addition to WAIHA.4
An unusual subcategory of WAIHA is as-
sociated with IgM agglutinins in the plasma
that are reactive at 37 C.4,19 This type of WAIHA
is characterized by severe hemolysis, and the
prognosis for these patients can be poor. The
red cells are typically spontaneously aggluti-
nated in the DAT; that is, the washed red cells
432 䡲 AABB TECHNICAL MANUAL
are reactive with all reagents tested, including
a control, such as 6% albumin (see “Serologic
Problems” section below). Complement is
usually detected on the red cells; IgG or IgM
may or may not be detected. IgM agglutinins
are often detected in an eluate (eg, acid) when
it is inspected for agglutination after the 37 C
incubation and before the antiglobulin test is
conducted. Some serum IgM warm autoagglu-
tinins may be difficult to detect; some are en-
hanced in the presence of albumin or at low
pH. Optimal reactivity of the agglutinin some-
times occurs between 20 C and 30 C rather
than at 37 C. These antibodies have low titers
at 4 C, usually <64, which easily differentiates
this IgM warm antibody from those in CAD. To
prevent misinterpretation of titration results,
titrations at different temperatures (eg, 37 C,
30 C, room temperature, and 4 C) need to be
carried out with separate sets of tubes to avoid
carry-over agglutination.4,19
Serologic Problems
Warm autoantibodies can cause technical dif-
ficulties during red cell testing. Spontaneous
agglutination can occur if the red cells are
heavily coated with IgG and the reagent con-
tains a potentiator, such as albumin. This has
been observed when high-protein Rh typing
sera are used. If the control reagent provided
by the manufacturer for these antisera is reac-
tive, the typing is invalid. IgG can less com-
monly cause spontaneous agglutination in
lower protein reagents (eg, monoclonal typing
sera); this reactivity is often weaker or more
fragile than true agglutination and may not be
detected by a 6% albumin control.20
Warm-reactive IgM agglutinins can also
cause spontaneous agglutination, resulting in
ABO and Rh typing problems and/or reactivi-
ty with the negative control reagent for the
DAT.19 In these cases, treatment with dithio-
threitol (DTT) or 2-mercaptoethanol (2-ME)
(Method 2-18) to disrupt the IgM agglutinin is
required to accurately interpret typing and
DAT results. When the spontaneous agglutina-
tion is disrupted, the control reagent is nonre-
active.
When the DAT result is positive due to
IgG, antiglobulin-reactive typing reagents can-
not be used unless the red-cell-bound IgG is
first removed (see Methods 2-20 and 2-21). An
alternative is to use low-protein antisera (eg,
monoclonal reagents) that do not require an
antiglobulin test (refer to the manufacturer’s
instructions for the detection of spontaneous
agglutination). It is helpful to know which of
the common red cell antigens are lacking on
the patient’s red cells to predict which clinical-
ly significant alloantibodies the patient may
have produced or may produce in the future.
Antigens absent from autologous cells could
well be the target of present or future alloanti-
bodies.
The presence of autoantibody in the se-
rum increases the complexity of the serologic
evaluation and the time needed to complete
pretransfusion testing. If a patient who has
warm-reactive autoantibodies in the serum
needs a transfusion, it is important to deter-
mine whether alloantibodies are also present.
Some alloantibodies may make their presence
known by reacting more strongly or at differ-
ent phases than the autoantibody, but quite
often, routine testing may not suggest the exis-
tence of masked alloantibodies.21,22
Methods to detect alloantibodies in the
presence of warm-reactive autoantibodies are
used to attempt to remove, reduce, or circum-
vent the autoantibody. Antibody detection
methods that use PEG, enzymes, column ag-
glutination, or solid-phase red cell adherence
usually enhance autoantibodies. Antibody de-
tection tests using low-ionic-strength saline
(LISS) or saline tube methods may not detect
autoantibodies but they do detect most clini-
cally significant alloantibodies. Other proce-
dures involve adsorption; two widely used ad-
sorption approaches are discussed below.
Adsorption with Autologous Red Cells
In a patient who has not been transfused re-
cently, adsorption with autologous red cells
(autologous adsorption; see Method 4-8) is the
best way to detect alloantibodies in the pres-
ence of warm-reactive autoantibodies. Only
 CHAPTER 17
autoantibodies are removed, and alloantibod-
ies, if present, remain in the serum.
Autologous adsorption typically requires
some initial preparation of the patient’s red
cells. At 37 C, in-vivo adsorption has occurred,
and all antigen sites on the patient’s own red
cells may be blocked. A gentle heat elution at
56 C for 3 to 5 minutes can dissociate some of
the bound IgG. This can be followed by treat-
ment of the autologous red cells with proteo-
lytic enzymes to increase their capacity to ad-
sorb autoantibody. Treatment of the red cells
with ZZAP, a mixture of papain or ficin and
DTT, accomplishes both of these actions in
one step. It is proposed that the sulfhydryl
component makes the IgG molecules more
susceptible to the protease and dissociates the
antibody molecules from the cell.23 Multiple
sequential autologous adsorptions with new
aliquots of red cells may be necessary if the se-
rum contains high levels of autoantibody.
Once autoantibody has been removed, the ad-
sorbed serum is tested for alloantibody reac-
tivity.
Autologous adsorption is not recom-
mended for patients who have been trans-
fused within the last 3 months because a blood
sample may contain some transfused red cells
that might adsorb alloantibody. Red cells nor-
mally survive for about 110 to 120 days. In pa-
tients with AIHA, autologous and transfused
red cells can be expected to have shortened
survival. However, determining how long
transfused red cells remain in circulation in
patients who need repeated transfusions is not
feasible. It has been demonstrated that very
small amounts (<10%) of antigen-positive red
cells are capable of removing alloantibody re-
activity in in-vitro studies.24 Therefore, it is
recommended to wait for 3 months after
transfusion before performing autologous ad-
sorptions.
Adsorption with Allogeneic Red Cells
The use of allogeneic red cells for adsorption
(allogeneic adsorption) may be helpful when
the patient has been recently transfused or in-
sufficient autologous red cells are available.
The goal is to remove autoantibody and leave
DAT/Immune Hemolysis 䡲 433
the alloantibody in the adsorbed serum. The
adsorbing red cells must not have the antigens
against which the alloantibodies are reactive.
Because alloantibody specificity is unknown,
red cells of different phenotypes are usually
used to adsorb several aliquots of the patient’s
serum.
Given the number of potential alloanti-
bodies, the task of selecting the red cells may
appear formidable. However, red cell selection
is based only on those few antigens for which
alloantibodies of clinical significance are like-
ly to be present. These include the common
Rh antigens (D, C, E, c, and e), K, Fya and Fyb,
Jka and Jkb, and S and s. Red cell selection is
made easier by the fact that some of these an-
tigens can be destroyed by appropriate pre-
treatment (eg, with enzymes or ZZAP) before
use in adsorption procedures (see Table 16-4).
Antibodies to high-prevalence antigens can-
not be excluded by allogeneic adsorptions be-
cause the adsorbing red cells are expected to
express the antigen and adsorb the alloanti-
body along with autoantibody.
When the patient’s phenotype is not
known, group O red cell samples of three dif-
ferent Rh phenotypes (R1R1, R2R2, and rr)
should be selected (see Method 4-9). One sam-
ple should lack Jka and another, Jkb. As shown
in Table 17-5, ZZAP or enzyme pretreatment of
the adsorbing red cells reduces the phenotype
requirements. Untreated red cells may be
used, but the adsorbing red cells must include
at least one sample that is negative for the S, s,
Fya, Fyb, and K antigens in addition to the Rh
and Kidd requirements stated above.
If the patient’s phenotype is known or can
be determined, adsorption with a single sample
of red cells may be possible. Red cells can be
selected that match the patient’s phenotype or
at least match the Rh and Kidd phenotypes if
ZZAP treatment is used. For example, if a pa-
tient’s phenotype is E– K– S– Fy(a–) Jk(a–),
untreated adsorbing red cells need to lack all
five antigens, but enzyme-treated red cells
only need to be E– K– Jk(a–) and ZZAP-treated
red cells only need to be E– Jk(a–). Adsorption
using untreated red cells in the presence of
PEG (Method 4-10) or LISS25,26 are modifica-
tions that have been used to decrease the
434 䡲 AABB TECHNICAL MANUAL
TABLE 17-5. Selection of Red Cells for Allogeneic Adsorption
Step 1. Select red cells for each Rh phenotype.
R1R1
R2R2
rr
Step 2. On the basis of the red cell treatment, or lack of treatment (below), at least one of the Rh-phenotyped
cells should be negative for the antigens listed below.
ZZAP-Treated Red Cells Enzyme-Treated Red Cells
Jk(a–)
Jk(b–)
incubation time for adsorptions and increase
efficiency.
Testing of Adsorbed Serum
In some cases, each aliquot of serum may
need to be adsorbed two or three times to re-
move the autoantibody. The fully adsorbed ali-
quots are then tested against reagent red cells
known to either lack or carry common anti-
gens of the Rh, MNS, Kell, Duffy, and Kidd
blood group systems (eg, antibody detection
cells). If an adsorbed aliquot is reactive, the ali-
quot should be tested to identify the antibody.
Adsorbing several aliquots with different red
cell samples provides a battery of potentially
informative specimens. For example, if the ali-
quot adsorbed with Jk(a–) red cells subse-
quently is reactive only with Jk(a+) red cells,
the presence of alloanti-Jka can be inferred
confidently.
Occasionally, autoantibody is not re-
moved by three sequential adsorptions. Addi-
tional adsorptions can be performed, but the
performance of multiple adsorptions has the
potential to dilute the serum. If the adsorbing
cells do not appear to remove the antibody, the
Untreated Red Cells
Jk(a–) Jk(a–)
Jk(b–) Jk(b–)
K– K–
Fy(a–)
Fy(b–)
S–
s–
autoantibody may have an unusual specificity
that is not reactive with the red cells used for
adsorption. For example, autoantibodies with
Kell, LW, or EnaFS specificity are not removed
by ZZAP-treated red cells (see Table 16-4 for a
list of antigens altered by various agents). The
possibility that the sample contains an auto-
or alloantibody to a high-prevalence antigen
should always be considered when adsorption
fails to remove the reactivity.
Autoantibodies sometimes have patterns
of reactivity that suggest the presence of allo-
antibody. For example, the serum of a D– pa-
tient may have apparent anti-C reactivity. The
anti-C reactivity may reflect warm-reactive au-
toantibody even if the patient’s red cells lack C.
The apparent alloanti-C would, in this case, be
adsorbed by C– red cells, both autologous and
allogeneic. This is unlike the behavior of a true
alloanti-C, which would be adsorbed only by
C+ red cells. In one study, the serum adsorbed
with autologous red cells often retained auto-
antibodies that mimicked alloantibodies in
addition to the true alloantibody(ies) present,
whereas serum adsorbed with allogeneic red
cells most often contained only alloantibod-
ies.27 This reflects an inefficiency of autologous
 CHAPTER 17
adsorption that is primarily caused by limited
volumes of autologous red cells available for
removing all of the autoantibody reactivity
from the serum.
Specificity of Autoantibody
In many cases of WAIHA, no autoantibody
specificity is apparent. The patient’s serum re-
acts with all of the red cell samples tested. If
testing is performed with cells of rare Rh phe-
notypes, such as D– – or Rhnull, some autoanti-
bodies are weakly reactive or are nonreactive,
and the autoantibody appears to have broad
specificity in the Rh system. Apparent specific-
ity for simple Rh antigens (D, C, E, c, and e) is
occasionally seen, especially in saline or LISS
indirect antiglobulin tests. A “relative” speci-
ficity based on stronger reactivity with cells of
certain phenotypes may also be seen; relative
specificity may also be apparent after adsorp-
tion. Autoantibody specificities are clearer in
the serum than in the eluate.
Apart from Rh specificity, warm autoanti-
bodies with many other specificities have been
reported (eg, specificities in the LW, Kell, Kidd,
Duffy, and Diego systems).28,29 Patients with
autoantibodies of Kell, Rh, LW, Ge, Sc, Lu, and
Lan specificities may have transiently de-
pressed expression of the respective antigen,
and the DAT result may be negative or very
weakly positive.29 In these cases, the autoanti-
body may initially appear to be alloantibody.
Proof that it is truly autoantibody is demon-
stration that after the antibody and hemolytic
anemia subside, the antigen strength returns
to normal and stored serum containing anti-
body is reactive with the patient’s red cells.
Tests against red cells of a rare phenotype
and by special techniques to determine auto-
antibody specificity have limited clinical or
practical application. If the autoantibody re-
acts with all red cells except those of a rare Rh
phenotype (eg, Rhnull), compatible donor
blood is unlikely to be available. Such blood, if
available, should be reserved for alloimmu-
nized patients of that uncommon phenotype.
DAT/Immune Hemolysis 䡲 435
Selection of Blood for Transfusion
The most important consideration is to ex-
clude the presence of potentially clinically sig-
nificant alloantibodies before selecting Red
Blood Cell (RBC) units for transfusion. There
are multiple reports in the literature demon-
strating that patients who have warm autoan-
tibodies in their sera have a higher rate of allo-
immunization (eg, 12% to 40%, with a mean of
32%).21,30-33 Although these patients present a
serologic challenge, they deserve the same
protection from hemolytic transfusion reac-
tions as any other patient. Autoantibodies that
react with all reagent red cells, even weakly, are
capable of masking alloantibody reactivity (ie,
reactivity of red cells with both alloantibody
and autoantibody may not be any stronger
than with autoantibody alone).21,22
It is the exclusion of newly formed alloan-
tibodies that is of concern. Because of the
presence of autoantibodies, all crossmatches
are incompatible. This is unlike the case of
clinically significant alloantibodies without
autoantibodies, where a compatible cross-
match with antigen-negative red cells is possi-
ble. Monitoring for evidence of red cell de-
struction caused by alloantibodies is difficult
in patients who already have AIHA; these pa-
tients’ own red cells and transfused red cells
have shortened survival.
If no alloantibodies are detected in ad-
sorbed serum, random units of the appropri-
ate ABO group and Rh type may be selected for
transfusion. If clinically significant alloanti-
bodies are present, the transfused cells should
lack the corresponding antigen(s). For patients
facing long-term transfusion support, it is pru-
dent to obtain an extended phenotype or gen-
otype. Consideration can then be given to
transfusion with donor units that are antigen
matched for clinically significant blood group
antigens to avoid additional alloimmunization
and potentially decrease the number of
adsorptions required and the complexity of
pretransfusion workup.
If the autoantibody has clear-cut specific-
ity for a single antigen (eg, anti-e) and active
hemolysis is ongoing, blood lacking that anti-
gen should be selected. There is evidence that
436 䡲 AABB TECHNICAL MANUAL
such red cells survive longer than the patient’s
own red cells.4 In the absence of hemolysis or
evidence of compromised survival of trans-
fused cells, autoantibody specificity is not im-
portant. However, donor units that are nega-
tive for the antigen may be chosen because
this is a simple way to circumvent the autoan-
tibody and detect potential alloantibodies.
If the autoantibody shows broader reac-
tivity—reacting with all cells but showing
some relative specificity (eg, preferentially re-
acting with e+ red cells)—whether to transfuse
blood lacking the corresponding antigen is de-
batable. It may be undesirable to expose the
patient to Rh antigens absent from autologous
cells, especially D and especially in females of
childbearing potential, merely to improve se-
rologic compatibility testing results with the
autoantibody. (For example, when a D– pa-
tient has autoanti-e, available e– units are like-
ly to be D+; D–e– units are extremely rare.)
Referral of the sample for molecular investiga-
tion to determine the risk for allo- or autoanti-
body production will aid in decision making in
these complex cases and potentially improve
patient care.
Some laboratories use the adsorbed se-
rum to screen and select nonreactive units
(units that are antigen-negative for clinically
significant alloantibodies, if detected) for
transfusion. Other laboratories do not perform
a crossmatch with the adsorbed serum be-
cause all units will be incompatible in vivo due
to the autoantibody. Issuing a unit that is sero-
logically compatible with adsorbed serum
may provide some assurance that the correct
unit has been selected and avoid incompati-
bility because of additional antibodies (eg,
anti-Wra), but this practice can also provide a
false sense of security about the safety of the
transfusion for these patients.
A transfusion management protocol us-
ing prophylactic antigen-matched units for
patients with warm autoantibodies, where fea-
sible, in combination with streamlined ad-
sorption procedures has been described.34 The
same antigens for the commonly occurring,
clinically significant antibodies (D, C, E, c, e, K,
Fya, Fyb, Jka, Jkb, and S and s) are taken into ac-
count, as discussed in the previous section on
adsorptions. The ability to implement such a
protocol depends on the ability of the transfu-
sion service, and more often the blood suppli-
er, to maintain an adequate inventory of phe-
notyped units to meet the antigen-matching
needs.
In recent years, molecular technologies
have been applied to red cell genotyping for
patients with warm autoantibodies to deter-
mine which alloantibodies the patient can
make. DNA tests are attractive for determining
the predicted phenotype of patients with a
positive DAT (IgG) result because IgG is not al-
ways successfully removed and some red cell
antigens are sensitive to IgG removal treat-
ment.35-37 Recent transfusions do not interfere
with molecular testing. It must be remem-
bered that genotyping may not accurately pre-
dict the phenotype if uncommon or rare si-
lencing mutations are present or the patient
has received a stem cell transplant.
Some experts propose that an electronic
crossmatch can be safely used for patients
with autoantibodies when the presence of
common, clinically significant alloantibodies
has been excluded.38,39 This approach circum-
vents the need to issue units that are labeled
“incompatible”; however, as discussed above,
this practice can also lead to a false sense of se-
curity.
Although resolving serologic problems for
these patients is important, delaying transfu-
sion in the hope of finding serologically com-
patible blood may, in some cases, cause
greater danger to the patient. Only clinical
judgment can resolve this dilemma; therefore,
dialogue with the patient’s physician is impor-
tant.
Transfusion of Patients with Warm-
Reactive Autoantibodies
Patients with warm-reactive autoantibodies
may have no apparent hemolysis or may have
life-threatening anemia. Patients with little or
no evidence of significant hemolysis tolerate
transfusion quite well. The risk of transfusion
is somewhat increased in these patients be-
cause of the difficulties with pretransfusion
testing. The duration of survival of the trans-
 CHAPTER 17
fused red cells is about the same as that of the
patient’s own red cells.
In patients with active hemolysis, transfu-
sion may increase hemolysis, and the trans-
fused red cells may be destroyed more rapidly
than the patient’s own red cells. This is related
to the increased red cell mass available from
the transfusion and the kinetics of red cell de-
struction.4 Destruction of transfused cells may
increase hemoglobinemia and hemoglobin-
uria. Disseminated intravascular coagulation
can develop in patients with severe posttrans-
fusion hemolysis.
The transfusion of patients with AIHA is a
clinical decision that should be based on the
balance between the risks and clinical need.
Transfusion should not be withheld solely be-
cause of serologic incompatibility. The vol-
ume transfused should usually be the smallest
amount required to maintain adequate oxygen
delivery and not necessarily the amount re-
quired to reach an arbitrary hemoglobin level.4
The patient should be carefully monitored
throughout the transfusion.
DAT-Negative AIHA
Clinical and hematologic evidence of WAIHA
is present in some patients whose DAT result is
negative. The most common causes of AIHA
associated with a negative DAT result are red-
cell bound IgG below the detection threshold
of the antiglobulin test, red-cell-bound IgM
and IgA that are not detectable by routine AHG
reagents, and low-affinity IgG that is washed
off the red cells during the washing phase for
the DAT.4,40
Nonroutine tests can be applied in these
situations. Unfortunately, these assays require
standardization and many have a low predic-
tive value. One of the easier tests is for low-
affinity antibodies. Washing with ice-cold (eg,
4 C) saline or LISS may help retain antibody on
the cells; a control (eg, 6% albumin) is neces-
sary to confirm that cold autoagglutinins are
not causing the positive results.4,40 Comple-
ment fixation antibody consumption assay,
enzyme-linked antiglobulin test, radiolabeled
anti-IgG, flow cytometry, solid-phase, direct
PEG test, direct Polybrene test, column agglu-
DAT/Immune Hemolysis 䡲 437
tination, and concentrated eluates are all
methods that have been used to detect lower
levels of red cell-bound IgG.40
Anti-IgG, anti-C3d, and the combined
anti-C3b,-C3d reagents are the only licensed
products available in the United States for use
with human red cells. AHG reagents that react
with IgA or IgM are available commercially but
probably have not been standardized for use
with red cells in agglutination tests. They must
be used cautiously, and their hemagglutina-
tion reactivity must be carefully standardized
by the user.4 In countries outside the United
States, AHG reagents for the detection of IgM
and IgA in tube tests or column agglutination
tests may be available.
Cold Agglutinin Disease
CAD, which is less common than WAIHA, is
the hemolytic anemia that is most commonly
associated with autoantibodies that react pref-
erentially in the cold. CAD occurs as an acute
or chronic condition. The acute form is often
secondary to Mycoplasma pneumoniae infec-
tion. The chronic form is often seen in elderly
patients and is sometimes associated with
lymphoma, chronic lymphocytic leukemia, or
Waldenström macroglobulinemia. Acrocyano-
sis and hemoglobinuria may occur in cold
weather. CAD is often characterized by aggluti-
nation, at room temperature, of red cells in an
EDTA specimen, sometimes to the degree that
the red cells appear to be clotted.
Serologic Characteristics
Complement is the only protein detected on
red cells in almost all cases of CAD. If the red
cells have been collected properly and washed
at 37 C, there will be no immunoglobulin on
the cells and no reactivity in the eluate. If other
proteins are detected, a negative control for
the DAT (eg, 6% albumin or saline) should be
tested to ensure that the cold autoagglutinin is
not causing a false-positive result. The cold-re-
active autoagglutinin is usually IgM, which
binds to red cells in the lower temperature of
the peripheral circulation and causes comple-
ment components to attach to the red cells. As
438 䡲 AABB TECHNICAL MANUAL
the red cells circulate to warmer areas, the IgM
dissociates but the complement remains.
IgM cold-reactive autoagglutinins associ-
ated with immune hemolysis usually react at
30 C, and 60% have a titer of 1000 when test-
ed at 4 C.4 If 22% to 30% bovine albumin is in-
cluded in the test system, pathologic cold ag-
glutinins will react at 30 C or 37 C.4 On
occasion, pathologic cold agglutinins have a
lower titer (ie, <1000), but they have a high
thermal amplitude (ie, reactive at 30 C with or
without the addition of albumin). The thermal
amplitude of the antibody has greater signifi-
cance than the titer. Hemolytic activity against
untreated red cells can sometimes be demon-
strated at 20 C to 25 C. Except in rare cases
with Pr specificity, enzyme-treated red cells
are hemolyzed in the presence of adequate
complement.
To determine the true thermal amplitude
or titer of the cold autoagglutinin, the speci-
men is collected and maintained strictly at 37
C until the serum and red cells are separated to
avoid in-vitro autoadsorption. Alternatively,
plasma can be used from an EDTA-anticoagu-
lated specimen that has been warmed for 10 to
15 minutes at 37 C (with repeated mixing) and
then separated from the cells, ideally at 37 C.
This process should release autoadsorbed an-
tibody back into the plasma.
In chronic CAD, the IgM autoagglutinin is
usually a monoclonal protein with kappa light
chains. In the acute form induced by Myco-
plasma or viral infections, the antibody is
polyclonal IgM with normal kappa and lamb-
da light-chain distribution. Rare examples of
IgA and IgG cold-reactive autoagglutinins have
also been described.4
Serologic Problems
Problems with ABO and Rh typing and other
tests are not uncommon. Often, it is only nec-
essary to maintain the blood sample at 37 C
immediately after collection and to wash the
red cells with warm (37 C) saline before test-
ing. Alternatively, an EDTA sample can be
warmed to 37 C for about 10 minutes, after
which the red cells are washed with warm sa-
line. It is helpful to perform a parallel control
test with 6% bovine albumin to determine
whether autoagglutination persists. If the con-
trol test result is nonreactive, the results ob-
tained with anti-A and anti-B are usually valid.
If autoagglutination still occurs, it may be nec-
essary to treat the red cells with sulfhydryl re-
agents. Because cold-reactive autoagglutinins
are almost always IgM and sulfhydryl reagents
denature IgM molecules, reagents (such as 2-
ME or DTT) can be used to abolish autoagglu-
tination (see Method 2-18). Treating the red
cells with ZZAP reagent, as in the preparation
for adsorptions, can also be used (see Method
4-8).
When the serum agglutinates group O re-
agent red cells, ABO serum tests are invalid.
Repeating the tests using prewarmed serum
and group A1, B, and O red cells and allowing
the red cells to “settle” after incubation at 37 C
for 1 hour (instead of centrifuging the sample)
often resolves any discrepancy (see Method 2-
11). By eliminating the centrifugation step, in-
terference by cold-reactive autoantibodies
might be avoided. Weak anti-A and/or -B in
some patients’ sera may not react at 37 C. Al-
ternatively, adsorbed serum (either autoad-
sorbed or adsorbed with allogeneic group O
red cells) can be used. Rabbit erythrocyte stro-
ma should not be used for ABO serum tests be-
cause anti-B and anti-A1 may be removed.41,42
Detection of Alloantibodies in the
Presence of Cold-Reactive Autoantibodies
Cold-reactive autoagglutinins rarely mask
clinically significant alloantibodies if serum
tests are conducted at 37 C and IgG-specific
reagents are used for the antiglobulin phase.
The use of potentiators (eg, albumin or PEG) is
not recommended because they may increase
the reactivity of the autoantibodies. In rare in-
stances, it may be necessary to perform autol-
ogous adsorption at 4 C (see Method 4-5).
Achieving the complete removal of potent
cold-reactive autoagglutinins is very time con-
suming and usually unnecessary. Removal of
sufficient cold autoagglutinins may be facili-
tated by treating the patient’s cells with en-
zymes or ZZAP before adsorption. One or two
cold autologous adsorptions should remove
 CHAPTER 17
enough autoantibody to make it possible to
detect alloantibodies at 37 C that were other-
wise masked by the cold-reactive autoanti-
body. As an alternative, the allogeneic adsorp-
tion process used for WAIHA can be
performed at 4 C. Rabbit erythrocyte stroma,
which removes autoanti-I and -IH from sera,
should be used with caution because this
method can remove clinically significant allo-
antibodies—notably anti-D, -E, -Vel, and IgM
antibodies regardless of blood group specifici-
ty.43,44
Specificity of Autoantibody
The autoantibody specificity in CAD is most
often anti-I but is usually of academic interest
only. Anti-i is found less commonly, and it is
usually associated with infectious mononucle-
osis. On rare occasions, other specificities are
seen.
Autoantibody specificity is not diagnostic
for CAD. Autoanti-I may be seen in healthy in-
dividuals as well as in patients with CAD. The
nonpathologic forms of autoanti-I, however,
rarely react at titers above 64 at 4 C and are
usually nonreactive with I– (cord i and adult i)
red cells at room temperature. In contrast, the
autoanti-I of CAD may react quite strongly
with I– red cells in tests at room temperature,
and equal or even stronger reactions occur
with I+ red cells. Autoanti-i reacts in the oppo-
site manner, demonstrating stronger reac-
tions with I– red cells than with red cells that
are I+. Anti-IT, originally thought to recognize a
transition state of i to I (explaining the desig-
nation “IT”), reacts strongly with cord red cells,
weakly with normal adult I red cells, and most
weakly with the rare adult i red cells. In rare
cases, the cold agglutinin specificity may be
anti-Pr, which reacts equally well with untreat-
ed red cells of I or i phenotypes but does not
react with enzyme-treated red cells. Proce-
dures to determine the titer and specificity of
cold-reactive autoantibodies are given in
Methods 4-6 and 4-7. Typical reactivity pat-
terns of cold autoantibodies are shown in the
table in Method 4-6.
DAT/Immune Hemolysis 䡲 439
Mixed-Type AIHA
Although about one-third of patients with
WAIHA have nonpathologic IgM antibodies
that agglutinate at room temperature, another
group of patients with WAIHA have cold agglu-
tinins that react at or above 30 C. This latter
group is referred to as having “mixed” or “com-
bined warm and cold” AIHA and can be subdi-
vided into patients with high-titer, high-ther-
mal-amplitude IgM cold antibodies (the rare
WAIHA plus classic CAD) and patients with
normal-titer (<64 at 4 C), high-thermal-ampli-
tude cold antibodies.45-47 Patients with mixed-
type AIHA often present with hemolysis and
complex serum reactivity in all phases of
testing.
Serologic Characteristics
In mixed-type AIHA, both IgG and C3 are usu-
ally detectable on patients’ red cells; however,
C3, IgG, or IgA alone may be detectable on the
red cells.4 An eluate contains a warm-reactive
IgG autoantibody. Both warm-reactive IgG au-
toantibodies and cold-reactive, agglutinating
IgM autoantibodies are present in the serum.
These autoantibodies usually result in reactivi-
ty at all phases of testing and with virtually all
cells tested. The IgM agglutinating autoanti-
body reacts at 30 C or above. If adsorptions are
performed to detect alloantibodies, it may be
necessary to perform them at both 37 C and 4 C.
Specificity of Autoantibodies
The unusual cold-reactive IgM agglutinating
autoantibody can have specificities that are
typical of CAD (ie, anti-I or -i) but often has no
apparent specificity.45,46 The warm-reactive
IgG autoantibody often appears to be serologi-
cally indistinguishable from autoantibodies
encountered in typical WAIHA.
Transfusion of Patients with Mixed-Type
AIHA
If blood transfusions are necessary, the con-
siderations for the exclusion of alloantibodies
and the selection of blood for transfusion are
identical to those described for patients with
440 䡲 AABB TECHNICAL MANUAL
acute hemolysis caused by WAIHA and CAD
(see above).
Paroxysmal Cold Hemoglobinuria
PCH is the rarest form of DAT-positive AIHA.
Historically, PCH was associated with syphilis,
but this association is now unusual.48 More
commonly, PCH presents as an acute transient
condition that is secondary to a viral infection,
particularly in young children. In such cases,
the biphasic hemolysin may be only transient-
ly detectable. PCH can also occur as an idio-
pathic chronic disease in older people.
Serologic Characteristics
PCH is caused by a cold-reactive IgG comple-
ment-binding antibody. As with IgM cold-re-
active autoagglutinins, reactivity occurs with
red cells in colder areas of the body (usually
the extremities) and causes C3 to bind irre-
versibly to red cells. The antibody then dissoci-
ates from the red cells as the blood circulates
to warmer parts of the body. Red cells washed
in a routine manner for the DAT are usually
coated only with complement, but IgG may be
detectable on cells that have been washed
with cold saline and tested with cold anti-IgG
reagent.4 Keeping the test system close to its
optimal binding temperature allows the cold-
reactive IgG autoantibody to remain attached
to its antigen. Because complement compo-
nents are usually the only globulins present on
circulating red cells, eluates prepared from the
red cells of patients with PCH are almost al-
ways nonreactive.
The IgG autoantibody in PCH is classical-
ly described as a biphasic hemolysin because
binding to red cells occurs at low temperatures
but hemolysis does not occur until the com-
plement-coated red cells are warmed to 37 C.
This is the basis of the diagnostic test for the
disease, the Donath-Landsteiner test (see
Method 4-11). The autoantibody may aggluti-
nate normal red cells at 4 C but rarely to titers
greater than 64. Because the antibody rarely
reacts above 4 C, pretransfusion antibody de-
tection tests are usually nonreactive and the
serum is usually compatible with random do-
nor cells by routine crossmatch procedures.
Specificity of Autoantibody
The autoantibody of PCH has most frequently
been shown to have P specificity. The autoan-
tibody reacts with all red cells by the Donath-
Landsteiner test (including the patient’s own
red cells), except those of the very rare p or Pk
phenotypes.
Transfusion of Patients with PCH
Transfusion is rarely necessary for adult pa-
tients with PCH, unless their hemolysis is se-
vere. In young children, the thermal amplitude
of the antibody tends to be much wider than in
adults and hemolysis is often more brisk, so
transfusion may be required as a lifesaving
measure. Although there is some evidence that
p red cells survive longer than P+ (P1+ or P1–)
red cells, the prevalence of p blood is approxi-
mately 1 in 200,000, and the urgent need for
transfusion usually precludes attempts to ob-
tain this rare blood. Transfusion of donor
blood should not be withheld from patients
with PCH whose need is urgent. Transfusion of
red cells that are negative for the P antigen
should be considered only for those patients
who do not respond adequately to randomly
selected units of donor blood.4
DRUG-INDUCED IMMUNE
HEMOLY TI C ANEMIA
Drugs rarely cause immune hemolytic anemia;
the estimated incidence is 1 in 1 million peo-
ple.49 Many drugs have been implicated in he-
molytic anemia over the years, as can be seen
in the list provided in Appendix 17-1 and re-
viewed elsewhere.12
Drugs sometimes induce the formation of
antibodies—against the drug, red cell mem-
brane components, or an antigen formed by
the drug and the red cell membrane. These
antibodies may cause a positive DAT result,
immune red cell destruction, or both.12,40 In
some instances, a positive DAT result can be
caused by nonimmunologic protein adsorp-
tion (NIPA) onto the red cell, which is caused
by the drug.12
 CHAPTER 17 DAT/Immune Hemolysis 䡲 441

Theoretical Mechanisms of Drug- Serologic Classification

Induced Antibodies
Numerous theories have been suggested to ex-
plain how drugs induce immune responses
and what relation such responses may have to
the positive DAT result and immune-mediated
cell destruction observed in some patients.4
For many years, drug-associated positive DAT
results were classified by four mechanisms:
drug adsorption (penicillin-type), immune
complex formation, autoantibody produc-
tion, and NIPA. This classification has been
useful serologically, but many aspects lack de-
finitive proof. In addition, some drugs demon-
strate serologic reactivity that appears to in-
volve more than one mechanism. A more
comprehensive approach, termed a “unifying
hypothesis,” is shown in Fig 17-1. One or more
populations of antibodies may be present. In
addition, NIPA, which is independent of anti-
body production, appears to play a role in
drug-induced immune hemolytic anemia.12
Drug-induced antibodies can be classified into
two groups: drug dependent (those that re-
quire the presence of the drug in the test sys-
tem to be detected) and drug independent
(those that do not require the in-vitro addition
of the drug for detection).4 Drug-dependent
antibodies are subdivided into those that react
with drug-treated red cells (eg, antibodies to
penicillin and some cephalosporins) and
those that react with untreated red cells in the
presence of a solution of the drug (eg, antibod-
ies to quinine and ceftriaxone). Drug-indepen-
dent antibodies (eg, autoantibodies induced
by methyldopa and fludarabine) have serolog-
ic reactivity that is independent of the drug de-
spite the fact that the drug originally induced
the immune response. Because the drug does
not need to be added to the test system, drug-
independent antibodies behave like autoanti-
bodies that are serologically indistinguishable
from idiopathic warm autoantibodies.

FIGURE 17-1. Proposed unifying theory of drug-induced antibody reactions (based on a cartoon by Habibi as
cited by Garratty28). The thicker lines represent antigen-binding sites for the Fab region of the drug-induced
antibody. Drugs (haptens) bind loosely or firmly to cell membranes, and antibodies may be made to 1) the drug
[producing in-vitro reactions typical of a drug adsorption (penicillin-type) reaction]; 2) membrane components
or mainly membrane components (producing in-vitro reactions typical of autoantibody); or 3) part-drug, part-
membrane components (producing an in-vitro reaction typical of the so-called immune complex
mechanism).28(p55)
442 䡲 AABB TECHNICAL MANUAL
If a patient is suspected of having drug-
induced immune hemolytic anemia (DIIHA),
then the suspected drug should be stopped.
Laboratory testing to detect drug-dependent
antibodies can be performed, but DIIHA
caused by drug-independent antibodies or
NIPA can only be suggested by showing a tem-
poral association of the drug administration
and hemolysis.
The historical details of penicillin- and
methyldopa-induced antibodies are not de-
scribed in this chapter but have been exten-
sively reviewed elsewhere.4,49 DIIHA caused by
high-dose intravenous penicillin therapy is no
longer seen, and methyldopa, the prototype
for drug-independent antibodies, is not used
as frequently as in the past. Currently, the
drugs most commonly associated with DIIHA
are piperacillin, ceftriaxone, and cefotetan.50
Drug-Dependent Antibodies that Are
Reactive with Drug-Treated Red Cells
Some drugs (eg, penicillin, ampicillin, and
many cephalosporins) covalently bind to red
cells, thus making it possible to coat red cells
in the laboratory with the drug. Antibodies di-
rected to these drugs will react with the drug-
treated red cells but not with untreated red
cells (unless the patient also has alloantibod-
ies to red cell antigens present on these cells).
Penicillin and the cephalosporins are
beta-lactam antibiotics. It was thought for
some time that antibodies to any drug in the
penicillin and cephalosporin families could be
detected by testing red cells with drug-treated
cells using methods previously described for
penicillin and cephalothin. It is now known
that this is not the case. Synthetic penicillins
and newer cephalosporins cannot be as-
sumed to have the same red cell binding char-
acteristics as penicillin and cephalothin (a
first-generation cephalosporin). Cefotetan (a
second-generation cephalosporin) binds very
well to red cells, and antibodies caused by ce-
fotetan typically react to very high titers with
cefotetan-treated red cells. However, ceftriax-
one (a third-generation cephalosporin) does
not bind well to red cells; therefore, antibodies
to ceftriaxone cannot be tested by this meth-
od.50 Piperacillin, a semisynthetic penicillin,
binds to red cells at high pH. However, a large
percentage of plasma from healthy blood do-
nors and patients react with piperacillin-treat-
ed red cells, so this method is not recommend-
ed for testing for piperacillin antibodies.51 For
drug antibodies detected using drug-treated
red cells, the following are expected:
䡲 The DAT result is usually positive for IgG,
but complement may also be present.
䡲 The serum contains an antibody that reacts
with the drug-treated red cells but not the
untreated red cells.
䡲 Antibody eluted from the patient’s red cells
reacts with drug-treated red cells but not
untreated red cells.
Hemolysis develops gradually but may be
life threatening if the etiology is unrecognized
and drug administration is continued. The pa-
tient may or may not have been previously ex-
posed to the drug and, in the case of cefotetan,
only a single dose given prophylactically can
result in severe hemolysis. Normal plasma has
been shown to react with some drug-treated
red cells (eg, red cells treated with cefotetan,4
piperacillin,51 or oxaliplatin52), suggesting prior
exposure to these drugs through environmen-
tal routes.
Drug-Dependent Antibodies that Are
Reactive with Untreated Red Cells in the
Presence of Drug
Antibodies to many drugs that have been re-
ported to cause immune hemolytic anemia are
detected by testing untreated red cells in the
presence of the drug. Piperacillin and some of
the second- and third-generation cephalospo-
rins react by this method; anti-ceftriaxone has
been detected only by testing red cells in the
presence of drug.49 The following observations
are characteristic:
䡲 Complement may be the only protein easily
detected on the red cells, but IgG may be
present.
䡲 The serum antibody can be IgM, IgG, or
IgM with IgG.
 CHAPTER 17
䡲 A drug (or metabolite) must be present in
vitro for the antibody in the patient’s serum
to be detected. Antibodies may cause he-
molysis, agglutination, and/or sensitization
of red cells in the presence of the drug.
䡲 The patient need only take a small amount
of the drug (eg, a single dose).
䡲 Acute intravascular hemolysis with hemo-
globinemia and hemoglobinuria is the usu-
al presentation. Renal failure is quite com-
mon.
䡲 Once antibody has been formed, severe he-
molytic episodes may recur after exposure
to very small quantities of the drug.
On occasion, it appears that a patient’s se-
rum contains an “autoantibody” in addition to
a drug antibody reacting in the presence of the
drug. Rather than a true autoantibody, it is be-
lieved that this reactivity is due to the presence
of circulating drug or drug plus antibody com-
plexes.50,53 In these cases, an eluate is usually
nonreactive when the drug is not present in
the system. However, in some cases involving
piperacillin, the eluate reacts while the patient
is still taking the drug. A sample collected sev-
eral days after the drug has been discontinued
will be nonreactive. A true warm autoantibody
is expected to be reactive in an eluate prepared
from the patient’s red cells, and autoantibody
in the serum persists. Consequently, DIIHA
caused by piperacillin can be misdiagnosed as
WAIHA, especially if the eluate reacts. Differ-
entiation of warm-reactive autoantibody from
drug-induced immune hemolytic anemia is
important for clinical management.53
Drug-Independent Antibodies:
Autoantibody Production
Some drugs induce autoantibodies that ap-
pear serologically indistinguishable from
those of WAIHA. Red cells are coated with IgG,
and the eluate as well as the serum react with
virtually all cells tested in the absence of the
drug. The antibody has no direct or indirect in-
vitro interaction with the drug. The prototype
drug for such cases is methyldopa, which is
now used much less frequently than in the
past. Currently, fludarabine, used to treat
DAT/Immune Hemolysis 䡲 443
chronic lymphocytic leukemia, is the most
commonly used drug to produce drug-inde-
pendent antibodies and AIHA.49
Non-Immunologic Protein Adsorption
The positive DAT result associated with some
drugs is caused by modification of the red cell
membrane by the drug and is independent of
antibody production. Hemolytic anemia asso-
ciated with this mechanism is rare.
Cephalosporins (primarily cephalothin)
are the drugs with which positive DAT results
and NIPA were originally associated. In vitro,
red cells coated with cephalothin in pH 9.8
buffer and incubated with normal plasma ad-
sorb albumin, IgA, IgG, IgM, C3, and other
proteins in a nonimmunologic manner. For
this reason, the indirect antiglobulin test result
with virtually all plasma will be positive. Other
drugs that cause NIPA and a positive DAT re-
sult include diglycoaldehyde, cisplatin, oxali-
platin, and beta-lactamase inhibitors (clavu-
lanic acid, sulbactam, and tazobactam).12
NIPA should be suspected when a pa-
tient’s plasma/serum and most normal plas-
ma/sera are reactive in an indirect antiglobu-
lin test with drug-treated red cells but the
eluate from the patient’s red cells is nonreac-
tive with the drug-treated cells.
Laboratory Investigation of Drug-
Induced Immune Hemolysis
The drug-related problems that are most com-
monly encountered in the blood bank are
those associated with a positive DAT result and
a nonreactive eluate. Recent red cell transfu-
sions and/or dramatic hemolysis may result in
a weak DAT result by the time hemolysis is sus-
pected. When other, more common causes of
immune-mediated hemolysis have been ex-
cluded and a temporal relationship exists be-
tween the administration of a drug and the
hemolytic anemia, a drug antibody investiga-
tion should be pursued.
The patient’s serum should be tested for
unexpected antibodies by routine procedures.
If the serum does not react with untreated red
cells, the tests should be repeated with the
444 䡲 AABB TECHNICAL MANUAL
drug(s) suspected of causing the problem.
Some drug formulations contain inert ingredi-
ents (eg, pill or capsule forms), and other
drugs are combinations of two drugs (eg,
piperacillin plus tazobactam). Although it
would seem logical to test the patient’s serum
with the actual drug that the patient received,
inert ingredients or drug combinations can
make preparation of drug-treated red cells dif-
ficult or make the results confusing. It is pref-
erable to test serum using pure drug formula-
tions as well as separate components of
combination drugs.
If the drug has already been reported to
cause hemolytic anemia, testing methods may
be described in the case reports. Far more drug
antibodies are detected by testing serum in the
presence of drug; therefore, when a previous
report of antibodies to a drug is not available,
an initial screening test can be performed with
a solution of the drug at a concentration of ap-
proximately 1 mg/mL in phosphate-buffered
saline (see Method 4-13). Serum, rather than
plasma, is the preferred specimen for testing
for hemolysis to be observed; this also allows
for the addition of fresh normal serum (as a
source of complement) to the test system. The
addition of the fresh complement increases
the sensitivity of the test for the detection of
in-vitro hemolysis resulting from complement
activation.
If these tests are not informative, at-
tempts can be made to coat normal red cells
with the drug. The patient’s serum and an elu-
ate from the patient’s red cells can be tested
against the drug-treated red cells (see Method
4-12). This is the method of choice when ceph-
alosporins (except for ceftriaxone) are thought
to be implicated. Results that are definitive for
a drug-induced positive DAT result are reactiv-
ity of the eluate with drug-treated red cells and
absence of reactivity with untreated red cells.
KEY POINTS
1. The DAT is used to determine whether red cells have been coated in vivo with immunoglob-
ulin, complement, or both. The DAT is used primarily for the investigation of hemolytic
transfusion reactions, HDFN, AIHA, and drug-induced immune hemolysis.
2. The DAT should be used to determine whether a hemolytic anemia has an immune etiology.
Drug-treated red cells should always be
tested with saline and normal serum (or plas-
ma) as negative controls. This approach en-
sures that the observed reactivity with the
patient’s serum/plasma is appropriately inter-
preted. Antibodies reactive with red cells treat-
ed with some drugs (eg, beta-lactams and plat-
inums) have been detected in the plasma from
blood donors and patients without hemolytic
anemia thought to be due to environmental
exposure. Therefore, misinterpretation of re-
activity in a patient’s serum is possible.51-52
Whenever possible, a positive control
should be tested with drug-treated red cells.
Negative results of a patient’s serum and elu-
ate without a positive control can only be in-
terpreted as showing that antibodies to that
drug were not detected. The drug may or may
not be bound to the test red cells.
If the drug in question is known to cause
NIPA, the patient’s serum and the controls
(negative and positive) should also be tested at
a dilution of 1 in 20. Normal sera at this dilu-
tion do not usually contain enough protein for
NIPA to be detected.
An immune response may be caused by a
metabolite of a drug rather than the drug itself.
If the clinical picture is consistent with im-
mune-mediated hemolysis and the above tests
are noninformative, it may be helpful to test
metabolites of the parent drug that are present
in the serum or urine of an individual who is
taking that drug.54 Antibodies to some nonste-
roidal antiinflammatory drugs have required
testing in the presence of metabolite.55 The
metabolism and half-life of the drug deter-
mines when the drug metabolite should be
collected. Pharmacology information for the
metabolite(s) detectable in serum or urine and
to previous reports for the drug under investi-
gation should be consulted.
 CHAPTER 17 DAT/Immune Hemolysis 䡲 445

3. A positive DAT result may or may not be associated with hemolysis.

4. Performance of the DAT on postreaction specimens is part of the initial investigation of a
transfusion reaction. The DAT result may be positive if sensitized red cells have not been de-
stroyed, or may be negative if hemolysis and rapid clearance have occurred.
5. The DAT is performed by testing freshly washed red cells directly with antiglobulin reagents
containing anti-IgG and anti-C3d. False-negative or weaker results can be obtained if the
washed red cells are allowed to sit before testing with anti-IgG or if the reading is delayed.
6. When the DAT result is positive with both anti-IgG and anti-C3, the red cells should be test-
ed with an inert control reagent (eg, 6% albumin or saline). If the control is reactive, the DAT
result is invalid, possibly indicating spontaneous agglutination from heavy coating of IgG or
rare warm-reactive IgM. The invalid DAT result could also be due to IgM cold autoaggluti-
nins that were not dissociated during routine washing.
7. A positive DAT result alone is not diagnostic of hemolytic anemia. The interpretation of the
significance of this positive result requires additional patient-specific information. Dia-
logue with the attending physician is important. Clinical considerations together with labo-
ratory data should dictate the extent to which a positive DAT result is evaluated.
8. The following situations may warrant further investigation of a positive DAT:
a. Evidence of in-vivo red cell destruction.
b. Recent transfusion.
c. Administration of drugs that have previously been associated with immune-mediated
hemolysis.
d. History of hematopoietic progenitor cell or organ transplantation.
e. Administration of IVIG or intravenous anti-D.
9. Elution frees antibody from sensitized red cells and recovers antibody in a usable form. Elu-
tion is useful in certain situations for implicating an autoantibody, detecting specific anti-
bodies that may not be detectable in the serum, and deciding to test patient’s serum for
drug-related antibodies.
10. AIHAs are subdivided into the major types: WAIHA, CAD, mixed- or combined-type AIHA,
and PCH. Drugs may also induce immune hemolysis.

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448 䡲 AABB TECHNICAL MANUAL

䡲 APPENDIX 17-1
Drugs Associated with Immune Hemolytic Anemia
Drug Method of Detection

Aceclofenac +Drug

Acetaminophen + Drug

Acyclovir DT

Aminopyrine DT
Amoxicillin DT
Amphotericin B + Drug

Ampicillin DT + Drug

Antazoline + Drug
Azapropazone AA DT

Butizide + Drug

Carbimazole AA DT + Drug

Carboplatin AA DT + Drug NIPA

Carbromal DT
Cefamandole DT
Cefazolin DT
Cefixime DT + Drug
Cefotaxime DT + Drug

Cefotetan AA DT + Drug NIPA

Cefoxitin AA DT + Drug

Ceftazidime AA DT + Drug

Ceftizoxime DT + Drug

Ceftriaxone + Drug
Cefuroxime DT
Cephalexin DT
Cephalothin DT + Drug NIPA

Chloramphenicol AA DT

Chlorinated hydrocarbons AA DT + Drug

Chlorpromazine AA + Drug
Chlorpropamide + Drug

Cimetidine DT +Drug
 CHAPTER 17 DAT/Immune Hemolysis 䡲 449

䡲 APPENDIX 17-1
Drugs Associated with Immune Hemolytic Anemia (Continued)
Drug Method of Detection

Ciprofloxacin +Drug
Cisplatin DT +Drug NIPA
Cladribine AA
Clavulanate NIPA
Cyanidanol AA DT + Drug
Cyclofenil AA + Drug

Cyclosporin DT

Diclofenac AA DT + Drug

Diethylstilbestrol + Drug

Diglycoaldehyde NIPA
Dipyrone DT + Drug
Erythromycin DT
Etodolac + Drug
Fenoprofen AA + Drug

Fluconazole DT + Drug
Fludarabine AA
Fluorescein DT + Drug

Fluorouracil + Drug

Furosemide + Drug
Hydralizine DT
Hydrochlorothiazide DT + Drug

Hydrocortisone DT + Drug

9-Hydroxy-methyl-ellipticinium + Drug

Ibuprofen +Drug
Imatinib mesylate DT
Insulin DT
Isoniazid DT + Drug
Levodopa AA
Levofloxacin DT +Drug
Mefenamic acid AA
(Continued)
450 䡲 AABB TECHNICAL MANUAL

䡲 APPENDIX 17-1
Drugs Associated with Immune Hemolytic Anemia (Continued)
Drug Method of Detection

Mefloquine DT + Drug

Melphalan + Drug

6-Mercaptopurine DT
Methadone DT

Methotrexate AA DT + Drug
Methyldopa AA
Nabumetone +Drug
Nafcillin DT
Naproxen + Drug

Oxaliplatin DT + Drug NIPA

p-Aminosalicylic acid + Drug

Penicillin G DT
Phenacetin + Drug
Phenytoin DT

Piperacillin DT + Drug

Probenicid + Drug

Procainamide AA
Propyphenazone + Drug
Pyrazinamide DT + Drug

Pyrimethamine DT
Quinidine DT + Drug
Quinine + Drug

Ranitidine DT + Drug

Rifabutin + Drug

Rifampicin DT + Drug

Sodium pentothal/thiopental + Drug

Stibophen + Drug

Streptomycin AA DT + Drug

Sulbactam NIPA
Sulfamethoxazole + Drug
 CHAPTER 17 DAT/Immune Hemolysis 䡲 451

䡲 APPENDIX 17-1
Drugs Associated with Immune Hemolytic Anemia (Continued)
Drug Method of Detection

Sulfasalazine + Drug

Sulfisoxazole +Drug

Sulindac AA DT + Drug

Suprofen AA + Drug

Tazobactam NIPA
Teicoplanin AA + Drug
Teniposide AA + Drug

Tetracycline DT
Ticarcillin AA DT
Tolbutamide DT
Tolmetin AA + Drug

Triamterene DT + Drug

Trimethoprim + Drug

Vancomycin + Drug

Zomepirac AA + Drug
AA = drug-independent autoantibody; DT = testing with drug-treated red cells; + Drug = testing in the presence of drug;
NIPA = nonimmunologic protein adsorption.
 C h a p t e r 1 8

Platelet and Granulocyte Antigens

and Antibodies

Brian R. Curtis, PhD, D(ABMLI), MT(ASCP)SBB

THIS CHAPTER DISCUSSES anti- multiple ligand-receptor interactions involv-

gens expressed on platelets and granulo-
cytes together with antibodies that arise when
individuals are sensitized to these markers.
These antigens and the immune responses to
them are of importance in alloimmune, auto-
immune, and drug-induced immune syn-
dromes involving platelets and granulocytes.
PL AT ELET ANTIGENS AND
ANTIBODIES
Platelets express a variety of antigenic markers
on their surface. Some of these antigens are
shared with other cells, as with ABO and HLA,
whereas others are essentially platelet specific,
like human platelet alloantigens (HPAs).
HPAs
Platelets reportedly play roles in inflamma-
tion; innate, adaptive, and autoimmunity; car-
diovascular disease; and even cancer.1,2 How-
ever, they are most well known for their
function in forming clots to stem bleeding.
Platelets perform these functions through
ing glycoproteins (GPs) expressed on their
cell-surface membranes.
Platelet membrane GPs are expressed in
different forms due to single nucleotide poly-
morphisms (SNPs) in the genes that encode
them. The amino acid changes resulting from
these SNPs induce changes in the glycoprotein
structure to form antigens that can elicit anti-
bodies through exposure from pregnancy or
platelet transfusions. Currently, 33 different
HPAs expressed on six different platelet mem-
brane glycoproteins—GPIIb, GPIIIa, GPIb,
GPIb, GPIa, and CD109—have been identi-
fied (Table 18-1).3 These antigens are often re-
ferred to as “platelet specific,” but some are
found on cells other than platelets (especially
leukocytes and endothelial cells), although
their chief clinical importance appears to be
linked to their presence on platelets.
Twelve antigens are clustered into six bi-
allelic groups (HPA-1, HPA-2, HPA-3, HPA-4,
HPA-5, and HPA-15). The nomenclature for
HPAs consists of numbering the antigens in
their order of discovery, with the higher-

Brian R. Curtis PhD, D(ABMLI), MT(ASCP)SBB, Director, Platelet and Neutrophil Immunology Lab, Blood-
Center of Wisconsin, Milwaukee, Wisconsin
B. Curtis has disclosed a financial relationship with Gen-Probe Incorporated.

453
454 䡲 AABB TECHNICAL MANUAL

TABLE 18-1. Human Platelet Alloantigens

Phenotypic Amino Acid Encoding Nucleotide

Antigen Frequency* Glycoprotein (GP) Change Gene Change

HPA-1a 72% a/a GPIIIa Leu33Pro ITGB3 176T>C

HPA-1b 26% a/b
2% b/b
HPA-2a 85% a/a GPIb Thr145Met GPIBA 482C>T
HPA-2b 14% a/b
1% b/b
HPA-3a 37% a/a GPIIb Ile847Ser ITGA2B 2621T>G
HPA-3b 48% a/b
15% b/b
HPA-4a >99.9% a/a GPIIIa Arg143Gln ITGB3 506G>A
HPA-4b < 0.1% a/b
< 0.1% b/b
HPA-5a 88% a/a GPIa Glu505Lys ITGA2 1600G>A
HPA-5b 20% a/b
1% b/b
HPA-6bw < 1% b/b GPIIIa Arg489Gln ITGB3 1544G>A
HPA-7bw < 1% b/b GPIIIa Pro407Ala ITGB3 1297C>G
HPA-8bw < 1% b/b GPIIIa Arg636Cys ITGB3 1984C>T
HPA-9bw < 1% b/b GPIIb Val837Met ITGA2B 2602G>A
HPA-10bw < 1% b/b GPIIIa Arg62Gln ITGB3 263G>A
HPA-11bw < 1% b/b GPIIIa Arg633His ITGB3 1976G>A
HPA-12bw < 1% b/b GPIb Gly15Glu GPIBB 119G>A
HPA-13bw < 1% b/b GPIa Met799Thr ITGA2 2483C>T
HPA-14bw < 1% b/b GPIIIa Lys611del ITGB3 1909_1911delAAG
HPA-15bw 35% a/a CD109 Ser682Tyr CD109 2108C>A
42% a/b
23% b/b
HPA-16bw < 1% b/b GPIIIa Thr140Ile ITGB3 497C>T
HPA-17bw < 1% b/b GPIIIa Thr195Met ITGB3 662C>T
HPA-18bw < 1% b/b GPIa Gln716His ITGA2 2235G>T
HPA-19bw < 1% b/b GPIIIa Lys137Gln ITGB3 487A>C
HPA-20bw < 1% b/b GPIIb Thr619Met ITGA2B 1949C>T
 CHAPTER 18 Platelet and Granulocyte Antigens and Antibodies 䡲 455

TABLE 18-1. Human Platelet Alloantigens (Continued)

Phenotypic Amino Acid Encoding Nucleotide

Antigen Frequency* Glycoprotein (GP) Change Gene Change

HPA-21bw < 1% b/b GPIIIa Glu628Lys ITGB3 1960G>A

HPA-22bw < 1% b/b GPIIb Lys164Thr ITGA2B 584A>C
HPA-23bw < 1% b/b GPIIIa Arg622Trp ITGB3 1942C>T
HPA-24bw < 1% b/b GPIIb Ser472Asn ITGA2B 1508G>A
HPA-25bw < 1% b/b GPIa Thr1087Met ITGA2 3347C>T
HPA-26bw < 1% b/b GPIIIa Lys580Asn ITGB3 1818G>T
HPA-27bw < 1% b/b GPIIb Leu841Met ITGA2B 2614C>A
*Phenotypic frequencies are for people of European ancestry who live in North America. Human platelet alloantigen (HPA)
frequencies in other races and ethnic groups can be found in the IPD-Immuno Polymorphism Database.3

frequency antigens designated “a” and the
lower-frequency antigens designated “b.”4
HPAs for which antibodies against only one of
the two antithetical antigens have been de-
tected are labeled with a “w” for “workshop,”
such as HPA-6bw.
Platelet Alloantigens on GPIIb/IIIa
HPA-1a is the platelet alloantigen that was dis-
covered first and is most familiar.5 Originally
named “Zwa” and more commonly referred to
as “PlA1,” it is expressed on GPIIIa, the -sub-
unit of the integrin GPIIb/IIIa (2b/3) com-
plex.
Integrins are a broadly distributed family
of adhesion molecules consisting of an  and a
 chain held together by divalent cations in a
heterodimeric complex.6 Integrins are essen-
tial for platelet adhesion and aggregation be-
cause they serve as receptors for ligands, such
as fibrinogen, collagen, fibronectin, von Wille-
brand factor (vWF), and other extracellular
matrix proteins.
Binding of fibrinogen by GPIIb/IIIa re-
sults in platelet aggregation, which leads to the
formation of the “platelet plug” to stop bleed-
ing. The importance of GPIIb/IIIa in hemosta-
sis is demonstrated by the serious bleeding
that occurs in patients with the rare disorder
Glanzmann thrombasthenia, in which platelet
GPIIb/IIIa is absent or dysfunctional due to in-
herited mutations in ITGA2B and/or ITGB3.7
Patients with Glanzmann thrombasthenia
who are exposed to normal platelets by trans-
fusion or pregnancy can make isoantibodies
against GPIIb/IIIa.
GPIIb/IIIa is the most abundantly ex-
pressed (approximately 80,000 molecules/
platelet) glycoprotein complex on the platelet
membrane, making it highly immunogenic.
Antibodies against HPA-1a account for the
vast majority (>80%) of the HPA-specific plate-
let antibodies detected in the sera of alloim-
munized people of European ancestry. HPA-1a
antibodies are produced by the 2% of individu-
als with the platelet type HPA-1b/1b. Antibod-
ies specific for HPA-1b are commonly detected
in patients with posttransfusion purpura
(PTP).
Twenty of the 33 HPAs are carried by GPI-
Ib (6) and GPIIIa (14). Like HPA-1a/1b, the
HPA-4a/4b antigens are also expressed on
GPIIIa and have been implicated in fetal and
neonatal alloimmune thrombocytopenia
(FNAIT), PTP, and platelet transfusion refrac-
toriness. The low-frequency HPA-4b antigen is
more common in populations of Japanese and
Chinese ancestry.
456 䡲 AABB TECHNICAL MANUAL
The HPA-3a/3b antigens are expressed on
GPIIb, but despite the high rate of incompati-
bility for both antigens in the general popula-
tion, detection of HPA-3 antibodies is uncom-
mon. Some HPA-3 antibodies are difficult to
detect in monoclonal antigen capture assays,
such as the modified antigen capture enzyme-
linked immunosorbent assay (MACE) and
monoclonal antibody immobilization of plate-
let antigens (MAIPA), in which GPIIb is ex-
tracted from platelets with detergents that can
denature the antigenic epitopes recognized by
HPA-3 antibodies.8,9 X-ray crystallography
studies could not determine the portion of the
domain of GPIIb where the HPA-3 antigens are
located, which might explain the difficulties
with detection of HPA-3 antibodies.10
An additional 17 different low-frequency
platelet antigens are expressed on either GPIIb
or GPIIIa (Table 18-1). These antigens were all
discovered in cases of FNAIT when specific an-
tibodies in maternal sera were detected that
were reactive only with the fathers’ GPIIb/IIIa.
The vast majority of these antigens are private
antigens restricted to the single families in
which they were discovered. HPA-6bw and
HPA-21bw are exceptions, having antigen fre-
quencies of 1% and 2%, respectively, in people
of Japanese ancestry, and HPA-9bw has been
implicated in several cases of FNAIT.11-14
Platelet Alloantigens on GPIb/V/IX
The GPIb/V/IX complex forms the vWF recep-
tor on platelets, and platelets express approxi-
mately 25,000 copies of the complex. Follow-
ing vascular injury, binding of the GPIb/V/IX
complex to vWF facilitates platelet adhesion to
vascular subendothelium and initiates signal-
ing events within adherent platelets that lead
to platelet activation, aggregation, and hemo-
stasis. GPIb is composed of  (GPIb) and 
(GPIb) subunits that form noncovalent asso-
ciations with GPIX and GPV. GPIb carries
HPA-2a/2b, and GPIb carries HPA-12bw. An-
tibodies against HPA-2a, -2b, and -12bw have
all been implicated in causing FNAIT.
Deficiency of the entire GPIb/V/IX com-
plex can occur due to mutations in the encod-
ing genes GPIBA, GPIBB, or GP9, and is the
cause of Bernard Soulier syndrome (BSS). BSS
is a disorder characterized by prolonged
bleeding time, thrombocytopenia, and the
presence of “giant platelets” that affects ap-
proximately 1 in 1 million people.7,15 BSS pa-
tients whose platelets are devoid of GPIb/V/IX
can produce antibodies when they are ex-
posed to the protein complex on normal plate-
lets through transfusions or pregnancy.
Platelet Alloantigens on GPIa/IIa
The integrin GPIa/IIa, also known as integrin
21, is a major collagen receptor on platelets.
The GPIa protein carries the HPA-5a/5b anti-
gens. Antibodies against HPA-5 antigens are
the second most frequently detected, after
anti-HPA-1a, in patients with FNAIT and are
also frequently detected in patients with PTP.
About 3000 to 5000 molecules of the GPIa/IIa
heterodimeric complex are expressed on
platelets, and higher expression correlates
with the presence of both HPA-5b and threo-
nine at amino acid 807 in GPIa. 16 HPA-13bw,
-18bw, and -25bw are low-frequency antigens
that are also expressed on GPIa and have all
been implicated in FNAIT. Interestingly, the
HPA-13bw polymorphism has been reported
to cause functional defects that reduce platelet
responses to collagen-induced aggregation
and spreading on collagen-coated surfaces.4
Platelet Alloantigens on CD109
CD109 is a glycosylphosphatidylinositol (GPI)-
linked protein and a member of the 2-macro-
globulin/complement superfamily. Its func-
tion is still not completely understood, but
CD109 has been reported to bind to and nega-
tively regulate the signaling of transforming
growth factor beta. CD109 is also expressed on
activated T lymphocytes, CD34+ hematopoiet-
ic cells, and endothelial cells, and it carries the
HPA-15 antigens.
The clinical significance of HPA-15 anti-
bodies is uncertain because platelets express
only about 1000 molecules of CD109. Studies
show the presence of HPA-15 antibodies in
0.22% to 4% of maternal sera in patients with
suspected FNAIT, and one report suggests that
HPA-15 antibodies are more frequently detect-
 CHAPTER 18 Platelet and Granulocyte Antigens and Antibodies 䡲 457

ed in sera from patients with immune platelet
refractoriness.17-19
Other Antigens on Platelets
ABO and Other Blood Groups
Most of the ABO antigen on platelets is carried
on saccharides attached to the major platelet
membrane glycoproteins (Table 18-2). GPIIb
and platelet endothelial cell adhesion mole-
cule 1 (PECAM-1/CD31) carry the largest
amounts of A and B antigens.20 Platelet A and B
antigen levels are quite variable from individu-
al to individual, with 5% to 10% of non-group-
O individuals expressing extremely high levels
of A1 or B on their platelets.20,21 These “high ex-
pressers” have highly active glycosyltransfer-
ases, which are much more efficient at attach-
ing A or B antigens.20
Interestingly, although individuals with
the subgroup A2 red cell phenotype express
lower levels of A on their red cells than A1 indi-
viduals, they do not express detectable A anti-
gens on their platelets. As a result, A2 platelets
have been successfully transfused to group O
patients, even those with high-titer anti-A.22
Although platelets are often transfused
without regard to ABO compatibility, the use of
mismatched platelets frequently results in
lower posttransfusion recovery rates.23,24 In
some cases, high-titer immunoglobulin G (Ig
G) A,B antibodies in blood group O recipients
are reactive with transfused platelets carrying
large amounts of A or B antigens, resulting in
platelet transfusion refractoriness.21 Recovery
of transfused platelets can also be influenced
by the transfusion of group O platelets to
group A recipients. Anti-A and/or anti-B in the
donor plasma might be reactive with soluble A
or B in the recipient plasma to form immune
complexes that bind to transfused (and autol-
ogous) platelets via FcRIIa, thereby influenc-
ing the survival of the transfused platelets.25
Clinical trials comparing ABO-identical to
-unmatched platelets in patients with cancer
who require multiple platelet transfusions
have suggested that rates of refractoriness are
significantly higher when unmatched compo-
nents are used.22,25 Although other red cell an-
tigens (eg, Lea, Leb, I, i, P, Pk, and Cromer) are
also present on platelets, there is no evidence
that these antigens significantly reduce plate-
let survival in vivo.26,27

TABLE 18-2. Other Platelet Antigens

Phenotypic Glycoprotein Amino Acid Encoding Nucleotide

Antigen Frequency (GP)* Change† Gene Change‡

ABO Same as for red cells GPIIb, IIIa, IV, Ia/ Multiple ABO Multiple
IIa, GPIb/V/IX,
CD31
HLA-A, B, Same as for leukocytes Class I HLA Multiple MHC Multiple
and C
GPIV 90-97% (Africans) CD36 Tyr325Thr* CD36 1264T>G*
90-97% (Asians) Pro90Ser* 478C>T*
99.9% (Caucasians) Exons 1-3 del
GPVI N/A GPVI N/A GP6/N/A N/A
*ABO saccharides are attached to platelet GPs during their glycosylationj.
†
Only the most common changes are shown.
‡
Only the most common mutations are shown.
N/A = not applicable.
458 䡲 AABB TECHNICAL MANUAL
GPIV/CD36
Platelets, monocytes/macrophages, and nu-
cleated erythrocytes are the only blood cells
that express GPIV/CD36 (Table 18-2). GPIV be-
longs to the Class B scavenger receptor family
and binds a number of different ligands, in-
cluding low-density lipoprotein cholesterol,
thrombospondin, Types I and IV collagen, and
malaria-infected red cells. A number of muta-
tions in CD36 have been described that result
in a complete lack of protein expression on
both platelets and monocytes in populations
of Asian and African ancestry.28-30 CD36-defi-
cient individuals exposed to normal platelets
can produce antibodies to CD36 that have
been reported to cause FNAIT, PTP, and plate-
let refractoriness.29,31,32
GPVI
GPVI is a major collagen receptor on platelets
and a member of the Ig superfamily. GPVI in-
teractions with collagen exposed on the extra-
cellular matrix result in platelet activation and
aggregation. To date, no HPAs have been iden-
tified on GPVI, but platelet autoantibodies
formed against GPVI have been reported to
cause a mild form of autoimmune thrombocy-
topenia.33,34 Interestingly, GPVI autoantibod-
ies induce shedding of GPVI from platelets,
resulting in reduced collagen binding and clin-
ically significant bleeding.
HLA
HLA is present on all nucleated cells of the
body (see Chapter 19). HLA associated with
platelets is the main source of Class I HLA in
whole blood.35 Most Class I HLA on platelets is
expressed as integral membrane proteins,
whereas smaller amounts may be adsorbed
from surrounding plasma. HLA-A and -B locus
antigens are significantly represented, but
there appears to be only minimal platelet ex-
pression of HLA-C.36 With rare exceptions,
Class II HLA is not present on the platelet
membrane.
Several factors influence the likelihood
that HLA antibodies will develop after transfu-
sions, and sensitization may be clinically im-
portant in patients receiving multiple platelet
transfusions. Transfusion-associated HLA allo-
immunization appears to be influenced by the
underlying disease, immunosuppressive ef-
fects of treatment regimens, and whether the
blood components contain a significant
amount of leukocytes. With widespread use of
leukocyte-reduced (LR) blood components for
transfusion, associated HLA alloimmunization
has been reduced considerably. HLA antibod-
ies also commonly develop following pregnan-
cy and are present in the sera of more than
32% of women who have had four or more
pregnancies.37 HLA antibodies have also been
identified in 1.7% of never-pregnant or -trans-
fused women and men with no previous trans-
fusions.37 Sensitization to HLA antigens be-
comes important in managing patients
receiving platelet transfusions when HLA anti-
bodies cause destruction of transfused plate-
lets and, especially, when patients develop
platelet transfusion refractoriness.
Immune Platelet Disorders
Platelet Transfusion Refractoriness
A less-than-expected increase in platelet count
occurs in about 20% to 70% of multitransfused
patients with thrombocytopenia.38 Those
treated for malignant hematopoietic disorders
are particularly likely to become refractory to
platelet transfusions. Responses to platelet
transfusions are often determined 10 to 60
minutes after transfusion by calculating either
a corrected platelet count increment (CCI) or a
posttransfusion platelet recovery (PPR), both
of which normalize transfusion responses for
patient blood volume and platelet dose (see
Chapter 20, Table 5). Most experts would agree
that a 1-hour posttransfusion CCI of less than
5000 to 7500 after two consecutive transfu-
sions adequately defines the refractory state.
The CCI and PPR measurements are com-
monly used to assess the success of platelet
transfusions. However, these calculations may
be misleading, particularly when smaller dos-
es of platelets are administered, as can occur
during LR transfusions. Therefore, absolute
posttransfusion platelet count increments are
more accurate than CCI or PPR.23
 CHAPTER 18 Platelet and Granulocyte Antigens and Antibodies
HLA sensitization is the most common
immune cause of refractoriness and can be di-
agnosed by demonstration of significant lev-
els of antibodies to Class I HLA in the refracto-
ry patient’s serum (see Chapter 19 for more
information on detecting HLA antibodies).
Other immune causes to be considered in-
clude antibodies to HPA, ABO incompatibility,
and antibodies in the sera of patients with
congenital platelet glycoprotein deficiencies
(eg, Glanzmann’s thrombasthenia).
Although platelet alloimmunization is
one cause of refractoriness, there are multiple
nonimmune-related reasons why transfused
platelets may not yield the expected increase
in platelet count, such as sepsis, disseminated
intravascular coagulation, and the administra-
tion of certain drugs. It cannot be stressed
enough that these nonimmune factors are
more often implicated in transfusion refracto-
riness than alloimmunization.39 Some of the
most commonly cited nonimmune factors as-
sociated with refractoriness are listed in Table
18-3. Even when possible immune causes of
refractoriness are identified, nonimmune fac-
tors are often simultaneously present.
TABLE 18-3. Some Nonimmune Causes of
Platelet Refractoriness
䡲 Massive bleeding
䡲 Fever
䡲 Sepsis
䡲 Splenomegaly (splenic sequestration)
䡲 Disseminated intravascular coagulation
䡲 Allogeneic transplantation
䡲 Poor storage of platelets before transfusion
䡲 Effects of drugs (may include immune mecha-
nisms)
䡲 Intravenous amphotericin B
䡲 Thrombotic thrombocytopenic purpura
䡲 Treatment regimen (ie, total body irradiation or
chemotherapy)
䡲 Liver dysfunction
䡲 459
Selection of Platelets for Transfusion in
Patients with Alloimmune
Refractoriness
Several strategies may be considered when se-
lecting platelets for transfusion to patients
with alloimmune refractoriness. When HLA
antibodies are present, a widely used ap-
proach is to supply apheresis platelets from
donors whose Class I HLA closely matches
those of the patient. This approach is primarily
performed using molecular methods. A disad-
vantage of relying on HLA-matched platelets is
that a pool of 1000 to 3000 or more random,
HLA-typed, potential apheresis donors is gen-
erally necessary to find sufficient HLA-com-
patible donors to support a typical patient.40
Moreover, donor selection on the basis of HLA
type can lead to the exclusion of donors whose
HLA types, although different from that of the
recipient, may still be suitable for transfusion
to the patient.
An additional concern when using HLA-
selected platelets is that requesting “HLA-
matched” platelets does not necessarily lead
to receiving HLA-identical or even well-
matched platelets. It is important to under-
stand the degree of matching that may be
provided (Table 18-4). Platelets received fol-
lowing an HLA-matched request are typically
the closest match obtainable within the con-
straints of time and donor availability. In one
study, 43% of platelets provided as HLA
matched were relatively poor Grade B or C
matches.41 In alloimmune refractory patients,
the best increases in CCI occur with the subset
of Grade A and B1U or B2U HLA-matched
platelets, but platelets mismatched for some
antigens (eg, B44, 45) that are poorly expressed
on platelets can also be successfully trans-
fused.
According to AABB Standards for Blood
Banks and Transfusion Services, HLA-matched
platelets should be irradiated to prevent
transfusion-associated graft-vs-host disease
(GVHD).42(p40) HLA-matched platelets are select-
ed to minimize incompatible antigens. There-
fore, these components are more likely to cause
GVHD than random platelets because the re-
cipient’s immune system may fail to recognize
460 䡲 AABB TECHNICAL MANUAL
TABLE 18-4. Degree of Matching for HLA-Matched Platelets
Match
Grade Description
A 4-antigen match
B1U 1 antigen unknown or blank
B1X 1 cross-reactive group
B2UX 1 antigen blank and 1 cross-reactive
C 1 mismatched antigen present
D 2 or more mismatched antigens present
R Random
the donor T lymphocytes as foreign. Treating
the component with gamma irradiation effec-
tively eliminates this risk by inactivating the
donor lymphocytes so that they cannot prolif-
erate.
An alternative approach for supplying
HLA-compatible transfusions is to determine
the specificity of the patient’s HLA antibodies
and select donors whose platelets lack the cor-
responding antigens. This antibody specificity
prediction (ASP) method is as effective as se-
lection by HLA matching or platelet cross-
matching and is superior to random selection
of platelets.43 Furthermore, many more poten-
tial HLA-typed donors are identified by the
ASP method than are available using tradition-
al HLA-matching criteria. A variation of the
ASP method involves analysis of antibody
specificities detected in sensitive flow cytome-
try or Luminex-based assays, where single
HLAs are represented on discrete and identifi-
able populations of beads.44 Reactivity of the
patient’s serum with the specific bead popula-
tions yields both the specificity and the rela-
tive strength of the antibodies. Bead popula-
tions that lack reactivity with patient serum
are used to identify antigens that could be
used in donor selection, even though they may
not be matched to the patient’s HLA type using
classic criteria for HLA matching.
In the absence of information concern-
ing which specific platelet donor HLA to avoid,
permissive HLA-mismatched antigens can
also be identified using an HLA Matchmaker
Examples of Donor Phenotypes for a
Recipient Who Is A1, 3; B8, 27
A1, 3; B8, 27
A1, –; B8, 27
A1, 3; B8, 7
A1, –; B8, 7
A1, 3; B8, 35
A1, 32; B8, 35
A2, 28; B7, 35
algorithm that is based on knowledge of
shared HLA epitopes.45 The identification of
so-called permissive HLA epitopes is modeled
on a strategy used for identifying potentially
compatible deceased donor kidney grafts for
HLA-sensitized recipients.46 This is yet another
way to expand the donor pool to provide plate-
lets that are compatible with the patient’s HLA
antibodies.
Pretransfusion crossmatching of the pa-
tient’s serum against platelets from potential
donors is an additional approach to provide
effective platelet transfusions to patients who
are alloimmune refractory. Each potential
platelet product is tested in the crossmatch as-
say with a current sample of the patient’s se-
rum. The solid-phase red cell adherence
(SPRCA) test is the most widely used method.
Good correlation between test results and
posttransfusion platelet counts have been
achieved.47 Compared with HLA matching,
crossmatching can be more convenient and
cost effective. It avoids exclusion of HLA-mis-
matched but compatible donors and has the
added advantage of facilitating the selection of
platelets when platelet-specific antibodies are
present.
Platelet crossmatching, however, will not
always be successful, particularly when pa-
tients are highly alloimmunized, which can
make finding sufficient amounts of compati-
ble platelets problematic. Although the inci-
dence of platelet-specific antibodies causing
patients to be refractory to most or all platelet
 CHAPTER 18 Platelet and Granulocyte Antigens and Antibodies
transfusions is very small, this possibility
should be investigated when most of the
crossmatches are positive or HLA-matched
transfusions fail. If platelet-specific antibodies
are present, donors of known platelet antigen
phenotype or family members, who may be
more likely to share the patient’s phenotype,
should be tested. Platelet crossmatching or
HPA genotyping should be considered for pa-
tients who do not respond to ABO- and HLA-
compatible platelets.
Fetal and Neonatal Alloimmune
Thrombocytopenia
FNAIT (also known as neonatal alloimmune
thrombocytopenia and abbreviated as “NATP,”
“NAT,” “NAIT,” or “NIT”) is a syndrome involv-
ing immune destruction of fetal platelets by
maternal antibody that is analogous to red cell
destruction in hemolytic disease of the fetus
and newborn. During pregnancy, a mother
may become sensitized to an incompatible fe-
tal platelet antigen inherited from the father of
the fetus. IgG specific for the platelet antigen
crosses the placenta, causing thrombocyto-
penia.
FNAIT is the most common cause of se-
vere fetal/neonatal thrombocytopenia, and af-
fected infants are at risk of major bleeding
complications, especially intracranial hemor-
rhage. The most commonly implicated plate-
let antigen incompatibility in FNAIT is for
HPA-1a, but all HPAs identified to date have
been implicated.48 A serologic diagnosis of
FNAIT may be made by 1) testing maternal se-
rum for platelet antibodies using assays that
can differentiate platelet-specific from non-
platelet-specific reactivity and 2) performing
platelet genotyping on parental deoxyribonu-
cleic acid (DNA).49 Demonstration of both a
platelet-specific (HPA) antibody in the mater-
nal serum and the corresponding incompati-
bility for the antigen in the parental platelet
types confirms the diagnosis.
Treatment of acutely thrombocytopenic
newborns includes the administration of in-
travenous immune globulin (IVIG) with or
without antigen-compatible platelet transfu-
sions that are sometimes provided by the
䡲 461
mother as washed platelet products.50 Once
the diagnosis of FNAIT has been made in a
family, subsequent fetuses are at risk. Antena-
tal treatment with IVIG with or without ste-
roids has proven to be an effective means of
reducing fetal thrombocytopenia and prevent-
ing intracranial hemorrhage.51 (For a more in-
depth discussion of FNAIT, see Chapter 22.)
Posttransfusion Purpura
PTP is a rare syndrome characterized by the
development of dramatic, sudden, and self-
limiting thrombocytopenia 5 to 10 days after a
blood transfusion in patients with a previous
history of sensitization by pregnancy or trans-
fusion.52 Coincident with the thrombocytope-
nia is the development of a potent platelet-
specific alloantibody, usually anti-HPA-1a, in
the patient’s serum. Other specificities have
been implicated; these are almost always asso-
ciated with antigens on GPIIb/IIIa. PTP differs
from transfusion reactions caused by red cell
antibodies in that the patient’s own antigen-
negative platelets as well as any transfused
antigen-positive platelets are destroyed. The
pathogenesis of autologous platelet destruc-
tion in PTP is not fully understood; however,
mounting evidence suggests that distinct
platelet autoantibodies transiently arise, along
with the alloantibodies, and cause both autol-
ogous and transfused antigen-negative plate-
let destruction.52 These panreactive autoanti-
bodies often target the same glycoprotein that
expresses the HPA against which alloantibod-
ies were made.
Platelet antibody assays usually reveal an
antibody in the serum with HPA-1a specificity.
Genotyping documents the absence of HPA-1a
or other platelet-specific antigens. Plasma ex-
change—once the treatment of choice for
PTP—has now largely been supplanted by
IVIG as first-line therapy. Transfusion of anti-
gen-negative platelets may be of value during
the acute phase of PTP; however, such plate-
lets have reduced in-vivo survival due to de-
struction by the aforementioned platelet auto-
antibodies.53
Following recovery, future transfusions
should be from HPA-la-negative donors if
462 䡲 AABB TECHNICAL MANUAL
possible. Washed red cells may offer some pro-
tection against recurrence; however, this is
controversial and there is at least one report of
PTP being precipitated by a frozen deglycero-
lized red cell transfusion.54 Moreover, accord-
ing to recent data reported from the Serious
Hazards of Transfusion surveillance program,
the frequency of PTP has rather dramatically
decreased coincidently with the introduction
of leukocyte-reduced blood products.55 Al-
though no data have been reported to explain
this trend, use of leukocyte-reduced products
may also be of benefit in reducing the risk of
PTP recurrence.
Drug-Induced Thrombocytopenia
Thrombocytopenia caused by drug-induced
platelet antibodies is a recognized complica-
tion of drug therapy. Drugs commonly impli-
cated include quinine, sulfa drugs, vancomy-
cin, GPIIb/IIIa antagonists, and heparin.56,57
Both drug-dependent and non-drug-depen-
dent antibodies may be produced. Non-drug-
dependent antibodies, although stimulated by
drugs, do not require the continued presence
of the drug to be reactive with platelets and are
serologically indistinguishable from other
platelet autoantibodies.
Although several mechanisms for drug-
induced antibody formation have been de-
scribed, most clinically relevant drug-depen-
dent platelet antibodies are thought to result
when a drug interacts with platelet membrane
glycoproteins, inducing conformational chang-
es recognized by drug-dependent antibod-
ies.58,59 These antibodies can cause a thrombo-
cytopenia of sudden and rapid onset that
usually resolves within 3 to 4 days after the
drug is discontinued.
Among the drug-induced immune re-
sponses to platelets, those triggered by expo-
sure to heparin have particular clinical impor-
tance because of both the widespread use of
this anticoagulant and the devastating throm-
botic complications associated with the hepa-
rin-induced thrombocytopenia (HIT) syn-
drome.60 The incidence of HIT is unknown, but
it may develop in up to 5% of patients treated
with unfractionated heparin. Low-molecular-
weight heparin is less likely to be associated
with HIT than unfractionated heparin.
A reduction in baseline platelet count by
30% to 50% occurs generally within 5 to 14
days after primary exposure to heparin and
sooner if the patient has been exposed to hep-
arin within the last 3 months. The platelet
count is often less than 100,000/µL but usually
recovers within 5 to 7 days upon discontinua-
tion of heparin. More than 50% of patients
with HIT develop thrombosis, which can occur
in the arterial, venous, or both systems.61 Pa-
tients may develop stroke, myocardial infarc-
tion, limb ischemia, deep venous thrombosis,
or ischemia of other organs. The thrombotic
complications may lead to limb amputation or
prove fatal. Because the rate of HIT-associated
thrombosis is so substantial, it is of critical im-
portance to discontinue heparin therapy when
a diagnosis of HIT is suspected. Moreover,
strong consideration should be given to using
an alternative (nonheparin) anticoagulant (eg,
a direct thrombin inhibitor) to prevent throm-
bosis.61
The mechanism of HIT includes forma-
tion of a complex between heparin and plate-
let factor 4 (PF4), a tetrameric protein released
from platelet granules. Antibodies (IgG, IgA,
and some IgM) are produced to the complex,
and IgG in the complex attaches secondarily to
platelet receptor FcRIIa via its Fc, resulting in
platelet activation with subsequent thrombin
generation. The antibody may also bind to
complexes formed at other sites, notably on
endothelial cells and monocytes. Thus, HIT
might involve activation and damage not only
of platelets but also of endothelium and
monocytes/macrophages, causing increased
susceptibility to thrombosis. This understand-
ing of the mechanism of HIT antibodies is ex-
ploited by enzyme-linked immunosorbent as-
say (ELISA) tests in which microtiter wells are
coated with the complexes of PF4 and heparin
(or heparin-like molecules) rather than with
the platelets themselves.62
 CHAPTER 18 Platelet and Granulocyte Antigens and Antibodies
Autoimmune or Immune
Thrombocytopenic Purpura
Immune thrombocytopenic purpura (ITP) is
an immune platelet disorder in which autoan-
tibodies are directed against platelet antigens,
resulting in platelet destruction. Chronic ITP,
which is most common in adults, is character-
ized by an insidious onset and moderate
thrombocytopenia that may exist for months
to years before diagnosis. Females are twice as
likely to be affected as males.
Spontaneous remissions are rare, and
treatment is usually required to raise the plate-
let count. First-line therapy consists of steroids
or IVIG followed by more potent immunosup-
pressive agents or splenectomy in nonre-
sponders. Many other therapies have been
used in patients who do not respond to sple-
nectomy, with variable results.
Chronic autoimmune thrombocytopenia
may be idiopathic or associated with other
conditions, such as human immunodeficiency
virus infection, malignancy, or other autoim-
mune diseases. Acute ITP is mainly a child-
hood disease characterized by the abrupt on-
set of severe thrombocytopenia and bleeding
symptoms, often after a viral infection. The
majority of cases resolve spontaneously over a
2- to 6-month period. If treatment is required,
IVIG or anti-D immunoglobulin infusions giv-
en to D-positive patients are usually effective
in raising platelet counts. Steroids are used
less often because of their serious side effects
in children. Splenectomy, if used, is reserved
for children whose disease is severe and lasts
longer than 6 months; this condition is similar
to chronic ITP in adults. Rituximab and vari-
ous thrombopoietin receptor agonists have
been used as second-line therapies for acute
ITP.63
Studies of both sera and washed platelets
from patients with ITP have identified autoan-
tibodies of IgG, IgM, and IgA that are reactive
with a number of platelet surface-membrane
structures, most often including GP complexes
IIb/IIIa, Ia/IIa, and Ib/IX but that can also in-
clude GPIV, GPV, and GPVI.64 In the majority of
cases, platelet-associated autoantibodies are
reactive with two or more platelet glycopro-
䡲 463
teins.65 There is no compelling evidence to
date suggesting that a patient’s profile of auto-
antibody specificities correlates with the se-
verity of the disease or predicts that patient’s
response to therapy.
Testing for Platelet Antigens and
Antibodies
Detection of platelet antibodies in the labora-
tory provides important results to aid in mak-
ing a clinical diagnosis of an immune platelet
disorder. The leaders of the International Soci-
ety of Blood Transfusion (ISBT) platelet immu-
nology workshops, designed to establish best
practices in platelet antibody and antigen test-
ing, have determined that a comprehensive
work-up for platelet antibodies requires the
use of multiple test methods, including a gly-
coprotein-specific assay, a test employing in-
tact/whole platelets, and HPA genotyping.66
Glycoprotein-specific assays are the most sen-
sitive and specific for identifying the HPA
specificity of serum antibodies (Fig 18-1). The
inclusion of assays that use intact platelet tar-
gets is critical for detection of antibodies that
can be missed by glycoprotein-specific tests
because the process of platelet lysis with
detergent and capture of GP with a specific
monoclonal antibody can disrupt HPA epit-
opes recognized by some antibodies. HPA ge-
notyping by DNA methods is helpful to con-
firm the HPA specificity of the antibodies and
for prenatal typing of a fetus in suspected cas-
es of FNAIT. The test methods that follow are
examples of the current state-of-the-art meth-
ods used by reference laboratories. For in-
depth descriptions of platelet antibody and
antigen testing, readers should consult recent
reviews.49,67,68
Assays Using Intact Platelets
An assay that is widely used for the detection
of platelet-specific antibodies for platelet
crossmatching is the SPRCA.47 Intact platelets
are immobilized in round-bottomed wells of a
microtiter plate and are then incubated with
the patient’s serum. After washing, detector
red cells coated with antihuman IgG are
464 䡲 AABB TECHNICAL MANUAL
MACE MAIPA
Enz-AHG
HPA-Aby
GP
MoAb
G-AMIgG
Microtiter Plate Well
FIGURE 18-1. Antigen capture enzyme-linked
immunosorbent assay (ELISA) testing. Modified
antigen capture ELISA (MACE) involves incubation of
a patient’s serum with target platelets, washing and
solubilization of the platelets in nonionic detergent,
and addition of lysate to micrototer plate well for
capture of platelet glycoprotein (GP) by a specific
mouse immunoglobulin G (IgG) monoclonal antibody
(MoAb). Platelet-specific antibodies [human platelet
alloantigen (HPA)-Aby] in the patient’s serum bound
to the GP are detected by the addition of an enzyme-
labeled goat antihuman IgG (Enz-AHG). The
monoclonal antibody immobilization of platelet
antigens (MAIPA) assay is very similar to the MACE,
but the patient’s serum and MoAb are incubated with
platelets before washing and platelet solubilization,
and GP/HPA-Aby complex is captured by goat
antimouse IgG (G-AMIgG) adhered to the bottom of
the well.
added, centrifuged, and examined visually.
The method’s main limitations are its subjec-
tive endpoint and failure to distinguish plate-
let-specific from non-platelet-specific anti-
bodies.
Flow cytometry is commonly used for im-
munofluorescent detection of platelet anti-
bodies utilizing intact platelets.49 Following in-
cubation of platelets with the patient’s serum,
platelet-bound antibodies are detected with a
fluorescent-labeled antiglobulin reagent that
is specific for human IgG or IgM. The results
can be expressed as a ratio of the mean or me-
dian channel fluorescence of platelets sensi-
tized with patient serum to that of platelets in-
cubated with negative control serum. Platelet
autoantibodies coating the patient’s platelets
can also be detected in a direct flow-cytometry
assay.69
Flow cytometry has proven to be a very
sensitive method for detection of antibodies to
platelets. Alloantibodies that are specific for
labile epitopes and unreliably detected by an-
tigen capture assays (ACAs) can be detected
with intact platelets using flow cytometry.8
Flow cytometry does not differentiate between
platelet-specific (ie, platelet-glycoprotein-di-
rected/HPA) and non-platelet-specific anti-
bodies (ie, HLAs or autoantibodies). This is a
drawback when suspected FNAIT or PTP is in-
vestigated because the more relevant platelet-
specific antibodies that are characteristic of
these syndromes can be obscured by non-
platelet-specific reactivity.
Antigen Capture Assays
Platelet glycoprotein ACAs are used to deter-
mine the HPA that is recognized by platelet an-
tibodies in a patient’s serum. The assays devel-
oped for this purpose include MACE and
MAIPA (Fig 18-1).49,70 The assays require the
use of monoclonal antibodies that recognize
the target antigens of interest but do not com-
pete with the patient’s antibody. These assays
capture specific platelet glycoproteins on plas-
tic wells of a microplate after sensitization
with patient’s serum. The patient’s bound anti-
body is detected with an enzyme-labeled anti-
human Ig. Because only the glycoproteins of
interest are immobilized, interference by reac-
tions from non-platelet-specific antibodies,
especially anti-HLA, is eliminated.
 CHAPTER 18 Platelet and Granulocyte Antigens and Antibodies
Platelet Genotyping
Genotyping for the SNPs in the genes encod-
ing HPA can be performed by any of the myri-
ad molecular methods available. Polymerase
chain reaction (PCR) followed by restriction
fragment length polymorphism analysis and
allele-specific PCR are two methods that have
been used successfully.67 These techniques are
reliable, but they are also laborious and time
consuming. Higher-throughput methods have
been developed, such as real-time PCR and al-
lele-specific fluorescent probes.67
Testing for Platelet Autoantibodies
Numerous assays have been developed to de-
tect platelet autoantibodies in patients with
ITP. Although many tests are quite sensitive,
particularly in detecting cell-surface, platelet-
associated Igs, none has been sufficiently spe-
cific to be particularly useful in either the diag-
nosis or management of ITP. The American
Society of Hematology practice guidelines for
ITP state that serologic testing is unnecessary,
assuming that the clinical findings are com-
patible with the diagnosis.71 However, platelet
antibody tests may be helpful in the evaluation
of patients suspected of having ITP when non-
immune causes may be present. The goal of
serologic testing in ITP is to detect autoanti-
body bound to the patient’s own platelets with
or without demonstration of similar reactivity
in the patient’s plasma.
Newer assays are designed to detect im-
munoglobulin binding to platelet-specific epi-
topes found on platelet GPIIb/IIIa, GPIa/GPIIa,
and/or GPIb/IX complexes. These solid-phase,
GP-specific assays appear to have improved
specificity in distinguishing ITP from nonim-
mune thrombocytopenia, but this benefit is
often balanced by a decrease in sensitivity.65,72
One commercially available test uses eluates
prepared from the patient’s washed platelets.65
The eluates are tested against a panel of
monoclonal-antibody-immobilized platelet
glycoprotein complexes, and platelet antibod-
ies are detected using an enzyme-linked anti-
human Ig. In the indirect phase of the assay,
patient plasma is tested against the same gly-
coprotein panel. Although autoantibodies are
䡲 465
most often detected in the eluates, they are in-
frequently detected (in approximately 17% of
cases) in the plasma. Patients with ITP may
have antibodies that are reactive with one or
several GP targets.61
Testing for Drug-Dependent Platelet
Antibodies
Any platelet serology test that is used to detect
platelet-bound Ig can be modified to detect
platelet drug-dependent antibodies. Each pa-
tient serum or plasma sample must be tested
against normal platelets in the presence and
absence of the drug. Moreover, at least one
normal control serum sample should be tested
with and without the drug to control for the
nonspecific antibody binding that might be
induced by the drug’s presence. A positive con-
trol serum sample that is known to be reactive
with the drug being assayed should be tested
with and without the drug to complete the
evaluation. A positive result shows that the se-
rum is positive (or more positive) against nor-
mal platelets in the presence of the drug vs
without the drug and that the drug did not
nonspecifically cause a positive result with
normal serum controls. Flow cytometry is the
most sensitive and most commonly used
method to detect both IgG and IgM drug-
dependent antibodies.49,73 Limitations to
detection of drug-dependent platelet antibod-
ies include that 1) for many drugs, the optimal
concentration for antibody detection has not
been determined and hydrophobic drugs are
difficult to solubilize; 2) the presence of non-
drug antibodies can mask the antibodies; and
3) a patient may be sensitized to a drug metab-
olite and not the native drug.
Assays for heparin-dependent antibodies
include the PF4 ELISA, which involves the ad-
dition of the patient’s serum diluted at a 1:50
ratio alone and in the presence of high-dose
(100 U/mL) heparin to plastic microplate wells
to which bound complexes of PF4 and heparin
or heparin-like molecules (eg, polyvinyl sulfo-
nate) are affixed.62 Heparin-dependent anti-
bodies bind to the complexes and are detected
via enzyme-conjugated antihuman Ig. An op-
tical density value, generally above 0.4, in the
466 䡲 AABB TECHNICAL MANUAL
PF4-heparin well that can be inhibited by
high-dose heparin confirms the presence of
heparin-dependent antibodies. Although IgG
antibodies are the most clinically relevant an-
tibodies, a few patients with HIT appear to
have only IgM or IgA antibodies. PF4 ELISAs
are available in two forms: those that detect
but do not differentiate IgG, IgM, and IgA hep-
arin-dependent antibodies, and those that de-
tect only IgG.
The 14C-serotonin release assay (SRA) is a
functional assay that uses washed platelets for
detection of heparin-dependent antibodies.74
Normal, fresh platelets are incubated with
14
C-serotonin, which is taken up into their
dense granules. The platelets are then ex-
posed to the patient’s serum in the presence of
low (0.1 U/mL) and high (100 U/mL) concen-
trations of heparin. Release of at least 20% of
the radioactivity at the low dose of heparin and
inhibition of this release below 20% at the high
dose confirms the presence of heparin-depen-
dent antibodies.
Other functional tests used to detect hep-
arin-dependent antibodies include the hepa-
rin-induced platelet-aggregation test and the
heparin-induced platelet-activation test. The
PF4 ELISA and the SRA are both more sensitive
and specific than the platelet-aggregation test
for the detection of heparin-dependent plate-
let antibodies in patients for whom there is a
clinical suspicion that these antibodies are
present. However, in asymptomatic patients
receiving heparin or who have not yet received
the drug, neither test is sufficiently predictive
of HIT to warrant its use in screening.75
GRA NU LO C Y T E A NT I GE N S AN D
ANTIBODIES
Antibodies against granulocyte (neutrophil)
antigens are implicated in the following clini-
cal syndromes: neonatal alloimmune neutro-
penia (NAN), transfusion-related acute lung
injury (TRALI), immune neutropenia after
HPC transplantation, refractoriness to granu-
locyte transfusion, and chronic benign auto-
immune neutropenia of infancy (AIN). To
date, nine neutrophil antigens carried on five
different glycoproteins have been character-
ized and given human neutrophil alloantigen
(HNA) designations by the Granulocyte Anti-
gen Working Party of the ISBT (Table 18-5).76
This nomenclature system follows a similar
convention to that used for HPA nomencla-
ture. Several of the antigens on granulocytes
are shared with other cells and are not granu-
locyte specific.
HNA
Antigens on FcRIIIb
The first granulocyte-specific antigen detected
was NA1, later named “HNA-1a.” Three alleles
of HNA-1 have now been identified—HNA-1a,
HNA-1b, and HNA-1c—and are located on the
protein FcRIIIb (CD16b).77 FcRIIIb is a GPI-
linked protein receptor for the Fc of IgG and is
present only on the surfaces of neutrophils.
Neutrophils express 100,000 to 200,000 mole-
cules of FcRIIIb, but there are rare individuals
(approximately 0.1%) whose neutrophils ex-
press no FcRIIIb (CD16 null) and who can
produce antibodies that are reactive with
FcRIIIb when they are exposed to it through
transfusion or pregnancy.78,79 Antibodies to
HNA-1a and -1b have been implicated in
TRALI, NAN, and AIN.77
Antigens on CD177
HNA-2 (previously known as “NB1”) is not an
alloantigen because HNA-2 antibodies are iso-
antibodies that recognize common epitopes
on CD177 protein, which is missing from the
neutrophils of immunized individuals. Neu-
trophils from approximately 3% to 5% of peo-
ple lack expression of CD177 on neutrophils.77
Lack of CD177 is thought to be caused by a
messenger ribonucleic acid splicing defect
that results in a truncated protein that cannot
be expressed.80 Interestingly, CD177 is ex-
pressed only on a subpopulation of neutro-
phils in CD177-positive individuals.81 The pro-
portion of the CD177-positive neutrophil
population ranges from 0% to 100%.82 CD177
is expressed only on neutrophils and belongs
to the Ly-6/uPAR/snake-toxin family of
proteins.81
 CHAPTER 18 Platelet and Granulocyte Antigens and Antibodies 䡲 467

TABLE 18-5. Human Neutrophil Antigens

Phenotypic Amino Acid Nucleotide

Antigen Frequency* Glycoprotein Change Encoding Gene Change

HNA-1a 12% a/a CD16 Multiple† FCGR3B Multiple†

HNA-1b 54% a/b
46% b/b
HNA-1c 5%
HNA-2 97% CD177+ CD177 N/A CD177 N/A
3% CD177-
HNA-3a 56%-59% a/a CTL2 Arg152Gln SLC44A2 455G>A
HNA-3b 34%-40% a/b
3%-6% b/b
HNA-4a 78.6% a/a CD11b Arg61His ITGAM 230G>A
HNA-4b 19.3% a/b
2.1% b/b
HNA-5a 54.3% a/a CD11a Arg766Thr ITGAL 2466G>C
HNA-5b 38.6% a/b
7.1% b/b

Nucleotide Changes Amino Acid Changes

141 147 227 266 277 349 36 38 65 78 82 106

HNA-1a G C A C G G Arg Leu Asn Ala Asp Val
HNA-1b C T G C A A Ser Leu Ser Ala Asn Ile
HNA-1c C T G A A A Ser Leu Ser Asp Asn Ile
*Phenotypic frequencies are for people of European ancestry who live in North America.
†
HNA-1 amino acid and nucleotide changes are shown in a separate section of the table.
HNA = human neutrophil antigen; N/A = not applicable.

A recent report documented cation-de- Antigens on CTL2

pendent heterophilic interactions between
CD177 and the endothelial cell membrane
protein PECAM-1(CD31), suggesting a role for
CD177 in neutrophil transendothelial migra-
tion to sites of infection.83 Antibodies against
HNA-2 have been implicated in NAN, TRALI,
and neutropenia in marrow transplant recipi-
ents.84-86
HNA-3a and HNA-3b are carried on the cho-
line transporter-like protein 2 (CTL2), and a
SNP in the gene (SLC44A2) accounts for the
polymorphism (Table 18-5).87,88 CTL2 is also
expressed on both T and B lymphocytes, and
small amounts are present on platelets. HNA-
3a antibodies are usually agglutinins. They
468 䡲 AABB TECHNICAL MANUAL
occasionally develop in women after pregnan-
cy, and HNA-3a antibodies are the most fre-
quent cause of fatal TRALI.89 HNA-3b antibod-
ies are rarely detected, but several have been
found during screening of the serum of mul-
tiparous blood donors.
Antigens on CD11a and CD11b
HNA-4a and HNA-5a, both high-prevalence
antigens, are present on monocytes and lym-
phocytes as well as granulocytes. HNA-4a is
carried on the CD11b/18 (Mac-1, CR3, m2)
glycoprotein.77 CD11b/18 plays a role in neu-
trophil adhesion to endothelial cells and
phagocytosis of C3bi opsonized microbes.
There is some evidence showing that alloanti-
bodies against HNA-4a interfere with CD11b/
18-dependent neutrophil adhesion and en-
hance neutrophil respiratory burst.90 Antibod-
ies against HNA-4a have been implicated in
NAN, and autoantibodies against CD11b/18
have also been described.91,92
HNA-5a is carried on CD11a/18 (LFA-1,
L2) glycoprotein.93 CD11a/18, like CD11b/
18, plays a role in neutrophil adhesion to en-
dothelial cells. Antibodies that are reactive
with HNA-5a have been found in a chronically
transfused patient with aplastic anemia and
have also been reported to be associated with
NAN.94 The patient who made the original
HNA-5a antibody experienced prolonged sur-
vival of an HLA nonidentical skin graft that
was attributed to the HNA-5a antibody.93
Other Neutrophil Antigens
Neutrophils do not express ABH or other red
cell group antigens, but they do express mod-
est amounts of Class I and II HLA only upon
activation.
Immune Neutrophil Disorders
Neonatal Alloimmune Neutropenia
NAN is caused by maternal antibodies against
antigens on fetal neutrophils; the most fre-
quent specificities are against HNA-1a, HNA-
1b, and HNA-2 antigens. NAN may also occur
in the children of women who lack the
FcRIIIb protein. Neutropenia in NAN can oc-
casionally be life-threatening because of in-
creased susceptibility to infection.95 Manage-
ment with antibiotics, IVIG, granulocyte
colony-stimulating growth factor, and/or plas-
ma exchange may be helpful.
TRALI
TRALI is an acute, often life-threatening reac-
tion characterized by respiratory distress,
hypo- or hypertension, and noncardiogenic
pulmonary edema that occurs within 6 hours
of a blood component transfusion.96 TRALI has
been the most common cause of transfusion-
related death for more than 10 years.97 In
TRALI, the causative antibodies are most often
found in the plasma of the blood donor. When
these antibodies are transfused, they cause ac-
tivation of primed neutrophils that are seques-
tered in the lungs of certain patients. The acti-
vated neutrophils undergo oxidative burst,
releasing toxic substances that damage pul-
monary endothelium and resulting in capillary
leak and pulmonary edema. Class I and II HLA
and HNA antibodies have all been implicated
in TRALI. However, in a recent large clinical
study of TRALI, HNA and Class II HLA anti-
bodies, but not Class I HLA antibodies, were
significantly associated with TRALI.98 (For a
more in-depth discussion of TRALI, see Chap-
ter 27.)
Autoimmune Neutropenia
Autoimmune neutropenia may occur in adults
or in infants. When present in adults, it may be
idiopathic or be secondary to such diseases as
rheumatoid arthritis or systemic lupus erythe-
matosus or to bacterial infections.99 In autoim-
mune neutropenia of infancy, usually occur-
ring in children between the ages of 6 months
and 2 years, the autoantibody has neutrophil
antigen specificity (usually HNA-1a or -1b) in
about 30% of the patients. The condition is
generally self-limiting, with recovery usually
occurring in 7 to 24 months, and is relatively
benign and manageable with antibiotics.87
 CHAPTER 18 Platelet and Granulocyte Antigens and Antibodies 䡲 469

Testing for Granulocyte Antibodies
and Antigens
Granulocyte antibody testing is technically
complex and labor intensive. The inability to
maintain the integrity of granulocytes for test-
ing that are stored at room temperature, in re-
frigerated conditions, or by cryopreservation
requires that cells be isolated from fresh blood
on each day of testing. This demands that
readily available blood donors typed for the
various granulocyte antigens be available.
Class I HLA antibodies that are often present
in patient sera complicate detection and iden-
tification of granulocyte antibodies. For these
reasons, it is critical that granulocyte antibody
and antigen testing be performed by an expe-
rienced laboratory using appropriate controls.
GRANULOCYTE AGGLUTINATION TE ST . This
was one of the first tests developed for the de-
tection of granulocyte antibodies. It is typically
performed by overnight incubation of small
volumes of isolated fresh neutrophils with the
patient’s serum in a microplate. The wells are
viewed under an inverted phase microscope
for neutrophil agglutination or aggregation.
GRA NULOCYTE IMMUNOFLUORESCENCE
TE ST . This test also requires fresh target cells
that are incubated, usually at room tempera-
ture for 30 minutes, and washed in EDTA and
phosphate-buffered saline. Neutrophil-bound
antibodies are then detected with fluorescein
isothiocyanate-labeled antihuman IgG or IgM
with either a fluorescence microscope or a
flow cytometer.89,100 A combination of aggluti-
nation and immunofluorescence tests is bene-
ficial.79,88 Other methods include chemilumi-
nescence, SPRCA, and the monoclonal
antibody immobilization of granulocyte anti-
gens (MAIGA) assay, which is similar to the
MAIPA assay, but uses monoclonal antibodies
to capture the various glycoproteins that ex-
press HNA. The MAIGA assay is used to differ-
entiate between HLA- and HNA-specific anti-
bodies.
HNA TYPING. As with HPA, typing for HNA is
largely performed using molecular methods to
detect the allelic variants that determine the
antigens. Any methods used in HPA typing can
be applied to HNA typing with simple modifi-
cations to the primer and probe sequences.
Readers are referred to several publications on
this subject.101,102 Because the splicing defect
that results in CD177 deficiency is not known,
typing for HPA-2/CD177 requires testing for
CD177 on freshly isolated neutrophils using
specific monoclonal antibodies and the granu-
locyte immuno fluorescence test.

KEY POINTS

1. Platelets express a variety of antigenic markers on their surfaces. Some of these antigens,
such as ABH and HLA, are shared with other cells, whereas HPAs are essentially platelet spe-
cific. There are currently 33 HPAs carried on six platelet glycoproteins.
2. HLA sensitization is the most common immune cause of platelet refractoriness and can be
diagnosed by the demonstration of significant levels of antibodies to HLA Class I in a
patient’s serum. When antibodies to HLA antigens are demonstrated, a widely used
treatment approach is to supply apheresis platelets from donors whose Class I HLAs are
similar to those of the patient. HLA-matched platelets should be irradiated to prevent
transfusion-associated GVHD.
3. Compared with HLA matching for platelet-refractory patients, crossmatching can be both
more convenient and cost effective. It avoids exclusion of HLA-mismatched but compatible
donors and has the added advantage of selecting platelets when the antibodies involved are
directed at platelet-specific rather than HLA antigens.
4. Although blood group antibodies (anti-A and -B) and platelet-specific antibodies can be re-
sponsible for poor responses to platelet transfusion, they are less commonly implicated in
the refractory state than HLA antibodies.
470 䡲 AABB TECHNICAL MANUAL

5. Sensitization to HPA is the most common cause of FNAIT, a syndrome involving immune
destruction of fetal platelets by maternal antibody. Platelet-specific antibodies are also in-
volved in PTP, a rare syndrome characterized by severe thrombocytopenia that occurs 5 to
10 days after a blood transfusion. The most commonly implicated antibody in both condi-
tions is anti-HPA-1a. Serologic testing using intact platelets and antigen capture assays to-
gether with HPA genotyping is used to confirm both of these diagnoses.
6. Autoantibodies directed against platelet antigens may result in ITP. Chronic ITP, which is
most common in adults, is characterized by an insidious onset and moderate thrombocyto-
penia that may be present for months to years before diagnosis. Females are twice as likely
to be affected as males. The goal of serologic testing in ITP is to detect autoantibody bound
to the patient’s own platelets.
7. Granulocyte (neutrophil) antigens are implicated in the clinical syndromes NAN, TRALI,
immune neutropenia after hematopoietic progenitor cell transplantation, refractoriness to
granulocyte transfusion, and chronic benign autoimmune neutropenia of infancy.

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 C h a p t e r 1 9

The HLA System

Robert A. Bray, PhD; Marilyn S. Pollack, PhD; and

Howard M. Gebel, PhD

T H E H L A S Y S T E M is composed of a found on the surface of platelets and, with a

complex array of genes located within
the human major histocompatibility complex
(MHC) on the short arm of chromosome 6.
Their protein products, the HLA antigens, con-
tribute to the recognition of self and nonself,
the immune responses to antigenic stimuli,
and the coordination of cellular and humoral
immunity.
HLA antigens are cell-surface glycopro-
teins that are divided into two groups (Class I
and Class II) according to their coding gene lo-
cus, function, tissue distribution, and bio-
chemistry. HLA Class I molecules contain one
copy each of two polypeptides: a heavy chain
that is an integral cell-membrane protein and
a noncovalently associated light chain called
“2-microglobulin” (the 2-microglobulin gene
resides on chromosome 15). HLA Class II mol-
ecules are composed of one copy each of an -
chain and a -chain, both of which are integral
membrane proteins. Class I molecules are
few exceptions, on all nucleated cells of the
body. Mature red cells usually lack HLA anti-
gens that are identifiable by conventional
methods, whereas nucleated immature ery-
throid cells do express HLA antigens. MHC
Class II antigen expression is typically restrict-
ed to a few cell types, including B lympho-
cytes, monocytes, macrophages, dendritic
cells, and activated T lymphocytes.
HLA molecules play a key role in antigen
presentation and the initiation of the immune
response. The HLA system is generally viewed
as second in importance only to the ABO anti-
gens in influencing the survival of transplant-
ed solid organs. In hematopoietic progenitor
cell (HPC) transplantation, the HLA system is
paramount with regard to graft rejection and
graft-vs-host disease (GVHD). HLA antigens
and antibodies are also important in compli-
cations of transfusion therapy, such as platelet
refractoriness, febrile nonhemolytic transfu-

Robert A. Bray, PhD, Professor of Pathology, and Co-director, Histocompatibility and Molecular Immunoge-
netics Laboratory, Department of Pathology, Emory University, Atlanta, Georgia; Marilyn S. Pollack, PhD, Pro-
fessor of Pathology, University of Texas Health Science Center, and Director, University Health System
Histocompatibility and Immunogenetics Laboratory, San Antonio, Texas; and Howard M. Gebel, PhD, Profes-
sor of Pathology, and Co-director, Histocompatibility and Molecular Immunogenetics Laboratory, Depart-
ment of Pathology, Emory University, Atlanta, Georgia
The authors have disclosed no conflicts of interest.

475
476 䡲 AABB TECHNICAL MANUAL
sion reactions (FNHTRs), transfusion-related
acute lung injury (TRALI), and transfusion-
associated GVHD (TA-GVHD).
Interest in HLA polymorphisms has ex-
tended beyond their role as transplantation
antigens. Studies correlating them with dis-
ease susceptibility and disease resistance be-
gan soon after serologic techniques for HLA
Class I typing were developed. Historically,
HLA antigen typing had been of value in rela-
tionship assessments and forensic investiga-
tions. However, with the development of de-
oxyribonucleic acid (DNA) typing, molecular
methods replaced the mixed leukocyte culture
(MLC) as the preferred method to select
matched donor-recipient pairs for HPC trans-
plantation. Molecular HLA testing also fos-
tered a resurgence in the investigation of dis-
ease associations that permitted the analysis
of peptide-binding restriction parameters
needed for effective vaccine development and
provided a more accurate tool for anthropo-
logic population studies. Because of the poly-
morphic nature of the HLA genes, a complex
nomenclature was developed (and is continu-
ally evolving) to define unique allele sequenc-
es based on the relationship of each allele’s
protein sequence to the serologic specificity of
the corresponding antigen.1,2
G E N E T I C S OF T HE M H C
Class I and II HLA antigens are cell-surface gly-
coproteins that are products of closely linked
genes mapped to the p21.3 band on the short
arm of chromosome 6 (Fig 19-1). That genom-
ic region is called the “MHC” and is usually in-
herited en bloc as a haplotype. Each of the
many loci has multiple alleles with codomi-
nant expression of the products from each
chromosome. With the exception of immuno-
globulin (Ig) idiotypes, the HLA system is the
most polymorphic genetic system described in
humans.
The genes HLA-A, HLA-B, and HLA-C
encode the corresponding Class I A, B, and C
antigens. The genes HL A-DRB1, -DRB3,
-DRB4, -DRB5; HLA-DQA1, -DQB1; and HLA-
DPA1, -DPB1 encode the corresponding Class
II antigens. Located between the Class I and
Class II genes is a group of non-HLA genes that
code for molecules that include the comple-
ment proteins C2, Bf, C4A, and C4B; a steroid
enzyme (21-hydroxylase); and a cytokine (tu-
mor necrosis factor). This non-HLA region is
often referred to as “MHC Class III” even
though it does not contain any HLA genes.
Organization of HLA Genetic Regions
The HLA Class I region contains (in addition to
the classical genes HLA-A, HLA-B, and HLA-C)
other gene loci designated HLA-E, HLA-F,
HLA-G, HLA-H, HFE, HLA-J, HLA-K, HLA-L,
MICA, and MICB. The latter genes encode
nonclassical, or Class Ib, HLA proteins, which
have limited polymorphism and low levels of
expression.4 Some Class Ib genes express non-
functional proteins or no proteins whatsoever.
Genes that are unable to express a functional
protein product are termed “pseudogenes”
and presumably represent an evolutionary
dead end. In contrast, other nonclassical HLA
proteins that are expressed have been associ-
ated with a variety of functions. For example,
HLA-E is associated with the surveillance sys-
tem of one subset of natural killer cells. HLA-G
is expressed by the trophoblast and may be in-
volved in the development of maternal im-
mune tolerance of the fetus. Hereditary hemo-
chromatosis (HH), an iron overload disorder
with a 10% carrier frequency in people of
Northern European ancestry, is associated
with two missense mutations in a Class I-like
gene.5 The gene that causes HH was initially
named “HLA-H”; however, the HLA-H desig-
nation had already been assigned to an HLA
Class I pseudogene by the World Health Orga-
nization (WHO) Nomenclature Committee.6
The gene that is responsible for HH is now
called “HFE.” Additional Class I-like genes that
code for molecules, such as CD1, are also lo-
cated outside the MHC. These molecules pres-
ent nonprotein antigens (such as lipids) to T
cells.
The genomic organization of the MHC
Class II (HLA-D) region is more complex. An
MHC Class II molecule consists of a noncova-
lent complex of two structurally similar
chains: the -chain and the -chain. Both of
 CHAPTER 19
The HLA System
䡲

FIGURE 19-1. The HLA complex is located on the short arm of chromosome 6. The centromere is to the top left of the figure, the telomere to the bottom right. The organi-
zation of the Class I, II, and III regions is shown.3
477
478 䡲 AABB TECHNICAL MANUAL
these chains are encoded within the MHC. The
polymorphism of HLA Class II molecules re-
sults from differences in both the -chain and
the -chain; this polymorphism depends on
the Class II isoform. For example, with HLA-
DR, the -chain is essentially monomorphic,
but the -chain is very polymorphic. Multiple
loci code for the - and -chains of the Class II
MHC proteins.
Different haplotypes have different num-
bers of Class II genes and pseudogenes. The
proteins coded by DRA1 and DRB1 result in
HLA-DR1 through HLA-DR18 antigens. The
products of DRA1 and DRB3 (if present) ex-
press HLA-DR52; those of DRA1 and DRB4 (if
present) express HLA-DR53; and those of
DRA1 and DRB5 (if present) express HLA-
DR51. The HLA-DQ1 through DQ9 antigens
are expressed on the glycoproteins coded by
DQA1 and DQB1 in the DQ cluster. Many of
the other genes of the DQ cluster are probably
pseudogenes. A similar organization is found
in the HLA-DP gene cluster.
Although not generally considered part of
the HLA system, the MHC Class III region con-
tains four complement genes with alleles that
are typically inherited together as a unit,
termed a “complotype.” More than 10 different
complotypes are inherited in humans. Two of
the Class III genes, C4A and C4B, encode for
variants of the C4 molecule and antigens of the
Chido/Rodgers blood group system. These
variants have distinct protein structures and
functions; the C4A molecule (if present) car-
ries the Rg antigen, and the C4B molecule (if
present) carries the Ch antigen. Both of these
antigens are adsorbed onto the red cells of in-
dividuals who possess the gene(s).
Patterns of Inheritance
Although MHC organization is complicated,
its inheritance follows the established princi-
ples of Mendelian genetics. Every person has
two different copies of chromosome 6 and
possesses two HLA haplotypes, one from each
parent. The expressed gene products consti-
tute the phenotype, which can be determined
by typing for HLA antigens or alleles. Because
HLA genes are autosomal and codominant,
the phenotype represents the combined ex-
pression of both haplotypes. However, to de-
fine haplotypes, parents (and possibly other
family members) must also be phenotyped to
determine which alleles are inherited together.
Figure 19-2 illustrates inheritance of haplo-
types.
Finding HLA-Identical Siblings
A child inherits one copy of chromosome 6
from each parent; hence, one MHC haplotype
is inherited from each parent. Because each
parent has two different copies of chromo-
some 6, four different combinations of haplo-
types are possible in the offspring (assuming
that no recombination occurs). The inheri-
tance pattern is important in predicting
whether family members will be compatible
donors for transplantation. The chance that
two siblings will be genotypically HLA identi-
cal is 25%. The chance that any one patient
with “n” siblings will have at least one HLA-
identical sibling is 1 – (3/4)n. Having two sib-
lings provides a 44% chance, and having three
siblings provides a 58% chance, that one sib-
ling will be HLA identical. Moreover, each time
a new sibling is tested, that new sibling has
(only) a 25% chance of being a match, no mat-
ter how many siblings have previously been
tested.
Absence of Antigens
Before the advent of molecular-based HLA
typing, the absence of an antigen in serologic
phenotyping results was attributed to homo-
zygosity at a locus (eg, inheritance of A1 from
both parents, which in reality represented only
an apparent absence of the antigen as a result
of limitations of phenotyping methods) or to a
null (nonexpressed) allele. With DNA sequenc-
ing and other molecular HLA typing methods,
homozygosity can now be presumed with a
higher degree of confidence. However, homo-
zygosity still can be proven only through fami-
ly studies or methods permitting hemizygous
typing (ie, typing of an individual haplotype).
A null allele is characterized by one or more
substitutions that are within or outside the
gene’s coding region and that prevent expres-
 CHAPTER 19
FIGURE 19-2. The designations a/b and c/d represent paternal and maternal HLA haplotypes, respectively.
Except for crossovers, the HLA complex is transmitted en bloc from parent to offspring.
sion of a functional protein at the cell surface.
Such inactivation of a gene may be caused by
nucleotide substitutions, deletions, or inser-
tions that lead to a premature cessation in the
antigen’s synthesis. In the absence of a family
study, a phenotyping study revealing a single
allele at any locus offers only presumptive evi-
dence for homozygosity. In this situation, the
allele should be listed only once because it is
unknown whether that allele is present twice
(a true homozygote) or there is another allele
not detected by the available method.
Crossovers
The genes of the HLA region occasionally
demonstrate chromosome crossover, in which
segments containing linked genetic material
are exchanged between the two chromosomes
during meiosis or gametogenesis. The recom-
binant chromosomes are then transmitted as
new haplotypes to the offspring. Crossover fre-
quency is related partly to the physical dis-
tance between the genes and partly to the re-
sistance or susceptibility of specific A, B, and
The HLA System 䡲 479
DR antigens to recombination (see below).
The HLA-A, HLA-B, and HLA-DR loci are close
together, with 0.8% crossover between the A
and B loci and 0.5% between the B and DR loci.
Crossovers between the HLA-B and HLA-C loci
or between the HLA-DR and the HLA-DQ loci
are extremely rare, whereas crossovers be-
tween the DQ and DP loci are relatively com-
mon.7,8 In family studies and relationship eval-
uations, the possibility of recombination
should always be considered.
Linkage Disequilibrium
The MHC system is so polymorphic that the
number of possible unique HLA phenotypes is
theoretically greater than that of the global hu-
man population. Moreover, new HLA alleles
are constantly being discovered and character-
ized. As of March 2014, 2579 HLA-A alleles,
3285 HLA-B alleles, 1512 DRB1 alleles, and 509
DQB1 alleles had been identified.9 However,
because HLA genes are inherited as an entire
chromosome, in reality, many HLA haplotypes
are overrepresented compared with what
480 䡲 AABB TECHNICAL MANUAL
would be expected if the distribution of HLA
genes were random. The phenomenon of link-
age disequilibrium accounts for the discrepan-
cy between expected and observed HLA hap-
lotype frequencies.
Expected frequencies for HLA haplotypes
are derived by multiplication of the frequen-
cies of each allele. For example, in individuals
of European ancestry, the overall frequency of
the gene coding for HLA-A1 is 0.15 and that for
HLA-B8 is 0.10; therefore, 1.5% (0.15 × 0.10) of
all HLA haplotypes in people of European eth-
nicity would be expected to contain genes
coding for both HLA-A1 and HLA-B8 if the
haplotypes were randomly distributed. The
actual haplotype frequency of the A1 and B8
combination, however, is 7% to 8% in that
population.
Certain allelic combinations occur with
increased frequency in different racial groups
and constitute common haplotypes in those
populations. These common haplotypes are
called “ancestral haplotypes” because they ap-
pear to be inherited from a single common an-
cestor or to be conserved within the popula-
tion because of resistance to recombination or
survival advantage. The most common ances-
tral haplotype in people of Northern European
ancestry—A1, B8, DR17 (DRB1*03:01), DQ2—
includes both Class I and Class II regions.
Some haplotypes in apparent linkage dis-
equilibrium may represent relatively young
haplotypes that have not had sufficient time to
undergo recombination, whereas some old
haplotypes are resistant to recombination be-
cause of selection or physical limitations. For
example, the A1, B8, DRB1*03:01 haplotype
appears to be resistant to recombination be-
cause that sequence of DNA has a different
length than most other comparable sequenc-
es due to the deletion of the complement gene
C4A. Linkage disequilibrium in the HLA sys-
tem is important in relationship studies be-
cause haplotype frequencies in the relevant
population make the transmission of certain
gene combinations more likely than others.
Linkage disequilibrium also affects the likeli-
hood of finding suitable unrelated donors for
HLA-matched platelet transfusions and HPC
transplantation.
B I OC HE M I ST RY, T IS S U E
DISTR IBUTION, AND
S TRUC T U R E
Characteristics of Class I and Class II
Antigens
Class I antigens (HLA-A, -B, and -C) have a
molecular weight of approximately 57,000 Dal-
tons and consist of two protein chains: a glyco-
protein heavy chain (45,000 Daltons) encoded
on the short arm of chromosome 6 and, as a
light chain, the 2-microglobulin molecule
(12,000 Daltons) encoded by a gene on chro-
mosome 15. The heavy chain penetrates the
cell membrane, whereas 2microglobulin does
not. Rather, 2microglobulin associates (non-
covalently) with the heavy chain through the
latter’s nonvariable (3) domain (see Fig 19-3).
The external portion of the heavy chain con-
sists of three amino acid domains (1, 2, and
3), of which the outermost 1 and 2 do-
mains contain the majority of polymorphic re-
gions conferring serologic HLA antigen speci-
ficity.
Class I molecules are present on platelets
and most nucleated cells in the body, with
some exceptions that include neurons, corneal
epithelial cells, trophoblasts, and germinal
cells. Only vestigial amounts remain on ma-
ture red cells, with certain allotypes better ex-
pressed than others. These Class I types were
independently recognized as red cell antigens
by serologists and designated as “Bennett-
Goodspeed” (Bg) antigens. The specificities
called “Bga,” “Bgb,” and “Bgc” are actually HLA-
B7, HLA-B17 (B57 or B58), and HLA-A28 (A68
or A69), respectively. Platelets express primari-
ly HLA-A and HLA-B antigens. HLA-C antigens
are present at very low levels, and Class II anti-
gens are generally not present.
Class II antigens (HLA-DR, -DQ, and -DP)
have a molecular weight of approximately
63,000 Daltons and consist of two structurally
similar glycoprotein chains ( and ), both of
which traverse the membrane (see Fig 19-3).
The extramembranous portion of each chain
has two amino acid domains, of which the out-
ermost one contains the variable regions of
the Class II alleles. The expression of Class II
 CHAPTER 19 The HLA System 䡲 481

FIGURE 19-3. Stylized diagram of Class I and Class II major histocompatibility complex molecules showing 
and  polypeptide chains, their structural domains, and attached carbohydrate units.

antigens is more restricted than that of Class I nor leukocytes and the duration of storage.11
antigens. Class II antigens are expressed con- Soluble HLA in blood components may be in-
stitutively on B lymphocytes, monocytes, mac- volved in the immunomodulatory effect of
rophages, dendritic cells, intestinal epitheli- blood transfusion.
um, and early hematopoietic cells. There is
also constitutive expression of Class II anti- Configuration
gens on some endothelial cells, especially
A representative three-dimensional structure
those lining the microvasculature. However, in
of Class I and Class II molecules can be ob-
general, endothelium, particularly that of larg-
tained by x-ray crystallographic analysis of pu-
er blood vessels, is negative for Class II antigen
rified HLA antigens (see Fig 19-4). The outer
expression, although Class II antigen expres-
domains, which contain the regions of amino
sion can be readily induced (for instance, by
acid variability and the antigenic epitopes of
interferon- during immune activation). Rest-
the molecules, form a structure known as the
ing T lymphocytes are negative for Class II an-
“peptide-binding groove.” Alleles that are de-
tigen expression but can become positive
fined by polymorphisms in the HLA gene se-
when activated.
quences encode unique amino acid sequences
Soluble HLA Class I and Class II antigens
and therefore form unique binding grooves,
shed from cells are present in blood and body
each of which is able to bind peptides of differ-
fluids and may play a role in modulating im-
ent sequences. The peptide-binding groove is
mune reactivity.10 Levels of soluble HLA in-
critical for the functional aspects of HLA mole-
crease with infection [including with human
cules (see “Biologic Function” section below).
immunodeficiency virus (HIV)], inflammato-
ry disease, and transplant rejection, but HLA
Nomenclature for HLA Antigens
levels decline with progression of malignancy.
Levels of soluble HLA in blood components An international committee sponsored by the
are proportional to the number of residual do- WHO establishes the nomenclature of the HLA
482 䡲 AABB TECHNICAL MANUAL
FIGURE 19-4. Ribbon diagram of HLA Class I and Class II molecule. Note the peptide in the groove of each
molecule.
system. This nomenclature is updated regular-
ly to incorporate new HLA alleles.2 HLA anti-
gens are designated by a number following the
letter that denotes the HLA series (eg, HLA-A1
or HLA-B8). Previously, antigenic specificities
that were not fully confirmed carried the prefix
“w” (eg, HLA-Aw33) for “workshop.” When the
antigen’s identification became definitive, the
WHO Nomenclature Committee dropped the
“w” from the designation. (The Committee
meets regularly to update nomenclature by
recognizing new specificities or genetic loci.)
The “w” prefix is no longer applied in this
manner and is now used only for the following:
1) Bw4 and Bw6, to distinguish such “public”
antigens (see “‘Public’ Antigens” section be-
low) from other B locus alleles; 2) all serologi-
cally defined C locus specificities, to avoid
confusion with components of the comple-
ment system; and 3) Dw specificities that were
defined by mixed leukocyte reactions but are
now known to be caused by HLA-DR, HLA-DQ,
and HLA-DP polymorphisms. The numeric
designations for the HLA-A and HLA-B speci-
ficities were assigned according to the order of
their discovery.
Splits and Cross-Reactive Groups
Refinement of serologic methods permitted
antigens that were previously believed to rep-
resent a single specificity to be “split” into
specificities that were serologically (and, later,
genetically) distinct. The designation for an in-
dividual antigen that was split from an earlier
recognized antigen often includes the number
of the parent antigen in parentheses [eg, HLA-
B44 (12)].
In addition to “splits,” certain apparently
distinct HLA antigens may have other epitopes
in common. Antibodies that are reactive with
these shared determinants often cause cross-
reactions in serologic testing. The collective
term for a group of HLA antigens that exhibit
such cross-reactivity is “cross-reactive group”
(CREG).
“Public” Antigens
In addition to splits and CREGs, HLA proteins
have reactivity that is common to many differ-
ent HLA specificities. Called “public” antigens,
these common amino acid sequences appear
to represent the less variable portion of the
HLA molecule. Two well-characterized public
antigens, HLA-Bw4 and HLA-Bw6, are present
in almost all HLA-B molecules.12 The HLA-A
locus molecules A23, A24, A25, and A32 also
have a Bw4-like epitope.
Public antigens are clinically important
because patients exposed to them through
pregnancy, transfusion, or transplantation can
 CHAPTER 19
make antibodies to these antigens if the pa-
tients do not express the epitopes themselves.
A single antibody, when directed against a
public antigen, can resemble multiple discrete
alloantibodies, and this has significant conse-
quences for identifying compatible donors for
transplantation and platelet transfusion.
Nomenclature for HLA Alleles
Because nucleotide sequencing is used to in-
vestigate the HLA system, increasing numbers
of HLA alleles are being identified, many of
which share a common serologic phenotype.
The minimum requirement for designation of
a new allele is the sequence of exons 2 and 3
for HLA Class I and exon 2 for HLA Class II
(DRB1). These exons encode the variable ami-
no acids that confer HLA antigen specificity.
A uniform nomenclature has been adopt-
ed that takes into account the locus, major se-
rologic specificity, and allele group deter-
mined by molecular typing techniques. For
example, although many alleles have been se-
quenced only for exon 2, nucleotide sequenc-
ing has identified at least 165 unique amino
acid sequence variants (alleles) of HLA-DR4 as
of March 2014. The first HLA-DR4 variant is
designated “DRB1*04:01,” indicating the locus
(DR), protein (1 chain), major serologic spec-
ificity (04 for HLA-DR4), and sequence 2 varia-
tion allele number (variant 01). The asterisk
indicates that an allele name follows (and that
TABLE 19-1. HLA Nomenclature 2013
Antigen
Species Locus Equivalent Allele
HLA DRB1 * 04 : 01 :
Examples:
DR4 - Serology
DRB1*04:xx - Serologic Equivalent
DRB1*04:02 - Allele
DRB1*04:01:01; DRB1*04:01:02 - Silent Mutations
A*02:15N; DRB4*01:03:01:02N - Null Alleles (exon, intron)
A*24:02:01:02L
B*44:02:01:02 - - Expression Modifiers
B*32:11Q
The HLA System 䡲 483
the typing was determined by molecular tech-
niques).
A similar system is used for naming Class
I alleles. The name of the locus—for example,
“HLA-B”—is followed by an asterisk and then
by several digits separated by colons. The first
two digits correspond to the antigen’s serologic
specificity in most cases. The next group of
digits makes up the code for a unique amino
acid sequence in exons 2 and 3, with numbers
being assigned in the order in which the
DNA sequences were determined. Therefore,
B*27:04 represents the HLA-B locus, has a se-
rologic specificity of B27, and was the fourth
unique sequence 2 and 3 allele described in
this family (see Table 19-1). A third “place” in
the allele name is added for alleles that differ
only by synonymous (“silent”) nucleotide sub-
stitutions in exons 2 and 3 for Class I or in exon
2 for Class II. For example, A*01:01:02 differs
from A*01:01:01 only in that the codon for iso-
leucine in position 142 is ATT instead of ATC. A
fourth “place” in the allele name can be added
for alleles that differ only in sequences within
introns or in 3 or 5 untranslated regions. Fi-
nally, the nomenclature accommodates al-
leles with null or low expression or other char-
acteristics by the addition of an “N” or “L,”
respectively, or another letter as appropriate to
the end of the allele name. The other official
expression modifiers are as follows: S (secret-
ed, not on cell surface), Q (expression level
Silent Outside Expression
Mutation Exon Modifier
01 : 01:02 N,L,S,Q
484 䡲 AABB TECHNICAL MANUAL
questionable), A (unknown but aberrant ex-
pression, perhaps null), and C (cytoplasmic
expression only). The last two have not been
used to date.
Biologic Function
The essential function of the HLA system is
self/nonself discrimination, which is accom-
plished by the interaction of T lymphocytes
with peptide antigens presented by HLA pro-
teins. T lymphocytes interact with peptide an-
tigens only when the T-cell receptor (TCR) for
antigen engages both an HLA molecule and
the antigenic peptide contained within the
TCR’s peptide-binding groove. This limitation
is referred to as “MHC restriction.”13
In the thymus, T lymphocytes with TCRs
that bind to a self HLA molecule are selected
(positive selection), with the exception of
those with TCRs that also bind to a peptide de-
rived from a self-antigen, in which case the T
lymphocytes are deleted (negative selection).
Some self-reactive T cells escape negative se-
lection. If not functionally inactivated (for in-
stance, by the mechanism of anergy), such
self-reactive T cells may become involved in
an autoimmune process.
Role of Class I Molecules
Class I molecules are synthesized, and peptide
antigens are inserted into the peptide-binding
groove, in the endoplasmic reticulum. Peptide
antigens that fit into the Class I peptide-bind-
ing groove are typically eight or nine amino ac-
ids in length and are derived from proteins
made by the cell (endogenous proteins). Such
endogenous proteins—which may be normal
self-proteins; altered self-proteins, such as
those in cancer cells; or viral proteins, such as
those in virus-infected cells—are degraded in
the cytosol by a large multifunctional protease
(LMP) and are transported to the endoplasmic
reticulum by a transporter associated with an-
tigen processing (TAP). The LMP and TAP
genes are both localized to the MHC.
Class I molecules are transported to the
cell surface, where the molecules are available
to interact with CD8-positive T lymphocytes. If
the TCR of a CD8 cell can bind the antigenic
peptide in the context of the specific Class I
molecule displaying it, then TCR binding acti-
vates the cytotoxic properties of the T cell,
which attacks the cell, characteristically elicit-
ing an inflammatory response. The presenta-
tion of antigen by Class I molecules is especial-
ly important in host defense against viral
pathogens and malignant transformation. Tu-
mor cells that do not express Class I antigens
escape this immune surveillance.
Role of Class II Molecules
Like Class I molecules, Class II molecules are
synthesized in the endoplasmic reticulum, but
peptide antigens are not inserted into the pep-
tide-binding groove there. Instead, an invari-
ant chain (Ii) is inserted as a place holder. The
Class II-invariant chain complex is transport-
ed to an endosome, where the invariant chain
is removed by a specialized Class II molecule
called “DM.” The DM locus is also localized to
the MHC. A Class II antigenic peptide is then
inserted into the peptide-binding groove.
Peptide antigens that fit into the Class II
peptide-binding groove are typically 12 to 25
amino acids in length and are derived from
proteins that are taken up by the cell through
endocytosis (of exogenous proteins). Exoge-
nous proteins, which may be normal self-pro-
teins or proteins derived from pathogens, such
as bacteria, are degraded to peptides by en-
zymes in the endosomal pathway. Class II mol-
ecules are then transported to the cell surface,
where the molecules are available to interact
with CD4-positive T lymphocytes, which se-
crete immunostimulatory cytokines in re-
sponse. That mechanism is especially impor-
tant for the production of antibodies.
I D E NT I F I C AT IO N O F HL A
ANTIGENS AND ALLELES
Methods for the identification of HLA antigens
and alleles fall into two categories: 1) molecu-
lar (DNA based) and 2) serologic (antibody
based). Historically, cell-based assays were
also used.
Detailed procedures for commonly used
assays are provided in the ASHI Laboratory
 CHAPTER 19 The HLA System 䡲 485

Manual from the American Society for Histo-
compatibility and Immunogenetics.14 De-
pending on the clinical situation, a particular
HLA antigen/allele detection or typing meth-
od may be preferable (see Table 19-2).
DR16), whereas high-resolution typing distin-
guishes individual alleles (eg, DRB1*01:01:01
from DRB1*01:02:01). Several PCR-based
methods have been developed; three general
approaches are described below.

DNA-Based Assays OLIGONUCLEOTIDE PROBE S. This method

DNA-based typing has several advantages over
serologic assays: 1) high sensitivity and speci-
ficity, 2) use of small sample volumes, and 3)
no need for cell-surface-antigen expression or
cell viability. Although serologic methods can
distinguish among a limited number of
HLA specificities, high-resolution DNA-based
methods have the capability to identify all
known alleles.
Polymerase Chain Reaction Testing
Polymerase chain reaction (PCR) technology
allows amplification of large quantities of a
particular target segment of genomic DNA.
Low- to intermediate-resolution typing de-
tects the HLA serologic equivalents with great
accuracy (eg, it distinguishes DR15 from
for establishing HLA genotypes features ar-
rays of oligonucleotide probes that have been
individually attached to a solid-phase matrix—
for example, each probe is attached to a differ-
ent microbead or well of an enzyme-linked
immunosorbent assay (ELISA) plate. DNA for
an entire locus is then amplified and the bind-
ing to different probes is evaluated. The micro-
bead array assay is a sequence-specific oligo-
nucleotide probe method for HLA Class I and
Class II low-to-high-resolution, DNA-based,
tissue typing. This method allows high
throughput with a reduction in sample pro-
cessing time.15
SEQUENCE-SPECIFIC PRI MERS. A second
major technique uses sequence-specific prim-
er (SSP) pairs that target and amplify a particu-

TABLE 19-2. HLA Typing Methods and Appropriate Applications

Method Clinical Application Resolution

Microlymphocytotoxicity Solid-organ transplantation, evaluation Serologic specificity

SSP (PCR)
Forward SSOP Hybridization
Reverse SSOP Hybridization
DNA Sequencing
SSP = sequence-specific primer; PCR = polymerase chain reaction; HPC = hematopoietic progenitor cell; SSOP = sequence-
specific oligonucleotide probe.
of platelet refractoriness, HLA typing
(Class I only) of platelet recipients and
platelet donors
Solid-organ, related and unrelated Serologic to allele level, higher
HPC transplantation resolution with large number of
primers
Solid-organ and HPC transplantation Serologic to allele level
(can accommodate high-volume
testing)
Solid-organ, related and unrelated Serologic, higher resolution with
HPC transplantation larger number of probes
Unrelated HPC transplantation, resolu- Allele level
tion of typing problems with other
methods, characterization of new alleles
486 䡲 AABB TECHNICAL MANUAL
lar DNA sequence.16 The sequence-specific
method requires the performance of multiple
PCR assays in which each reaction is specific
for a particular allele or group of alleles. The
amplified alleles are directly visualized after
agarose gel electrophoresis. Because SSPs have
such specific targets, the amplified material
indicates the presence of the allele or alleles
that have that sequence. The pattern of
positive and negative PCR amplifications is
examined to determine the actual HLA
allele(s) present. Primer pair sets are commer-
cially available that can determine a complete
HLA-A, -B, -C, -DR, -DQA1, -DQB1, and -DPB1
allele-level phenotype.
SEQUENCE-BASED T YPING. High-resolution
nucleic acid sequencing of HLA alleles gener-
ates allele-level sequences. Sequence-based
typing (SBT) can be used to characterize new
allele(s). Although SBT is considered the “gold
standard” for HLA typing, ambiguities often
occur when two different base pairs at the
same position can result in two different possi-
ble combinations of alleles. These ambiguities
occur because SBT evaluates both maternal
and paternal HLA genes (haplotypes) simulta-
neously, and which haplotype to assign each
base pair to is not always certain. New tech-
niques for high-resolution SBT allow separa-
tion of HLA haplotypes before sequencing,
thereby avoiding such ambiguities.
Serologic (Lymphocytotoxicity) Assays
The microlymphocytotoxicity test can be used
to detect HLA-A, -B, -C, -DR, and -DQ anti-
gens. Lymphocytes are used for testing be-
cause they are readily obtained from anticoag-
ulated peripheral blood and are a more
reliable target than granulocytes. Lymphocytes
obtained from lymph nodes or spleen may
also be used. HLA-typing sera are obtained
primarily from multiparous women. Some
mouse antihuman HLA monoclonal antisera
are also available.
HLA sera of known specificities are placed
in different wells of a microdroplet test plate. A
suspension of lymphocytes is added to each
well. Rabbit complement is then added; if suf-
ficient antibody has bound to the lymphocyte
membranes, the complement cascade is acti-
vated through the membrane attack complex,
leading to lymphocytotoxicity. Damage to the
cell membrane can be detected by the addi-
tion of dye. Cells that have no attached anti-
body, activated complement, or damage to the
membrane keep the vital dyes from penetrat-
ing; however, cells with damaged membranes
allow the dye to enter. The cells are examined
for dye exclusion or uptake under phase con-
trast microscopy. If a fluorescent microscope is
available, fluorescent vital dyes can also be
used.
Because HLA-Class II antigens (DR, DQ,
and DP) are expressed on B cells and not on
resting T cells, typing for these antigens usual-
ly requires manipulation of the initial lympho-
cyte preparation to yield an enriched B-cell
population before testing.
The interpretation of serologic reactions
requires skill and experience. The extreme
polymorphic nature of the HLA system; varia-
tion in antigen frequencies among different
racial groups; reliance on biologic antisera and
living target cells; and complexities introduced
by splits, CREGs, and “public” antigens all con-
tribute to difficulties in accurate serologic HLA
typing. Given these problems, most US labora-
tories now rely primarily on DNA-based meth-
ods for HLA typing. However, although the
more common “null alleles,” such as DRB4*01:
03:01:02N and C*04:09N, can now be identified
by routine molecular typing methods, rare null
alleles may not be as easily identified. It is im-
portant to stay current on the ever-changing
array of expressed and nonexpressed HLA
polymorphisms. A very useful resource is the
International Immunogenetics Project’s IMGT/
HLA Database.2,9
Cellular Assays
Historically, the MLC [also called “mixed leu-
kocyte reaction” (MLR)] and primed lympho-
cyte typing (PLT) assays were used to detect
genetic differences in the Class II region. In the
MLC, mononuclear cells from different indi-
viduals are cultured together; the ability of one
population (the responder) to recognize the
 CHAPTER 19
HLA-D (combined DR, DQ, and DP) antigens/
alleles of the other (the target) as foreign can
be detected by measuring the proliferation of
the responders. In PLT, reagent cells previously
stimulated by specific Class II mismatched
types allow the identification of those types by
their accelerated proliferative responses to
stimulator cells sharing the original mis-
matches.
These classical cellular assays have been
largely replaced by molecular typing methods
for HLA antigens. However, these assays are
still used in some laboratories to monitor im-
mune function, assess relative functional
compatibility, or select a partially mismatched
unrelated donor for HPC transplantation.
CROSSM ATCHING AND
DE TE CT ION OF HL A
ANTIBODIES
Serologic Assays
The same microlymphocytotoxicity testing
used for serologic HLA typing can be used to
test serum specimens for antibodies against
selected target cells. This type of testing is
referred to as “lymphocyte crossmatching.”
Crossmatching consists of incubating serum
from a potential recipient with lymphocytes
(unfractioned or separated into T and B lym-
phocytes) from prospective donors. A varia-
tion of the microlymphocytotoxicity test uses
an antiglobulin reagent, which increases assay
sensitivity. Flow cytometry is yet another
crossmatch method with even greater sensitiv-
ity than the antiglobulin-enhanced cross-
match.
Testing the patient’s serum against a pan-
el of 30 to 60 (or more) different target cells
was historically used to assess the extent of
HLA alloimmunization. A positive result was
target cell death. The percentage of panel cells
that died after reacting with the cytotoxic anti-
bodies in patient serum was referred to as the
“panel-reactive antibody” (PRA) level in that
patient. Determination of cytotoxic PRA was
(and may still be) useful for following patients
awaiting deceased-donor solid-organ trans-
plantation, conducting the work-up of pa-
The HLA System 䡲 487
tients for platelet refractoriness, and investi-
gating FNHTR or TRALI cases. The PRA “HLA
antibody screen” not only detects the presence
of cytotoxic HLA antibodies but can often also
identify the specificity of those antibodies.
Although they have long been the gold
standard for antibody identification, comple-
ment-dependent cytotoxic assays are not opti-
mal tests. Their sensitivity and specificity are
marginal, but more importantly, most cytotox-
ic assays cannot identify all of the possible
HLA antibody specificities in highly sensitized
patients whose cells are reactive with 80% or
more of test panel cells or in patients with
Class II antibodies.
Solid-Phase Assays
The current approach to identify HLA antibod-
ies (especially for highly sensitized solid-organ
transplant candidates) relies on the use of
beads or microparticles (ie, solid-phase meth-
odology) coated either with clusters of HLA
Class I or Class II antigens (ie, an HLA pheno-
type) or with recombinantly expressed, indi-
vidually purified HLA antigens (single antigen
beads).17 Antibody binding is detected by
staining with a fluorescently labeled antihu-
man globulin (AHG). Flow cytometry and
microarray methods are more sensitive than
lymphocytotoxicity or ELISA testing and focus
on the detection of IgG antibodies. The use of
single-antigen bead assays are of particular
importance for highly sensitized patients
where multiple HLA antibody specificities
cannot be reliably distinguished and identified
with either cell-based cytotoxic assays or sol-
id-phase assays using clusters of HLA mole-
cules.
The clinical significance of the low-level
antibodies that are detectable by the more
sensitive solid-phase assays and that may not
cause a positive cytotoxicity or flow-cytomet-
ric crossmatch is controversial. Newer adapta-
tions of the solid-phase technology can deter-
mine whether these antibodies do or do not fix
complement. However, use of this method is
also controversial because the significance of
noncomplement-fixing antibodies is also un-
known.
488 䡲 AABB TECHNICAL MANUAL
TH E H L A S Y S T EM A N D
TR ANSF USI O N
HLA system antigens and antibodies play im-
portant roles in a number of transfusion-relat-
ed events, including platelet refractoriness,
FNHTRs, TRALI, and TA-GVHD. HLA antigens
are highly immunogenic. In response to preg-
nancy, transfusion, or transplantation, immu-
nologically competent individuals are more
likely to form antibodies to HLA antigens than
to any other antigens.
Platelet Refractoriness
The incidence of HLA alloimmunization and
platelet refractoriness among patients receiv-
ing repeated transfusions of cellular blood
components ranges from 20% to 71%.18 The re-
fractory state exists when a transfusion of suit-
ably preserved platelets fails to increase the re-
cipient’s platelet count. Platelet refractoriness
may be caused by clinical factors, such as
sepsis, high fever, disseminated intravascular
coagulopathy, medications, hypersplenism,
complement-mediated destruction, or a com-
bination of these factors; alternatively, it may
have an immune basis.
Antibody Development
Antibodies against HLA Class I antigens are a
common cause of immune-mediated platelet
refractoriness, but antibodies to platelet-spe-
cific or ABH antigens may also be involved.
HLA alloimmunization can follow pregnancy,
transfusion, or organ transplantation because
the foreign antigens are the donor MHC anti-
gens themselves. A common example is the
development of HLA antibodies directed
against Class I and Class II antigens that oc-
curs with platelet transfusion, even though
platelets express only Class I antigens. It is
likely that the presence of donor leukocytes
(bearing Class I and II antigens) elicits alloim-
munization. The likelihood of HLA immuniza-
tion can be lessened (but not eliminated) by
transfusion of leukocyte-reduced blood com-
ponents.
The threshold level of leukocytes required
to provoke a primary HLA alloimmune re-
sponse is unclear and probably varies among
different recipients. Some studies have sug-
gested that 5 × 106 leukocytes per transfusion
may represent an immunizing dose. In pa-
tients who have been sensitized by prior trans-
plantation, pregnancy, or transfusion, expo-
sure to even lower numbers of allogeneic cells
is likely to provoke an anamnestic antibody re-
sponse.
Identifying Compatible Donors
The HLA antibody response of transfused indi-
viduals may be directed against individual
specificities or public alloantigens. Precise
characterization may be difficult and is best
accomplished using a single-antigen, solid-
phase assay as described in the “Solid-Phase
Assays” section above. Platelet-refractory pa-
tients with broadly reactive antibodies may be
difficult to support with platelet transfusions.
HLA-matched platelets, obtained by platelet-
pheresis, benefit some, but not all, of those re-
fractory patients. Donors who are phenotypi-
cally matched with the immunized recipient
for four Class I antigens (two alleles each at the
HLA-A and HLA-B loci) are difficult to find,
and strategies to obtain HLA-compatible
platelets vary. Although selection of partially
mismatched donors (based on serologic
CREGs) has been emphasized, such donations
may fail to provide an adequate transfusion re-
sponse in vivo.
An alternative approach to the selection
of donors is based on matching for immuno-
genic epitopes as described by the “HLA
Matchmaker” program developed at the Uni-
versity of Pittsburgh. HLA Matchmaker is used
to consider epitopes on all the patient’s HLA
Class I (and if indicated, Class II) molecules re-
gardless of which allele encodes them. HLA
Matchmaker is an excellent tool to predict
compatibility between a specific donor-recipi-
ent pair, but, unfortunately, the tool does not
generate accurate predictions 100% of the
time. Use of single-antigen beads or microar-
rays to identify HLA antibody specificities can
also help improve the selection of donors with
acceptable mismatched antigens.19
 CHAPTER 19
Approaches such as HLA Matchmaker
can be referred to as “pull” approaches, where-
in donors are selected (or pulled into) the pro-
cess. In contrast, antibody specificity identifi-
cation strategies can be considered “push”
approaches that exclude (push away) inappro-
priate donors. Either approach has a similar
net effect: identification of donors who have
the greatest likelihood of optimizing post-
transfusion platelet counts.
As an alternative approach, HLA-alloim-
munized patients often respond to cross-
match-compatible platelets selected by using
patient serum and samples of apheresis plate-
lets in a platelet antibody assay. Crossmatch-
ing techniques may assess compatibility for
both HLA and platelet-specific antibodies.20
Histocompatible platelet components are dis-
cussed further in Chapter 18.
Febrile Nonhemolytic Transfusion
Reactions
HLA, granulocyte, and platelet-specific anti-
bodies have been implicated in the pathogen-
esis of FNHTRs. The recipient’s antibodies, re-
acting with transfused antigens, elicit the
release of cytokines (eg, interleukin-1) that are
capable of causing fever. Serologic investiga-
tion, if undertaken, may require multiple tech-
niques and target cells from a number of dif-
ferent donors (see Chapter 27).
TRALI
In TRALI (a potentially fatal transfusion reac-
tion that is recognized with increasing fre-
quency), acute noncardiogenic pulmonary
edema develops in response to transfusion
(see Chapter 27). The pathogenesis of TRALI
appears to reflect the presence of HLA or neu-
trophil antibodies in donor blood. Studies
have shown that up to one-third of blood com-
ponents can contain detectable amounts of
HLA antibodies.21 If present, such antibodies
can be reactive with and fix complement to the
recipient’s granulocytes, leading to severe cap-
illary leakage and pulmonary edema. Rarely,
the recipient’s HLA antibodies are reactive
with transfused leukocytes from the donor.
The HLA System 䡲 489
Cases of TRALI have been reported that
appear to be caused by donor antibodies
against Class I or Class II antigens in recipi-
ents. Because Class II antigens are not ex-
pressed on resting neutrophils, an alternate
explanation for activation of neutrophils is re-
quired in these instances. One hypothesis is
that Class II antigens on the recipient’s pulmo-
nary macrophages are targeted by the comple-
ment-activating antibodies. The subsequent
release of cytokines and chemokines results in
the recruitment and activation of neutrophils
in the lungs.22 Yet another explanation is that
activated neutrophils, which can express HLA
Class II antigens in response to a patient’s in-
flammatory state, could be further activated
with the binding of donor anti HLA Class II an-
tibodies, resulting in TRALI.
Chimerism and TA-GVHD
“Chimerism” refers to the presence of two cell
populations, such as transfused or transplant-
ed donor cells in a recipient, in an individual.
Persistent chimerism after blood transfusion
may lead to the development of TA-GVHD in
the recipient. The development of TA-GVHD
depends on the following factors: 1) the degree
to which the recipient is immunocompro-
mised, 2) the number and viability of lympho-
cytes in the transfused component, and 3) the
degree of HLA similarity between the donor
and recipient. The development of TA-GVHD
with the use of fresh blood components from
blood relatives has highlighted the pathogenic
role of the HLA system.
Figure 19-5 illustrates the conditions for
increased risk of TA-GVHD. The parents have
one HLA haplotype in common. Each child,
therefore, has one chance in four of inheriting
the same haplotype from each parent, and
child 1 is homozygous for the shared parental
HLA haplotype. Transfusion of blood from
child 1 to an unrelated recipient with different
haplotypes would have no untoward conse-
quences. If, however, child 1 were a directed
donor for a relative who was heterozygous for
that haplotype (eg, one of the parents or child
3), the recipient’s body would fail to recognize
the antigens on the transfused lymphocytes as
490 䡲 AABB TECHNICAL MANUAL

FIGURE 19-5. HLA haplotypes in a family at risk for transfusion-associated graft-vs-host disease (GVHD). In
contrast to the family shown in Fig 19-2, each parent shares a common HLA haplotype, HLA-A1,B8,DR17. Child
1 is homozygous for the haplotype shared by the parents and by child 3. The lymphocytes of child 1 are capable
of producing posttransfusion GVHD if they are transfused to either parent or to child 3.

foreign and would not eliminate them. The
donor cells would recognize the recipient’s
other haplotype as foreign and would become
activated, proliferate, and attack the host.
To avoid this situation, it is recommended
that all cellular components from blood rela-
tives be irradiated before transfusion. Other
specially chosen donor units, including HLA-
matched platelets, may also present an in-
creased risk of TA-GVHD. Rarely, TA-GVHD
has occurred after the transfusion of blood
from an unrelated donor, usually within popu-
lations with relatively limited genetic diversi-
ty, such as in Japan.
Chimerism is proposed to be responsible
for the maintenance of tolerance in some or-
gan transplant recipients as well as for the
maintenance of HLA sensitization.23,24 It has
been postulated that scleroderma is a form of
GVHD resulting from chimeric cells derived
from fetal cells transferred across the placenta
during pregnancy.25 Furthermore, the persis-
tence of donor lymphocytes originally present
in and transplanted with a solid-organ al-
lograft has been documented to cause fatal
GVHD in recipients of these organs.26
Hemolytic Transfusion Reactions
HLA incompatibility has rarely been implicat-
ed in shortened red cell survival in patients
with antibodies to HLA antigens, such as Bga
(B7), Bgb (B17-B57 or B58), and Bgc (A28-A68 or
A69). These antigens are expressed, although
weakly, on red cells. Such incompatibility may
not be detected by conventional pretransfu-
sion testing.
 CHAPTER 19
HL A TESTING AND
TR A N SP L AN TAT I O N
HLA testing is an integral part of solid-organ
and HPC transplantation. The extent of testing
differs depending on the type of transplanta-
tion (see Chapters 29 and 30).
Hematopoietic Progenitor Cell
Transplants
It has long been recognized that disparity
within the HLA system is an important barrier
to successful HPC transplantation.27 HLA simi-
larity and compatibility between the donor
and the recipient are required for engraftment
and to help prevent GVHD. However, some de-
gree of rejection or GVHD is a common prob-
lem for recipients of allogeneic HPCs, despite
immunosuppressive conditioning.
Candidate donors and recipients are
typed for their HLA-A, -B, -C, -DR, and -DQ al-
leles and, in some cases, for their HLA-DP al-
leles. The goal of HLA typing is to match the al-
leles of the prospective donor and recipient at
the HLA-A, -B, -C, and -DRB1 loci.28 Some
transplant programs also attempt to match
donors and recipients for HLA-DQ alleles,
HLA-DP alleles, or both.
Although HLA-identical sibling donors
remain the best choice for HPC transplanta-
tion, there is increasing use of unrelated do-
nors identified by searching the files of more
than 10 million HPC donors listed in the
National Marrow Donor Program’s registry of
volunteer donors or other international regis-
tries. The use of umbilical cord HPCs and HPC
grafts from mismatched donors that have
undergone T-cell depletion may allow an
increased number of donor-recipient mis-
matches.29,30
Kidney Transplants
ABO compatibility is the most important fac-
tor in determining the immediate outcomes of
kidney transplants. Because ABH antigens are
expressed in varying amounts on all of the
body’s cells, transplanted ABO-incompatible
tissue comes into continuous contact with the
recipient’s ABO antibodies. Of particular im-
The HLA System 䡲 491
portance is the expression of ABH antigens on
vascular endothelial cells because the vascular
supply in the transplant is a common site for
rejection. Currently, the use of non-A1 A blood
group organs for group B and group O recipi-
ents with low anti-A blood group titers has be-
come acceptable.31,32 In fact, several transplan-
tation programs have used ABO-incompatible
donors with higher anti-A titers following pro-
tocols that include various combinations of
rituximab, splenectomy, plasmapheresis with
intravenous Ig, and other treatments to re-
move preexisting antibodies and promote ac-
commodation of the transplanted organ.
Both recipients and donors are routinely
typed for ABO and HLA-A, -B, and -DR anti-
gens. HLA-C and -DQ typing is usually also
performed. Before surgery, a crossmatch be-
tween recipient serum and donor lymphocytes
is required. The ASHI Standards for Accredited
Laboratories requires that the crossmatch be
performed using a method that is more sensi-
tive than routine microlymphocytotoxicity
testing, such as prolonged incubation, wash-
ing, augmentation with AHG reagents, or flow
cytometry.33 Flow cytometry is the most sensi-
tive method and has been credited with pre-
dicting early acute rejection and delayed graft
function, both of which are strong predictors
of chronic rejection (if results are positive) and
long-term allograft survival (if results are nega-
tive).34
In a study of patients undergoing de-
ceased-donor kidney retransplantation, the 7-
year graft survival rate using a negative T-cell
flow crossmatch to select the donor kidney
was comparable to that of patients undergoing
primary deceased-donor transplantation (68%
vs 72%) and was significantly better than that
of regraft patients for whom only the antiglob-
ulin lymphocytotoxicity crossmatch was used
(45%).35
Because HLA antibody responses are dy-
namic, the serum used for the crossmatch is
often obtained within 48 hours of surgery for
sensitized potential recipients and is retained
in the frozen state for any required subsequent
testing. An incompatible crossmatch with un-
fractionated or T lymphocytes is typically a
contraindication to kidney transplantation. A
492 䡲 AABB TECHNICAL MANUAL
positive B-cell crossmatch is significant when
caused by donor-specific HLA Class I or Class
II antibodies.
Serum from a patient awaiting deceased-
donor kidney transplant surgery is tested at
regular intervals for the degree of alloimmuni-
zation by determining the percent PRA as well
as the specificities of the detected antibodies.
If an antibody with a defined HLA specificity is
identified in a recipient, a common practice is
to avoid donors who express the correspond-
ing HLA antigen(s). Such antigens are deemed
“unacceptable.” A more recent approach is the
use of solid-phase assays to identify HLA anti-
bodies and unacceptable antigens and then
calculate the patient’s PRA (cPRA) using a da-
tabase of more than 12,000 HLA-typed do-
nors.36 Frozen serum samples used for period-
ic PRA testing are often stored so that
“historic” samples with the highest PRA can be
used in addition to the preoperative sample
for pretransplantation crossmatching.
Prospective crossmatching is often not
performed for recipients who are conclusively
devoid of HLA antibodies (ie, cPRA = 0%).
Prompt transplantation with reduced cold-
ischemia time for the renal allograft may pro-
vide greater benefit to the patient than pro-
spective crossmatching, provided that 1) a
very sensitive method for antibody detection,
such as flow cytometry or microarrays, has
been used, and 2) it is certain that the patient
has had no additional sensitizing event (ie, im-
munizations or transfusions in the 2 weeks be-
fore or at any time after that serum was
screened).37
The approach to kidney transplants with
living donors is different. In the past, when
several prospective living donors were being
considered, MLC testing of recipients and do-
nors was sometimes performed, but this is
rarely (if ever) the case today. HLA matching of
recipients with kidney donors (both living and
deceased) contributes to long-term allograft
survival by decreasing the likelihood of chron-
ic rejection. According to the Scientific Regis-
try for Transplant Recipients, recent 1-year
graft survival rates from living and deceased
renal donors were 96.5% and 91.7%, respec-
tively, and the half-lives of living-donor and
deceased-donor renal allografts were 21.6
years and 13.8 years, respectively.38
The significantly better graft survival rate
for recipients of living- vs deceased-donor re-
nal allografts, even when donors and recipi-
ents are completely unrelated, coupled with
inadequate numbers of deceased-organ do-
nors has led to a relatively new practice, kid-
ney paired donation (KPD).39 Recently, KPDs
have been facilitated through local and na-
tional registries, which permit patients with
ABO- or HLA-incompatible potential living
donors to exchange these donors for the do-
nors of other patients in the same situation. As
a simple example, a blood group A transplant
candidate with an incompatible blood group B
potential living kidney donor could exchange
that donor for the incompatible blood group A
living kidney donor of a blood group B trans-
plant candidate. Patients with HLA-incompat-
ible potential donors have similar possibilities
for donor exchange, and multiple “pairs” can
be involved in one continuous exchange
(chain) process.
The introduction of altruistic donors (ie,
individuals who choose to donate a kidney
without having a specific intended recipient)
can significantly expand KPD options. Briefly,
the altruistic donor donates a kidney to a pa-
tient with an incompatible potential living do-
nor who then donates to a different recipient
with an incompatible donor, starting a “chain”
with the possibility of a large number of living-
donor transplants. One chain of 10 trans-
plants was recently reported.40 Currently, there
is a movement to establish a national KPD pro-
gram.
Other Solid-Organ Transplants
For liver, heart, lung, and heart/lung trans-
plants, ABO compatibility remains the prima-
ry immunologic concern for donor selection,
and determining pretransplant ABO compati-
bility between the donor and recipient is re-
quired. However, it has been shown that young
pediatric heart or liver transplant recipients,
who have low levels of ABO isoagglutinins,
have successful outcomes with ABO-incom-
patible hearts or livers.41,42 Although it is not a
 CHAPTER 19 The HLA System 䡲 493

requirement, HLA typing of potential recipi- TABLE 19-3. HLA-Associated Diseases

ents of the above organs is recommended.
Furthermore, a crossmatch should be avail- Disease HLA RR43-46
able before transplantation when the recipient
Celiac disease DQ2 >250
has demonstrated presensitization, except for
emergency situations. Although a degree of Ankylosing spondylitis B27 >150
HLA compatibility correlates with graft surviv- Narcolepsy DQ6 >38
al after heart, lung, small-intestine, and liver
transplantations, prospective HLA matching is Subacute thyroiditis B35 14
generally not performed for these proce- Type 1 diabetes DQ8 14
dures.43 Pancreas transplantation generally
Multiple sclerosis DR15, DQ6 12
follows the same guidelines as kidney trans-
plantation. Rheumatoid arthritis DR4 9
Juvenile rheumatoid DR8 8
Relationship and Other Forensic arthritis
Testing
Grave disease DR17 4
HLA typing (particularly DNA-based HLA typ-
RR = Relative Risk
ing) has been useful in forensic testing. Al-
though rarely used now for relationship test-
ing, DNA-based HLA typing alone can exclude
more than 90% of falsely accused males. Hap- susceptibilities have several features in com-
lotype frequencies, rather than gene frequen- mon. The susceptibilities are known or sus-
cies, are used in such calculations because pected to be inherited, display a clinical
linkage disequilibrium is very common in the course with acute exacerbations and remis-
HLA system. It is important, however, to con- sions, and usually have characteristics of auto-
sider the racial differences that exist in HLA immune disorders. Furthermore, their exact
haplotype frequencies in the calculations cause is often unknown.
used; the possibility of recombination events Evidence has been accumulating that im-
should also be considered. plicates the HLA molecules themselves, rather
More commonly used DNA-based assays than linked genes, in most cases of disease
for relationship testing and forensic detection susceptibility. One mechanism that could lead
of polymorphisms between individuals are to the association of HLA phenotype and dis-
employed to measure variations in the num- ease is the presence of a Class I or Class II het-
ber of short tandem repeats, which are used to erodimer encoded by a specific allele that pref-
assess other polymorphic, non-HLA genetic erentially presents autoantigens to the TCR.
regions, and single nucleotide polymorphisms The ancestral haplotype HLA-A1, B8, DR17
(SNPs). DNA-based assays, including HLA typ- (DRB1*03:01), DQ2, discussed in the “Linkage
ing, allow identification of individuals on the Disequilibrium” section above, is associated
basis of extremely small samples of fluid or tis- with susceptibility to Type I diabetes, systemic
sue, such as hair, epithelial cells, or semen. lupus erythematosus, celiac disease, common
variable immunodeficiency, IgA deficiency,
and myasthenia gravis.47 This haplotype is also
OTH E R C L I NI C A L LY
associated with an accelerated course of HIV
SI G NI F I C A N T A SPE C TS OF H L A
infection that is likely caused by the presence
For some conditions, especially those believed of multiple genes.
to have an autoimmune etiology, an associa- However, HLA typing has only limited val-
tion exists between HLA phenotype and the ue in assessing the risk of most diseases be-
occurrence of, or resistance to, clinical disease cause the association is incomplete. The asso-
(see Table 19-3).43-46 HLA-associated disease ciation of HLA-B27 and ankylosing spondylitis
494 䡲 AABB TECHNICAL MANUAL
in patients of European ancestry is instructive.
The test is highly sensitive; more than 90% of
patients with ankylosing spondylitis possess
the HLA-B27 antigen. Conversely, the test’s
specificity is low; only 20% of individuals with
the B27 antigen develop ankylosing spondyli-
tis. A second condition, narcolepsy, is strongly
associated with the HLA allele DQB1*06:02.48
As with HLA-B27 and ankylosing spondylitis,
more than 90% of individuals with narcolepsy
are positive for HLA-DQB1*06:02, but only a
minority of individuals with that HLA allele
develop the disease. For some autoimmune
diseases, the specific peptide that might trig-
ger the autoimmune response has been at
least tentatively identified: a gluten peptide,
gliadin, for celiac disease; cyclic citrullinated
peptides for rheumatoid arthritis; and a pep-
tide from glutamic acid decarboxylase for type
I diabetes.49-51 Resistance to cerebral malaria
seems to result from a strong cytotoxic T-cell
response to particular malarial peptides that
are restricted by (ie, fit into the peptide-bind-
ing grooves of) two specific HLA molecules.52
A similar peptide-binding specificity is
important to consider in the development of
vaccines. For example, a vaccine to enhance
immune responses to melanoma using a mel-
anoma-specific peptide that binds only to the
cells of individuals with the HLA type HLA-
A*0201 was selected for development because
A*02:01 is the most common allele in virtually
all populations.53
HLA typing is also important in pharma-
cogenomic applications. For example, the
presence of certain HLA alleles confers in-
creased risk for several hypersensitivity reac-
KEY POINTS
1. Genes encoded by the major histocompatibility complex (or HLA complex in humans) are
critical components of the immune system and play a major role in distinguishing self from
nonself.
2. HLA genes are located within multiple loci on chromosome 6. Each locus is extremely poly-
morphic.
3. HLA genes encode multiple Class I (eg, HLA-A, -B, and -C) and Class II (eg, HLA-DR, -DQ,
and -DP) cell-surface proteins.
4. Class I proteins are expressed ubiquitously, but Class II proteins have restricted tissue distri-
bution.
tions to certain drugs: HLA-B*57:01 confers
sensitivity to abacavir, HLA-B*15:02 to carba-
mazepine, and HLA-B*58:01 to allopurinol.54.55
The degree of association between a given
HLA type and a disease is often described in
terms of relative risk (RR), which is a measure
of how much more frequently a disease occurs
in individuals with a specific HLA type than in
individuals not having that HLA type. Calcula-
tion of RR is usually based on the cross-prod-
uct ratio of a 2 × 2 contingency table. However,
because the HLA system is so polymorphic,
there is an increased possibility of finding an
association between an HLA antigen and a dis-
ease by chance alone. Therefore, calculating
RRs for HLA disease associations is more com-
plex and is typically accomplished by use of
Haldane’s modification of Woolf’s formula.56,57
The RR values for some diseases associated
with HLA types are shown in Table 19-3.
SUMMARY
In conclusion, the HLA system is a complex
and highly polymorphic set of genes that are
collectively involved in all aspects of the im-
mune response. The recent development of
molecular tools to explore this genetic oasis is
providing additional information, such as the
elucidation of unrecognized polymorphisms
within the HLA complex (ie, SNPs). In the fu-
ture, the translation of this basic information
will undoubtedly lead to new clinical applica-
tions in transplantation, autoimmune diseas-
es, vaccine development, pharmacogenetics,
and infectious diseases.
 CHAPTER 19 The HLA System 䡲 495

5. Everyone inherits a set of HLA genes from his or her mother and father, referred to as a “ma-
ternal haplotype” and “paternal haplotype,” respectively.
6. Together, the maternal and paternal haplotypes are referred to as a “genotype.” The cell-sur-
face expression of proteins encoded by these HLA genes is referred to as a “phenotype.”
7. Class I and Class II HLA proteins are strongly immunogenic and can induce an immune re-
sponse, eg, formation of HLA antibodies.
8. Donor-directed HLA antibodies are associated with graft dysfunction and/or loss.
9. Solid-phase assays (eg, flow cytometry and Luminex) have become the gold standard for de-
tecting and identifying HLA antibodies.
10. Identification of donor-directed HLA antibodies can be used to perform a virtual (in-silico)
crossmatch.

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pathological diseases. Immunol Rev 1999;167:
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48. Pelin Z, Guilleminault C, Risch N, et al.
HLADQB1*0602 homozygosity increases rela-
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 C h a p t e r 2 0

Hemotherapy Decisions
and Their Outcomes

Theresa Nester, MD; Shweta Jain, MD; and Jessica Poisson, MD

A S W I T H O T H E R medical interven- example, a patient with acute anemia requires

tions, transfusion offers both benefits
and risks that must be balanced for each pa-
tient. Although the myriad situations in which
transfusion might be considered prevent the
promulgation of “one-size-fits-all” transfu-
sion guidelines, the accumulating data in the
medical literature can certainly help clinicians
make hemotherapy decisions based on in-
creasingly sound evidence.
This chapter provides a review of the cur-
rent literature on the indications for, and out-
comes of, transfusion of blood components.
When health-care providers use this chapter in
combination with information presented else-
where in the Technical Manual on the content
of blood components and the risks they pose,
they should have sufficient data to form evi-
dence-based decisions about hemotherapy.
RED CE LL TRA NS FUS I O N
At first glance, the decision to transfuse a Red
Blood Cell (RBC) unit appears to be easy; for
an RBC transfusion to restore the body to its
baseline state. For the trauma patient with
massive injuries, death will ensue without a
transfusion. For the critically ill patient with
anemia stemming from a variety of causes, it
would seem reasonable to maintain adequate
oxygen stores in the tissues through transfu-
sion.
Yet the decision to transfuse a stored RBC
unit has become more complex in the last de-
cade, with substantial evidence suggesting
that RBC transfusion may be detrimental to
some patients. For example, a sentinel 1999
study randomly assigned critically ill patients
(who are among the most frequent recipients
of RBC products) to a restrictive or liberal
transfusion strategy (thresholds of hemoglo-
bin = 7 vs 10 g/dL).1 The lower threshold re-
duced morbidity and mortality significantly
and was not associated with increased length
of stay or morbidity in patients with heart dis-
ease or with prolonged ventilation for those re-
quiring a respirator. The authors concluded

Theresa Nester, MD, Associate Medical Director, Puget Sound Blood Center, and Associate Professor of Labo-
ratory Medicine, University of Washington, Seattle, Washington; Shweta Jain, MD, Transfusion Medicine Fel-
low, Puget Sound Blood Center, Seattle, Washington; and Jessica Poisson, MD, Director of Transfusion
Services, USC Medical Center, Los Angeles, California
The authors have disclosed no conflicts of interest.

499
500 䡲 AABB TECHNICAL MANUAL
that a hemoglobin threshold of 7 g/dL was ap-
propriate for all critically ill patients, except
perhaps those with unstable cardiac condi-
tions. Similarly, a 2008 systematic review of the
critical care literature determined that in most
cohort studies in 272,586 patients, RBC trans-
fusions were an independent predictor of
death, infection, multiorgan dysfunction, and
acute respiratory distress syndrome.2
Most randomized, controlled trials (RCTs)
conducted to date have demonstrated that a
restrictive RBC transfusion strategy is not infe-
rior to a liberal transfusion strategy. A recent
RCT that evaluated transfusion strategies in
patients with severe upper gastrointestinal
bleeding showed a superior outcome with a
restrictive strategy for patients with cirrhosis
or Child-Pugh Class A or B disease.3 Because of
such evidence, many studies now compare the
risk of anemia to that of transfusion and the
degree to which each risk affects clinical out-
comes.
The appropriate RBC transfusion thresh-
old has become a central point of discussion.
The hemoglobin level in any individual patient
only tells a portion of the story and, by itself,
does not reflect the compensatory mecha-
nisms used to respond to the anemia. Stable
tissue oxygenation may be maintained by
increased cardiac output (depending on the
coronary arteries’ ability to dilate) and in-
creased oxygen extraction, which result in low-
er venous oxygen content.4 Clinicians are ap-
propriately looking for ways to monitor a
patient’s response to anemia before deciding
to administer a transfusion. At the same time,
hospital-utilization and blood-management
committees look for guidance on the levels of
hemoglobin, below which to consider transfu-
sion in a given patient population.
Parallel to the RCTs performed thus far is
the observation that patients without evidence
of cardiovascular disease who refuse transfu-
sion on religious grounds and undergo elective
surgery tolerate hemoglobin reductions of 6 to
7 g/dL with only a minimal rise in periopera-
tive mortality risk.5
Although many otherwise-healthy adults
can tolerate significant anemia equivalent to a
halving of their red cell mass, comorbidities in
many patients may limit their reserve. So what
should the indication be for RBC transfusion
in the setting of acute anemia? A recent meta-
analysis of 19 RCTs evaluated the effects of dif-
ferent RBC transfusion thresholds on clinical
outcomes between 1956 to 2011, a period
during which the definitions of liberal vs re-
strictive RBC transfusion strategies varied.6,7
Of the 6264 patients in these studies, most
were in surgical or intensive care units. The
lower transfusion threshold (hemoglobin =
7.0-10.0 g/dL, and most commonly 7.0-8.0 g/dL)
was not inferior to the higher hemoglobin level
(9.0-13.3 g/dL, and most commonly 9.5-10.0
g/dL). In the lower threshold groups, fewer
RBC units were transfused without an adverse
impact on mortality, morbidity, or time to
functional recovery. There were also no signifi-
cant differences in the incidence of major
complications, such as stroke, pulmonary ede-
ma, or infection. Caution must be used in ex-
tending these findings to all patient popula-
tions because some high-risk groups (eg,
patients with acute brain injury or renal fail-
ure) were not adequately represented.
Although the findings of this meta-analy-
sis suggest that a restrictive transfusion strate-
gy is appropriate for many hospitalized, stable
patients, many clinicians still pause at the
thought of withholding transfusion from a pa-
tient with coronary artery disease. In the past,
large observational studies gave conflicting re-
sults regarding whether a higher or lower red
cell transfusion threshold was beneficial.8-10
More recently, an RCT known as “FOCUS” in-
cluded 2016 patients (average age = 82 years)
with risk factors for, or a history of, cardiovas-
cular disease who required hip surgery.11 The
results showed that the restrictive strategy (he-
moglobin <8 g/dL) was not inferior to a liberal
transfusion strategy (hemoglobin <10 g/dL)
with respect to mortality, morbidity, or func-
tion. The primary outcome of death or inabili-
ty to walk independently across the room at 60
days was similar in the two groups (34.7% in
the restrictive vs 35.2% in the liberal groups).
Rates of in-hospital cardiac events or death as
well as other complications were also similar.
The 1999 multicenter Transfusion Re-
quirements in Critical Care (TRICC) trial in 838
 CHAPTER 20
critically ill patients, including 326 with ath-
erosclerotic disease but not acute, unstable
myocardial conditions showed that mortality
at 30 days was no different in the subgroup
with a primary or secondary diagnosis of car-
diac disease between the two strategies
(hemoglobin <7 g/dL vs <10 g/dL).1 Cardiac
events (pulmonary edema and myocardial in-
farction) were more frequent in the liberal
strategy group, although there were no signifi-
cant differences in rates of these events during
the 48 hours prior to death in the patients who
died. The subgroup analysis showed a trend
toward increased mortality in patients with
ischemic heart disease in the restrictive arm,
leading the investigators to suggest that pa-
tients with active coronary ischemic syn-
dromes may need a higher transfusion thresh-
old than 7 g/dL.
The FOCUS and TRICC trials account for
60% of the available data on the risk of myo-
cardial infarction following restrictive vs liber-
al RBC transfusion strategies in patients who
have coronary artery disease. An analysis of
combined data from these trials and six small-
er trials did not find an elevated risk [risk ratio
(RR) = 0.88; 95% confidence interval (CI) =
0.38, 2.04] for myocardial infarction using a re-
strictive strategy.12 The statistical power of the
combined data was sufficient to detect moder-
ate to large differences in risk of myocardial in-
farction, but the analysis could have missed a
twofold higher risk of myocardial infarction
with a restrictive transfusion strategy. In light
of the available data, the Clinical Transfusion
Medicine Committee of the AABB published
guidelines suggesting that transfusion should
be considered for hospitalized patients with
preexisting cardiovascular disease who have
symptoms or a hemoglobin level of 8 g/dL or
less.12
For patients with acute coronary syn-
drome, no clinical trials have compared re-
strictive and liberal transfusion strategies. For
this reason, the development of a guideline
based on hemoglobin levels is difficult for pa-
tients with this diagnosis.
The most appropriate hemoglobin
threshold for transfusion is a patient-specific,
and even situation-specific, parameter. A he-
Hemotherapy Decisions 䡲 501
moglobin level <6 g/dL almost always requires
a transfusion, whereas a level >10 g/dL rarely
does so. Of course, most patients have a hemo-
globin level between these limits, and many
have one or more comorbidities that affect
their tolerance of anemia. The assemblage of
experience and knowledge must be blended
into the art of medicine to address this need.
In addition to symptoms, research continues
to identify consistent signs, such as venous ox-
ygen saturation, that reflect how an individual
patient is tolerating anemia.13
Transfusion in Patients with
Hemorrhagic Shock
A brief history of the changes in transfusion
support over the last 40 years for patients ex-
periencing hemorrhagic shock can be re-
viewed elsewhere.14 Currently, in the civilian
setting, such patients represent 2% to 3% of all
trauma admissions. Signs of acute blood loss
in such patients include tachycardia, hypoten-
sion, increased respiratory rate, and mental
status changes (Table 20-1). When these
symptoms are not addressed quickly, the mor-
tality rate in patients experiencing hemorrhag-
ic shock ranges from 40% to 70%.
Before 1995, aggressive resuscitation us-
ing crystalloid and RBCs was the standard of
TABLE 20-1. Signs and Symptoms of Acute
Blood Loss
Tachycardia
Palpitations
Cooling of extremities
Pallor
Hypotension
Reduced arterial pressure
Reduced central venous (jugular) pressure
Acidosis
Increased respiratory rate
Decline in urinary output
Mental status changes
502 䡲 AABB TECHNICAL MANUAL
care in civilian hospitals. In the late 1990s,
trauma surgeons started to recognize the po-
tential deleterious effects of too much crystal-
loid, including increased risk of acute respira-
tory distress syndrome, multiple-organ failure,
and abdominal compartment syndrome.
Gradually, and after new data became avail-
able from the military experience supporting
combat casualties in the Iraq and Afghanistan
wars, a new approach to hemorrhagic shock,
damage control resuscitation, was adopted.
This approach included the early transfusion
of plasma and platelets in addition to RBCs
while minimizing crystalloid use. More em-
phasis was also placed on addressing the lethal
triad of acidosis, hypothermia, and coagulopa-
thy early in patient resuscitation.
Since publication in 2007 of a seminal
study involving 252 military personnel with
combat casualties and using almost a 1:1 ratio
of plasma to RBCs, many publications from ci-
vilian hospitals have echoed the idea that us-
ing an increased ratio of plasma or platelets to
RBCs can improve outcomes.15 In 2012, the
Prospective, Observational, Multi-Center Mas-
sive Transfusion Study evaluated 905 patients
in 10 Level I US civilian trauma centers who
survived for 30 minutes after admission and
received at least 1 RBC unit within the first 6
hours and at least three other blood products
within 24 hours of admission.16 An increased
ratio of plasma to RBCs or platelets to RBCs
was independently associated with decreased
6-hour mortality. Furthermore, patients with
plasma to red cell ratios less than 1:2 during
the first 6 hours were three to four times more
likely to die than patients with ratios of 1:1 or
higher.
The Army Surgeon General has estab-
lished a clinical policy requiring transfusions
of Fresh Frozen Plasma (FFP), platelets, and
RBCs in a 1:1:1 ratio for patients injured in
combat who require a massive transfusion of 6
whole blood platelet units or 1 apheresis plate-
let unit following transfusion of 6 RBC units.
This emphasis on addressing coagulopathy
early in a massive resuscitation pushes labora-
torians to develop faster ways of evaluating co-
agulation results to provide optimal transfu-
sion support.17 The benefit of cryoprecipitate
infusion for low fibrinogen levels, although not
widely emphasized in these protocols, has also
been established.18
Transfusion in Patients with Chronic
Anemia
Transfusion is much less commonly indicated
when anemia has persisted for weeks or
months because compensatory mechanisms
have had time to work than in patients with
anemia of more recent onset. These longer-
term anemias are usually best treated by ad-
dressing their etiologies, such as providing
supplementation to treat a nutritional defi-
ciency (eg, of iron) or reducing the rate of au-
toimmune hemolysis. Congenital hemoglo-
binopathies, such as sickle cell disease, are
treated according to disease-related protocols
for purposes that are not necessarily related to
oxygen delivery. Hypoproliferative anemias
secondary to chemotherapy or end-stage renal
disease are often treated with marrow stimu-
lants, such as recombinant erythropoietin.
Any of these diseases could conceivably re-
quire a transfusion if patient symptomatology
requires more rapid reversal than would be
possible by treating the underlying mecha-
nisms, but transfusion is usually considered
only as a last resort in these patients.
Some patients become transfusion de-
pendent because of their inability to create
and maintain an adequate red cell mass. These
patients often “declare” the hemoglobin at
which their symptoms are best controlled. The
symptomatology reported by patients with
chronic anemia does not generally correlate
well with laboratory values in different pa-
tients but often corresponds well with these
values in an individual over time.
Selected Clinical Issues
Use of Whole Blood
Whole blood provides oxygen-carrying capaci-
ty, stable coagulation factors (concentrations
of Factors V and VIII decrease during storage),
and blood volume expansion. Thus, it is po-
tentially useful for patients with concomitant
red cell and volume deficits, such as actively
 CHAPTER 20
bleeding patients, and helps support coagula-
tion if coagulation factor consumption is the
primary cause of coagulopathy.19 However,
whole blood is rarely available for allogeneic
transfusion. Component therapy has evolved
as an improved way to expand the blood com-
ponent supply to meet the demand. Whole
blood is mostly commonly used in the United
States today for autologous transfusion. The
ABO type of transfused whole blood must be
identical to that of the recipient.
Duration of Storage
With the rapid disappearance of 2,3-diphos-
phoglycerate (DPG) from stored red cells and
the concomitant increase in hemoglobin’s af-
finity for O2, a concern is that RBC units stored
for more than 1 to 2 weeks may not provide the
intended increased availability of O2 at the tis-
sue level, at least for the first 12 to 24 hours af-
ter transfusion.20,21 Beyond 7 to 10 days of stor-
age, the P50 of hemoglobin decreases from 27
to 16 mmHg, markedly shifting the dissocia-
tion curve to the left.
There are other known changes to red
cells that occur with storage over time, some-
times referred to collectively as the “storage le-
sion.” These changes include shape changes,
such as decreased deformability of the red cell,
and decreased adenosine triphosphate. In ad-
dition, a loss of nitric oxide occurs within a few
hours after collection.22 Whether these chang-
es are clinically important is unclear.
The corollary question regarding whether
the age of blood matters for clinical outcomes
is an important and controversial one. A re-
cent meta-analysis that evaluated both obser-
vational studies and RCTs in which death was
the primary outcome showed that transfu-
sions of older RBC units (>21 days old) were
associated with a significantly increased risk of
death [odds ratio (OR) = 1.16, 95% CI = 1.07,
1.24]. The majority of studies in this analysis
were observational, and the three RCTs ana-
lyzed had only small numbers of patients.
Therefore, the conclusion that transfusions of
older blood components caused the increased
mortality is weakened by the confounding and
unintentional bias that arises with observa-
Hemotherapy Decisions 䡲 503
tional studies.23,24 At least one of the studies
was difficult to interpret because of clinical
differences in the two groups of patients.25 The
authors of an earlier meta-analysis that fo-
cused on homogeneous subgroups concluded
that the available data were inadequate to
prove causality between older stored blood
components and increased morbidity and
mortality.26
Three larger RCTs have been undertaken
to clarify the issue. The Age of Red Blood Cells
in Premature Infants trial included 377 very
low-birthweight premature infants requiring
transfusion. Transfusions of fresh RBC units
(average age = 5.1 days) vs standard-issue RBC
units (average age = 14.6 days) were compared,
with rates of major neonatal morbidities as the
primary outcome measure and the rate of no-
socomial infection as a secondary outcome. In
this study, there were no clinically meaningful
or statistically significant differences in either
outcome.27 The Red Cell Storage Duration
Study randomly assigned patients aged at least
12 years who required complex cardiac sur-
gery to receive RBCs less than 11 days old or
greater than 20 days old throughout their peri-
operative course.28 The primary outcome mea-
sure is an assessment of clinical outcomes us-
ing the Multiple Organ Dysfunction Score. One
ongoing RCT, the Age of Blood Evaluation
study, involves critically ill patients who re-
quire transfusion; this prospective random-
ized trial will compare all-cause mortality at 90
days in patients receiving RBCs less than 8
days old vs standard-issue RBC units.29
Certainly, if the storage lesion is detri-
mental to a patient’s clinical course, research
must determine whether and, if so, why cer-
tain patient populations are more affected
than others. With the tenuous balance that al-
ready exists between the safety and supply of
this human-derived resource, having fewer
blood components available to support pa-
tients may prove more detrimental than hav-
ing stored blood components available. Deter-
mining a better way to store RBCs—one that
prevents the storage lesion—seems to be a
prudent approach. Opportunities to improve
RBC storage, prevent cell damage, and
504 䡲 AABB TECHNICAL MANUAL
improve the preservation of 2,3-DPG continue
to be pursued.30
ABO Matching
Although matching patient and donor ABO
types ensures compatibility in that blood
group system, inventory considerations may
suggest that an ABO-compatible rather than
an ABO-identical RBC unit should be trans-
fused (Table 20-2). Although a compatible but
nonidentical RBC unit introduces some isoag-
glutinins directed against the recipient’s A
and/or B antigens, the small amount of plas-
ma in an additive system or packed RBC unit
(or even multiple units) is insufficient to cause
hemolysis.
Components containing larger amounts
of plasma, such as platelets (see below) or
whole blood units, raise the potential for iso-
agglutinins to cause hemolysis. Whole blood,
when used, would be transfused to an ABO-
identical recipient because of this issue. In the
setting of ABO-incompatible heart transplan-
tation in an infant, the transfusion service may
be asked to plasma-reduce or wash an RBC
unit, largely because the original protocol
called for this modification rather than be-
cause solid evidence indicates that incompati-
ble passive ABO isoagglutinins can impair a
new graft.31
Leukocyte Reduction
The most appropriate use of leukocyte reduc-
tion remains controversial. Ardent proponents
on both sides argue about whether all cellular
TABLE 20-2. Selection of ABO-Compatible Red
Blood Cell Units
Recipient Blood Compatible Red Blood
Group Cell Units*
A A, O
B B, O
AB AB, A, B, O
O O
*Red Blood Cells prepared as additive system or “packed”
units.
components should have their white cells
reduced.32 Although initially introduced in
several European countries to decrease the
transmission of prions, universal leukocyte re-
duction is most often used to reduce the risk of
postoperative infection and improve post-
transfusion survival by reducing transfusion-
related immunomodulation (TRIM).
The benefit of removing leukocytes for
patients who will be multitransfused and/or
transplanted is clearly evident in terms of re-
duced rates of alloimmunization, episodes of
refractoriness to platelet transfusion, and fe-
brile reactions.33-36 However, universal imple-
mentation of leukocyte reduction may not
cause a measureable drop in febrile reaction
rates, given the proportion of patients who are
susceptible to these reactions.37 A positive
clinical impact of universal leukocyte reduc-
tion has been difficult to identify and was not
seen in the only large-scale, prospective RCT
of its implementation.38 Even in more defined
situations, such as cardiac surgery, prospective
trials have reached contradictory conclusions,
including when they were performed by the
same research team.39-42 The many retrospec-
tive analyses ascribing benefits to leukocyte
reduction have often been limited by con-
founders.43,44 However, some support strong
arguments that removing leukocytes does lead
to reduced lengths of stay, reduced infection
rates, and improved outcomes.45,46 Other po-
tential concerns associated with the presence
of leukocytes in blood components, such as
increased red cell adhesive properties, raise
new questions about their impact.47 For a thor-
ough examination of this complex subject, the
reader is referred to several excellent meta-
analyses and other publications.48-51
Many studies have addressed the possi-
bility that leukocyte reduction can reduce the
incidence of clinical outcomes due to TRIM,
but the results have been contradictory.50 One
hypothesis was that the savings resulting from
reduced immunomodulation could offset the
costs of leukocyte reduction, but this was not
demonstrable in a large prospective RCT.42
Nonetheless, several countries maintain a leu-
kocyte-reduced blood supply, and the need for
leukocyte reduction remains controversial.43
 CHAPTER 20
The value of leukocyte reduction as a
means of preventing cytomegalovirus (CMV)
transmission has been well documented.52,53
Studies using polymerase chain reaction (PCR)
indicate that the highest risk of CMV transmis-
sion through blood transfusion may be from
recently seroconverted donors rather than do-
nors who have been seropositive for more
than a year.54,55 This information is interesting
but difficult to translate into an operational
policy. Because of a 1% to 2% risk of “break-
through” transmission with either seronega-
tive or leukoreduced components, patients at
high risk of disseminated CMV infection
should be actively monitored for evidence of
CMV using antigenemia assays or reverse-
transcription PCR techniques.56
The leukocyte content of units varies by
component type (Table 20-3).57,58 Current fed-
eral guidelines and AABB Standards for Blood
Banks and Transfusion Services define a leuko-
cyte-reduced component as one with <5 × 106
residual donor leukocytes per final product
(including RBCs; apheresis platelets, and
pooled platelets).59,60(p26) The AABB Standards
calls for <8.3 × 105 residual leukocytes in plate-
lets prepared from a single unit of whole
blood, and 5 × 106 in Pooled Platelets Leuko-
cytes Reduced.60(p29) The Food and Drug Ad-
TABLE 20-3. Approximate Leukocyte Content of Blood Components (Per Unit)
Whole blood
Red Blood Cells
Red Blood Cells, washed
Red Blood Cells, deglycerolized
Red Blood Cells, leukocyte reduced (by filtration)*
Apheresis Platelets
Apheresis Platelets, leukocyte reduced
Platelets†
Platelets, leukocyte reduced
Platelets, pooled, leukocyte reduced
Fresh Frozen Plasma, thawed
*Leukocyte reduction with third-generation leukocyte adsorption filter.
†
Derived from 1 unit of whole blood via platelet-rich plasma process.
Hemotherapy Decisions 䡲 505
ministration (FDA) recommends quality-con-
trol steps to indicate with 95% confidence that
at least 95% of units meet these criteria.59 By
comparison, European guidelines define leu-
kocyte-reduced components as those with
<1 × 106 residual leukocytes per unit and re-
quire no more than a 10% failure rate in the
leukoreduction process.61
Matching Units to Patient Hemoglobin
Targets
For adult patients, the discussion of when or
whether to transfuse RBCs has not usually pro-
ceeded to a consideration of how much to
transfuse. Although pediatricians commonly
prescribe blood components based on the pa-
tient’s size, the typical order for adult RBC
transfusion until recently had been 2 units, re-
gardless of patient size and usually without re-
gard to a desired target hemoglobin level. In
addition, the widely variable hemoglobin con-
tent of RBC units usually is not considered.62
A more physiologic approach would be to
identify the target hemoglobin desired after
transfusion and then base the dose (ie, num-
ber of units or g of hemoglobin to be trans-
fused) on the patient’s current hemoglobin,
any ongoing blood loss, and the calculated
blood volume. A pilot study, however, showed
109
108
107
106-107
<5 × 106
106-108
<5 × 106
107
<8.3 × 105
<5 × 106
0.6 × 106 - 1.5 × 107
506 䡲 AABB TECHNICAL MANUAL
that the number of units transfused using this
approach to a group of patients could be re-
duced, thus reaping benefits for inventory
management as well as donor exposure rates.63
Issues that remain to be resolved for this ap-
proach include the following:
䡲 Whether the approach could be imple-
mented successfully for all patients in an
institution.
䡲 Whether blood collection establishments
would be able to provide the needed hemo-
globin content for all RBC units.
䡲 Whether the approach could be sustained
in an era when most units are produced by
apheresis, with a standardized hemoglobin
content.
Emergency Transfusion
The unexpected need to transfuse possibly a
large amount of RBCs may require application
of alternative procedures. Release of group O
RBCs or antigen-negative, uncrossmatched
units may be necessary if waiting to complete
standard pretransfusion testing routines could
induce anemic morbidity (see Chapter 15).
An emergency-release protocol might be
integrated into one that allows rapid provision
of a large number of RBC units to accommo-
date the needs of patients who are bleeding
rapidly. Elements of such a block-release sys-
tem might include completing the paper work
for multiple units in advance and prepackag-
ing these units to facilitate immediate delivery
to the patient. The need for and components
of such a system vary according to the types of
patients served by the institution and the lo-
gistics of blood delivery. The expectations of
clinicians (particularly surgeons, anesthesiol-
ogists, and emergency physicians) for rapid
delivery of large volumes of RBCs should be
discussed with them so that appropriate re-
quirements can be met reliably. In evaluating
these clinical needs, attention should be given
not just to the time required to issue RBCs
from inventory to the laboratory but also for
the units to reach the patient.
Incompatible RBC Transfusion
Occasionally, transfusion of RBCs may be nec-
essary when no serologically compatible units
are available for a patient. This most often oc-
curs in patients with autoantibodies that typi-
cally are reactive with red cells from all donors;
however, as long as the presence of alloanti-
bodies can be ruled out, the transfused cells
should survive as long as autologous cells.
The key point in ensuring a safe and suc-
cessful transfusion in such a situation is the
exclusion of the presence of alloantibodies.
Because this may be difficult and the benefi-
cial effects of the transfusion may be tempo-
rally limited, a conservative transfusion strate-
gy is usually recommended for patients with
autoimmune hemolytic anemia. Determina-
tion of the patient’s phenotype before transfu-
sion simplifies subsequent investigations of
the presence of alloantibodies. (Chapters 15-
17 contain a more complete discussion of this
issue.)
Other situations in which all units appear
to be incompatible include the presence of al-
loantibodies to high-frequency antigens and/
or multiple antibody specificities. If serologic
testing fails to resolve the problem or the prob-
lem is identified but sufficient time is not
available to acquire compatible units, consul-
tation between the transfusion service physi-
cian and the patient’s clinician is advised to
weigh the risks and benefits of transfusion and
to consider which alternative therapies are
suitable. If the need is sufficiently urgent,
ABO-compatible but crossmatch-incompati-
ble RBCs may need to be used.
Depending on the alloantibody’s specific-
ity or possible specificities that have not been
ruled out, incompatible RBC transfusion does
not always result in immediate hemolysis, and
the incompatible cells may remain in the cir-
culation long enough to provide therapeutic
benefit.64
If time permits and equipment is avail-
able, the survival of a radiolabeled aliquot of
the incompatible RBCs can be determined.
However, this is beyond the capability of most
laboratories and is rarely needed. An in-vivo
crossmatch can be performed by cautiously
 CHAPTER 20
transfusing 25 to 50 mL of the incompatible
cells, watching the patient’s clinical response,
and checking a 30-minute posttransfusion
specimen for hemoglobin-tinged serum. Such
an assessment does not guarantee normal red
cell survival, but it can indicate whether an
acute reaction might occur. If no adverse
symptoms or hemolysis are observed, the re-
mainder of the unit can be transfused slowly
and with careful clinical monitoring. If the
condition requiring a transfusion intervention
is life threatening, RBC units may sometimes
be given without special testing, but clinical
staff should be prepared to treat any reaction
that may result.
RBC Substitutes
A variety of means have been explored to pro-
vide the O2-carrying capacity of hemoglobin
without utilizing red cells. Development has
proceeded furthest using Hb solutions derived
from red cell lysates in which the hemoglobin
molecules have undergone one or more chem-
ical modifications to optimize their O2 dissoci-
ation and prevent renal damage. Two of the
greatest challenges with cell-free hemoglobin
are its vasoconstrictive properties and its short
half-life within the intravascular circulation.
The goal of creating a hemoglobin substitute
that the FDA considers to be as safe as donor
red cells continues to be an elusive one.65,66
PL AT ELET TR ANSF USION
Prophylactic vs Therapeutic
Transfusion
Before the advent of platelet therapy, bleeding
exceeded infection as the most common cause
of death in patients with acute leukemia. The
availability of platelet concentrates led to the
natural consideration that prophylactic trans-
fusion to prevent or limit the most severe
thrombocytopenia would prevent the onset of
severe hemorrhage. Today, approximately 80%
of all platelet transfusions are administered to
patients with hypoproliferative thrombocyto-
penia.
Hemotherapy Decisions 䡲 507
However, the value of this approach has
never been definitively documented. Early
studies yielded equivocal results or utilized
statistical approaches that were less rigorous
than would be expected today.67,68 In fact, an
RCT in children with leukemia prior to the era
of leukocyte reduction indicated that the use
of prophylactic platelet transfusion from the
outset of chemotherapy increased the likeli-
hood of bleeding due to the development of
platelet refractoriness.67
The alternative strategy of directing plate-
let resources to those patients who are bleed-
ing may be a wiser and more effective use of a
limited resource.69 In fact, a recently published
Cochrane review showed that overall, a thera-
peutic platelet transfusion strategy might not
be inferior to a prophylactic strategy, but the
evidence supporting this approach is not ro-
bust.67 The majority of studies reviewed were
conducted in the 1970s, and the conclusions
did not take into account practice changes
since that time.
Overall, the role of prophylactic platelet
transfusions for the prevention and control of
thrombocytopenic bleeding is not yet clear.
When bleeding risk is identified in patients
with thrombocytopenia, several factors in ad-
dition to platelet count should be considered.
These include altered platelet function due to
intrinsic or acquired defects and hemostatic
defects involving the coagulation system. The
disease process itself must also be considered.
For example, in acute leukemia, intracranial
hemorrhage is more often associated with
high circulating blast counts than with low
platelet counts.70 The leukemic blasts may
cause sludging of cells in capillaries and possi-
ble hemorrhagic infarction, particularly in the
presence of a concomitant coagulopathy.
Transfusion Thresholds
If prophylactic platelet transfusions are to be
given to patients with hypoproliferative
thrombocytopenia, what is the most appropri-
ate threshold for transfusion? The first study to
address this question sought to define a clini-
cally useful threshold by observing and cate-
gorizing bleeding and associating the risks of
508 䡲 AABB TECHNICAL MANUAL
hemorrhage with patients’ platelet counts.71
The researchers were unable to identify a
platelet count threshold below which the risk
of hemorrhage increased rapidly. However,
this paper was often cited as the source of the
dictum that patients’ platelet counts should be
kept above 20,000/µL. In the 1960s, when this
study was conducted, aspirin was commonly
used to treat fever, pain, and transfusion reac-
tions among patients with neutropenia. But
with current knowledge about the platelet dys-
function induced by aspirin and associated
changes in hematology practice, the study re-
sults are less applicable today (Table 20-4).73
Over the last decade, several prospective
studies using either historical or randomized
control groups have documented the success-
ful application of 10,000/µL as a prophylactic
transfusion threshold in inpatients.67,74-76
These results parallel the finding that stool
blood loss does not accelerate until a platelet
count of approximately 5000/µL is reached.77
Many institutions have now adopted 10,000/
µL as their standard prophylactic platelet
transfusion threshold, but others have opted
to combine laboratory data with the patient’s
clinical status to determine the most appropri-
ate transfusion point72 (Table 20-4). Further-
more, because platelets adsorb circulating
thrombopoietin (TPO) and higher platelet
counts are associated with lower levels of free
TPO,78 maintaining patients at a lower platelet
count has been suggested to lead to shorter in-
tervals of thrombocytopenia, which is a poten-
tial additional benefit.
TABLE 20-4. Current Prophylactic Platelet Transfusion Thresholds
All patients
- or –
Stable patients
Patients with fever or recent hemorrhage
Patients with coagulopathy, on heparin, or with anatomic lesion
likely to bleed72
Note: These thresholds are most commonly applied to inpatients. Adjustment of the transfusion threshold may be necessi-
tated by unusual clinical situations.
For patients who are already bleeding or
are about to undergo a hemostatic challenge,
such as a surgical procedure, attempts are
made to keep the platelet count higher, usually
in the vicinity of 50,000/µL.79 Although there
are no trials to document that this is the most
appropriate level, it has become generally ac-
cepted as adequate.80 An even higher target, up
to 100,000/µL, is often used for intracerebral,
pulmonary, and ophthalmic hemorrhage. This
is to provide a greater “cushion” to ensure ade-
quate hemostasis in these vital and suscepti-
ble organs even if the platelet count should
drop. Higher cut points might also be used in
massive transfusion or disseminated intravas-
cular coagulation (DIC), where the platelet
count may drop rapidly.
The role of anemia should also be consid-
ered in the prevention or treatment of hemor-
rhage. In a 2001 study in healthy volunteers, a
reduction in hematocrit from 41% to 35% re-
sulted in almost a doubling of the bleeding
time, whereas a decrease in the platelet count
by one-third had no effect.81 Traditionally, this
result has been attributed to the rheologic
properties of flowing blood. Given their larger
size and higher density, red cells tend to occu-
py the central (axial) portion of the blood flow,
pushing platelets to the periphery in proximity
with the vessel wall. This makes teleologic
sense because it is only along the vessel wall
that a rent in the endothelium can occur
where the platelets would then perform their
hemostatic functions.
Recent research has focused on elucidat-
ing the mechanisms of red cell and platelet
10,000/L
5,000/L
10,000/L
20,000/L
 CHAPTER 20
communication, which might also explain the
hemostatic effect of a higher hematocrit.
These newer studies have shown, for example,
that the red cell membrane augments throm-
bin generation,82 adenosine diphosphate re-
leased from red cells may be a chemical mes-
senger for platelet activation,83 and red cell
phosphatidyl serine expression might be an-
other pathway for thrombin generation. Re-
gardless of the postulated mechanism, correc-
tion of anemia may be an additional tool to
use for bleeding prevention, particularly in pa-
tients with thrombocytopenia. In patients with
uremia, RBC transfusion or the administra-
tion of erythropoietin to increase the hemato-
crit improves hemostasis similarly.84 Thus, al-
though the patient’s cardiovascular system
might be tolerating the anemia associated
with chemotherapy (or hemorrhage), the he-
mostatic system may not.85
Transfusion to Correct
Thrombocytopathy
Patients whose platelets are not able to com-
plete all of the complex metabolic steps neces-
sary for activation, granular release, and aggre-
gation may have an increased likelihood of
bleeding and/or an inability to respond to
hemorrhage appropriately. These abnormali-
ties may be congenital (eg, Glanzmann throm-
basthenia) or acquired as the result of disease
(eg, myelodysplasia) or drug treatment (eg,
with aspirin or glycoprotein IIb/IIIa antago-
nists). In addition, patients who have recently
undergone extracorporeal circulation (eg, dur-
ing cardiac bypass surgery) may have platelet
counts that appear to be adequate for hemo-
stasis but, in fact, are dysfunctional due to pro-
longed exposure to roller pumps and foreign
surfaces, leading to partial activation and de-
granulation.86
In all of these circumstances, the decision
whether to transfuse platelets probably needs
to be based on the patient’s clinical status
rather than his or her platelet count. Patients
undergoing surgery while still under the effect
of previously ingested aspirin do not necessar-
ily bleed more than other patients, although
clinician knowledge that the patient has been
Hemotherapy Decisions 䡲 509
taking aspirin has been associated with in-
creased blood component usage.87-92 The pro-
phylactic use of platelets (and plasma) after
cardiac surgery has been shown to be neither
necessary nor beneficial, but a patient experi-
encing excessive blood loss postoperatively
whose heparin level has been reversed may
benefit from a dose of platelets even before his
or her postoperative platelet count is known.
Patients treated with irreversible platelet an-
tagonists (eg, clopidogrel) during cardiac cath-
eterization and who proceed directly to cardi-
ac surgery may need one or more doses of
platelets (as well as increased RBC transfu-
sions) because their own platelets are no lon-
ger functional.93,94 Antiplatelet therapies con-
tinue to be used in catheterization because
they appear to improve outcomes even if the
patient requires immediate surgery.95 The ef-
fect of these drugs can be antagonized through
pharmacologic intervention, including the use
of desmopressin acetate.96
Given the high proportion of patients
treated with aspirin for a variety of reasons,
patients who experience bleeding while tak-
ing aspirin may be encountered frequently. Al-
though the daily consumption of 81 mg aspi-
rin for cardiac prophylaxis is unlikely to
contribute to hemostatic difficulties, some pa-
tients are hyperresponders to aspirin and their
bleeding times may be dramatically extended.
Platelet transfusions are occasionally request-
ed to correct the effect of aspirin. In these cas-
es, only a small proportion—approximately
20%—of circulating platelets need to be func-
tional to correct the deficiency.97 Therefore,
neither achievement of donor platelet levels of
50,000/µL nor a full therapeutic dose of plate-
lets is required in most patients.
Patients with significant renal disease (ie,
a creatinine level exceeding 3 mg/dL) may also
have dysfunctional platelets due to the uremic
environment. Transfusion of platelets to these
patients is of little value, however, because
they, too, rapidly succumb to the same meta-
bolic derangement. Desmopressin acetate (1-
deamino-8-D-arginine vasopressin, or DDAVP)
treatment is usually recommended to aug-
ment the responsiveness of these platelets.98
Alternatively, cryoprecipitate may provide the
510 䡲 AABB TECHNICAL MANUAL
increased amount of von Willebrand factor
(vWF) that is believed to be helpful in activat-
ing these platelets if tachyphylaxis to multiple
doses of desmopressin acetate precludes fur-
ther treatment.99 Dialysis to decrease the ure-
mia is often indicated in clinical scenarios
where optimal platelet function is desired.
Dosage
The amount of platelets considered a thera-
peutic dose remains undecided, but recent re-
search is elucidating this issue.100 Much early
research was performed using platelet units
with significantly lower platelet counts than
units currently being produced. Initial platelet
separation efficiencies were significantly lower
than those achieved through evolutionary im-
provements in both component production
processes and larger whole-blood collections.
Accordingly, many blood centers now report
mean contents that are 20% to 40% above the
required minimum of 5.5 × 1010 platelets per
unit derived from whole blood. As a result,
fewer units need to be pooled to obtain the
same platelet dose. At the same time, accep-
tance of lower platelet counts in patients has
facilitated the progressive reduction in the
standard dose from 10 to 8, 6, or even 4 units
per pool (or transfusion).
The amount of platelets that can be col-
lected by apheresis also merits consideration.
The standard of 3.0 × 1011 apheresis platelets
per unit is the amount that could be practically
collected with early instruments and may also
have accounted for the maximum collection
time that donors would tolerate. These apher-
esis units yielded an increment similar to the
transfusion of a pool of 6 to 8 units of whole-
blood-derived platelet components. Today, the
increased efficiency of apheresis instruments
allows the collection of two or even three times
the standard quantity. Although blood centers
derive a significant economic boost from split-
ting platelet apheresis units, whether patients
are better served by receiving larger platelet
units remains to be determined.
The question of what the optimal platelet
dose is has been approached in several ways. A
mathematical model was used to calculate the
smallest number of platelets that would need
to be transfused if patients received just
enough platelets to achieve the desired target
level when they reached the transfusion
threshold.101 This analysis argued for the use of
small therapeutic doses administered more
frequently. The use of larger units in a clinical
study resulted in longer intertransfusion inter-
vals, although this temporal increase was
smaller than the increase in the number of
platelets transfused.102 In a paired study of
marrow transplant patients, larger units pro-
vided the expected lengthening of intertrans-
fusion intervals and, unexpectedly, resulted in
both count increments and corrected count
increments (CCIs) that were higher than those
achieved with smaller units.103
The Strategies for Transfusion of Platelets
study104 was a multicenter, prospective RCT
designed to show that a lower platelet dose for
prophylactic transfusions was not inferior to
the standard dose; the primary outcome was
incidence of WHO Grade 2 (or higher) bleed-
ing. The trial enrolled patients undergoing he-
matopoietic stem cell transplantation and
nontransplant patients with chemotherapy-
induced thrombocytopenia. The experimen-
tal arm received low-dose prophylactic plate-
let transfusions (1.5-2.9 × 1011 platelets per
transfusion, defined as half the standard dose)
and the control arm received a standard dose
(3.0-6.0 × 1011 platelets per transfusion). Al-
though sample size calculations indicated that
approximately 270 patients would be neces-
sary in each treatment arm, the study was
stopped after enrolling 130 patients based on a
preestablished safety threshold because the
cumulative incidence of Grade 4 bleeding ex-
ceeded 5% between the two study arms. The
main trends of this study did not support the
hypotheses that patients in the low-dose arm
would require fewer products and have a
shorter duration of thrombocytopenia.
Another recent multicenter, prospective,
RCT, the platelet dose (PLADO) study,105 did
proceed to completion. Patients with hypo-
proliferative thrombocytopenia secondary to
chemotherapy for hematologic malignancies
or undergoing either autologous or allogeneic
stem cell transplantation were randomly
 CHAPTER 20
assigned to a prophylactic platelet transfusion
dose of 1.1 (low dose), 2.2 (medium dose), or
4.4 × 1011/m2 platelets (high dose). The pa-
tients were transfused with their assigned dose
prophylactically for morning platelet counts of
10,000/µL. Additional platelet transfusions
could be given for active bleeding or planned
invasive procedures. The primary endpoint
was the percentage of patients in each group
with at least one episode of WHO Grade 2 or
higher bleeding. Of the 1272 patients who re-
ceived at least one platelet transfusion, the pri-
mary endpoint was achieved in 71%, 69%, and
70% in the low-, medium-, and high-dose
groups, respectively. These differences were
not statistically significant. Those in the low-
dose arm did receive an average of one more
platelet transfusion, however.
The lifespan of transfused platelets ap-
pears to be abnormally short in patients with
thrombocytopenia. Although autologous
platelets stored for 5 or 7 days and then re-
infused to healthy people usually survive for
more than 5 days, the lifespan of platelets in
patients with severe thrombocytopenia may
be only 2 days. This shortened lifespan may be
attributable to the fixed loss of platelets of ap-
proximately 7100/µL per day from circulation
for maintenance of normal hemostasis.106
When the patient’s platelet count is low (and,
of course, still subject to daily reductions due
to senescence), this obligatory loss represents
a large proportion of circulating platelets and
leads to the need for transfusions every 2 to 3
days even in patients achieving good incre-
ments from transfusions.107 More RCTs similar
to the PLADO study are needed to clarify
which dosage provides optimal hemostasis.
As with adult RBC transfusions, the size of
the patients (and their spleens) are usually not
considered in selecting the platelet unit dose
to be transfused. One of the dose studies men-
tioned above attempted to set the dose based
on patient weight, and the outcomes of the
two studies may be helpful in determining the
clinical importance of such a strategy. The pa-
tient’s size and the content of the unit are rou-
tinely assessed through the CCI (Table 20-5).
This measure, or a similar approach of calcu-
lating the recovery rate of transfused platelets
Hemotherapy Decisions 䡲 511
based on content and blood volume,108 can
provide a yardstick to determine whether spe-
cial efforts, such as selection of antigen-
negative units to combat alloimmunization,
may be helpful.
Unit Type and Age
The types of platelet units used for routine
transfusion varies by institution. Currently,
about 90% of platelet transfusions in the Unit-
ed States are derived from apheresis collec-
tions, and the usage of these platelets has in-
creased in a steady, linear manner for the past
two decades.109 Local pricing policies and the
preference of transfusion services to avoid the
extra processing steps associated with whole-
blood-derived units are probably significant
factors in this choice (Table 20-6). Some in-
vitro measures have identified a larger platelet
storage lesion in platelets produced via the
platelet-rich plasma (PRP) method than by
apheresis,110 and one paired reinfusion study
has confirmed these findings.111 However, the
radiolabeled autologous recovery and survival
rates of the two types of platelets do not ap-
pear dissimilar even after 7 days of stor-
age.112,113 Platelets derived from whole-blood
buffy coats may have the advantages of re-
duced activation during centrifugal prepara-
tion steps, but these are currently not available
in the United States, although they are proba-
bly the most widely used platelet component
around the world. From a clinical efficacy
standpoint, all three platelet preparations
yield good clinical results.
All platelet unit types accumulate a stor-
age lesion over time that leaves them less re-
sponsive to physiologic agonists. Typically,
these platelets also bear markers of platelet ac-
tivation, although none of these markers has
proven to be a useful predictor of recovery or
survival after transfusion.114-117 Studies in
which radiolabeled platelets at the limit of the
storage period are reinfused are commonly re-
quired for FDA licensure of new techniques of
collecting or storing platelets. Both recovery
and survival appear to become shorter with in-
creased storage time in such radiolabeling
studies. However, not all series of clinical
512 䡲 AABB TECHNICAL MANUAL

TABLE 20-5. Determination of Platelet Response

CCI
CI (per L) × BSA
CCI =
unit content
Platelet Recovery
CI × patient mass (kg) × blood volume (estimated at 75 mL/kg) × 100
Platelet recovery (%) =
unit content
Sample Calculations
Patient mass: 80 kg blood volume = 80 kg × 75 mL/kg = 6000 mL
Patient BSA: 2.0 m2 (determined from a table or nomogram, available in many textbooks)
Pretransfusion platelet count: 5000/L
 CI = 20,000/L
Posttransfusion platelet count: 25,000/L
Platelet count in unit: 1.5 × 106/L
Volume of unit: 267 mL  unit content = 4.0 × 1011 platelets

CCI = (20,000/L × 2.0 m2)/4.0 = 10,000

Successful transfusion: 7500
Refractory patient: Two or more transfusions with CCI <7500
Recovery = (20,000/L × 1000 L/mL × 6000 mL × 100%)/(4.0 × 1011) = 30%
Maximum achievable if the patient has a spleen: 65% to 70%
CCI = corrected count increment; CI = count increment; BSA = body surface area.

TABLE 20-6 Characteristics of Platelet Unit Types Available in the United States

Whole-Blood Derived

Prestorage
Platelet Unit Characteristic Individual Unit Pooled* Apheresis

Cost of preparation Lower Higher

Ease of bacterial testing Lower Higher Higher
Ease of leukoreduction Lower Higher Higher
Hospital preparation required More Less Less
Donor exposures Greater Fewer
HLA selection possible No Yes
Platelet content known No Yes
*Storage of pools of platelets for longer than 4 hours requires bacteria detection by a culture technique approved by the
Food and Drug Administration.
 CHAPTER 20
observations have shown a decrement in CCI
with increased storage time, perhaps due to
limited study sizes and the inherent variability
between patients and outcomes of transfu-
sions at different points during the course of
illness.118,119 Therefore, platelet-transfusion
policies in most institutions call for the most
efficient use of the short-lived and scarce re-
source by transfusing the most appropriate
units that are closest to their outdate. Some
patients may appear to be more sensitive to
the storage lesion than others and may benefit
from receipt of platelets that have been stored
for less time; this can only be determined em-
pirically.
Out-of-Group Transfusions
The importance of providing platelet transfu-
sions using units of the same ABO group as
that of the recipient is an unresolved issue.
There are several points to consider: 1) the
presence of ABH antigens on platelets that
could be targeted by the recipient’s isoaggluti-
nins,120 2) the presence of plasma in the unit
that could lead to hemolysis of the recipient’s
red cells, and 3) the potential that the incom-
patibility could have immunologic effects that
could affect patient outcomes. Typically, the
presence of ABO-incompatible donor red cells
in the platelet product is not a significant con-
cern because of their very low levels.
A recently published systematic review121
showed that ABO-identical and ABO-compati-
ble platelet transfusions (eg, group A platelets
in an AB recipient) result in a higher CCI than
ABO-incompatible platelet transfusions (eg,
group O platelets in a non-group O recipient).
Whether the benefits of transfusing ABO-
identical or -compatible platelets also trans-
lates into improved clinical outcomes in terms
of decreased bleeding severity or need for
transfusions is not clear from the existing data.
These data do indicate that a subset of recipi-
ents with a higher isoagglutinin titer, particu-
larly anti-A, have poorer recovery after trans-
fusion with a high-ABH-antigen-expression
unit, especially if the platelets comes from an
A1 donor.122-126 Any diminution in response ap-
pears to be more pronounced with repeated
Hemotherapy Decisions 䡲 513
out-of-group transfusions, possibly due to the
stimulation of higher isoagglutinin titers in the
recipients.127 Failure to achieve expected plate-
let increments with an out-of-group transfu-
sion should prompt a trial of ABO-identical
transfusions, particularly if the patient is not
alloimmunized to platelet-specific or HLA an-
tigens (Fig 20-1).128
An out-of-group platelet transfusion,
such as of a group O unit to a group A recipi-
ent, results in the transfusion of about 300 mL
of plasma containing isoagglutinins that are
directed against antigens present on the recip-
ient’s red cells. Although it is not standard
practice to transfuse a group O plasma unit to
a group A recipient, this practice is common
for platelet transfusions.80 Many patients show
no signs or symptoms of the mismatch, al-
though the majority may have a transiently
positive direct antiglobulin test result that, at
least, causes some immunohematologic con-
fusion regarding its cause.129
A high titer of isoagglutinins in a platelet
unit can cause hemolysis of recipient red cells
that may be clinically significant and even fa-
tal. This outcome may be more likely in situa-
tions where the recipient is smaller (and, thus,
the volume transfused represents a greater
proportion of the recipient’s plasma volume),
when apheresis units are used (and all the
plasma comes from the same donor and is not
“diluted” by plasma of lower isoagglutinin ti-
ters from other units in the pool or neutralized
by the presence of soluble ABO antigens in the
other units), and in patients undergoing multi-
ple transfusions.130 The risk of hemolysis with
apheresis platelet units is in the range of
1:3000 to 1:10,000.131,132 Whether ABO-compat-
ible but nonidentical plasma poses a risk
through circulating immune complexes has
been considered.133
Different approaches may be used to pre-
vent acute hemolysis when incompatible
plasma must be transfused as part of a platelet
transfusion. One approach is to limit the
amount of plasma being transfused by centri-
fuging the unit and expressing off the majority
of the plasma shortly before transfusion (see
Method 6-13). This reduces the potential se-
verity of any hemolysis but may not prevent it
514 䡲 AABB TECHNICAL MANUAL
FIGURE 20-1. One approach to management of patients with thrombocytopenia.
and, in any case, does result in the loss of some
proportion of the unit’s platelet content. An
approach that achieves a similar result is to
use platelet additive solutions, which allow
platelets to be stored in electrolyte solutions
that replace up to 65% of plasma, reducing the
amount of incompatible isoagglutinins. An-
other approach is to avoid out-of-group trans-
fusions of units having a dangerously high titer
of isoagglutinins. This approach usually in-
volves identifying units with amounts of anti-A
above a particular threshold, often a titer of
200. This approach lacks a solid evidence base,
its outcomes vary by method,134 and it does not
identify all potentially harmful units. 135 Avoid-
ance of out-of-group plasma or amelioration
of the situation through volume reduction is
especially important for pediatric recipients,
in whom the volume transfused is relatively
greater, and in neonates, where hyperbilirubi-
nemia may have particularly adverse conse-
quences.
The possibility exists that the transfusion
of incompatible plasma may also have other,
less-immediate but still untoward effects on
recipients’ immune systems. It is postulated
that these effects might be mediated by the
formation of circulating immune complex-
es.133 For example, minimization of exposure
to incompatible plasma was associated with
improved survival probability in marrow
transplant recipients in one retrospective
analysis,135 and another retrospective study
suggested that such a strategy reduced in-hos-
pital mortality after cardiac surgery by two-
thirds.136 This finding was not replicated in a
subsequent, larger study, however.137 There is
also some suggestion that transfusion with
ABO-incompatible platelets may hasten the
development of alloimmunization and plate-
let refractoriness138 and may reduce survival
after marrow transplantation.139
Any delayed immunologic impact of the
transfusion of mismatched plasma with plate-
lets remains to be fully elucidated, although
avoidance of out-of-group transfusion when
possible would obviate this concern. Weighed
against the practical issue of a human-derived
therapeutic product that expires in 4 to 5 days
issuing a platelet unit with incompatible plas-
ma generally carries a lower risk than with-
holding transfusion.
 CHAPTER 20
Rh Matching
Platelets themselves do not express or carry Rh
antigens. Despite improvements in apheresis-
collection and whole-blood-processing tech-
niques, a small but immunogenic dose of red
cells can be contained in a platelet unit. This is
more likely in whole-blood-derived than
apheresis platelet units. In one study, an inter-
nal quality control check of platelet units using
flow cytometry found that mean red cell con-
tent of platelet concentrates from whole-blood
donations and from apheresis were 0.036 mL
and 0.00043 mL, respectively.138
The risk of developing alloimmunization
to the D antigen from a platelet transfusion is
also dependent on a multitude of other plate-
let unit factors (eg, ABO compatibility and
whether the unit was leukoreduced) and recip-
ient factors (eg, gender, immunologic status,
whether the patient needs a massive transfu-
sion, and whether a concurrent febrile transfu-
sion reaction occurs). The development of
anti-D has been studied in many observation-
al and retrospective studies.140-142
The clinical magnitude of this issue is far
less than one might expect. Most patients re-
ceiving platelet transfusions are severely im-
munosuppressed, and a primary response to
the D or other red cell antigens is very uncom-
mon.140,142 In addition, for most patients, the
formation of anti-D has minimal impact on
their subsequent hemotherapy support. How-
ever, if the patient is a female of childbearing
potential, the formation of anti-D could have a
significant impact on her future pregnancies.
Therefore, attempts are usually made to pro-
vide Rh-negative recipients with Rh-negative
platelet units even though platelets do not ex-
press or carry Rh antigens. When an Rh-nega-
tive patient must receive an Rh-positive unit of
platelets, a dose of Rh Immune Globulin
(RhIG) may be administered to prevent Rh(D)
alloimmunization.
Rather than provide RhIG to all Rh-nega-
tive recipients of Rh-positive platelets, many
centers supply RhIG only to premenopausal
females. Given the 3-week half-life of IgG, a
single dose should provide prophylaxis for
multiple transfusions over a 2- to 4-week peri-
Hemotherapy Decisions 䡲 515
od and certainly for the period during which
anti-D is detectable serologically. Because the
recipient had, and probably still has, thrombo-
cytopenia, an intravenous form of RhIG may
be administered to avoid a hematoma, partic-
ularly if the platelet count remains below
50,000/µL.143
Alloimmunization: Prevention and
Response
Although patients who are receiving multiple
platelet transfusions are usually immunosup-
pressed by virtue of their underlying disease
and/or therapy, they are still able to mount an
immune response against antigens on plate-
lets. This response is usually in the form of an-
tibodies to HLA Class I antigens, but some pa-
tients may make antibodies to platelet-specific
antigens. The former require presentation of
the antigens via donor lymphocytes. Leuko-
cyte reduction of both platelet and RBC units
has proven to be highly effective in reducing
the risk of primary HLA alloimmunization.33
However, if a patient has previously been sen-
sitized, such as through pregnancy or a prior,
nonleukoreduced transfusion, transfusion of
the Class I antigens on the platelets may be
sufficient to provoke an anamnestic response
and the appearance of platelet refractoriness.
For patients who can be expected to re-
quire multiple platelet transfusions, such as
those who will undergo hematopoietic stem
cell transplantation, advance knowledge of
their (current) alloimmunization status may
help blood services prepare to provide special-
ly selected units. Usually, however, the hunt for
alloantibodies begins when alloimmunization
is suspected from poor responses to platelet
transfusions that cannot be explained by oth-
er, nonimmunologic factors, such as spleno-
megaly or sepsis. The availability of kits to
screen for HLA and other platelet-directed an-
tibodies by enzyme immunoassay or other
simple approaches has expanded the number
of laboratories able to perform this testing and
provide clinically useful information about the
results with a short turnaround time. Identifi-
cation of the specificities of multiple HLA anti-
516 䡲 AABB TECHNICAL MANUAL
bodies, however, may still require lymphocyto-
toxicity testing in some circumstances.
Before the ready availability of such test-
ing, the detection or supposition of immuno-
logic platelet refractoriness led to a call for
HLA-matched platelets.144,145 Even a large do-
nor registry may not be able to provide a com-
pletely matched unit for a given patient; many
“HLA-matched” units actually carry antigens
against which the patient may have an alloan-
tibody. (The presence of serologic cross-reac-
tive groups prevents recognition of some anti-
gens as foreign but, at the same time, reduces
the range of donors whose platelets are truly
compatible.) Platelet crossmatching is another
approach that can be utilized, with the lack of
in-vitro reactivity used as a predictor of good
in-vivo compatibility. This technique is often
still used when alloimmunization is directed at
a platelet antigen because few blood-collec-
tion agencies have phenotyped their donors
for these antigens. However, neither approach
can guarantee a good result more than 50% to
80% of the time, although a “good” HLA match
(A or BU grade) provides the best prediction of
transfusion success.146 An alternative, simpler
approach that parallels the practice with pa-
tients who are alloimmunized against red cell
antigens is the selection of platelet units that
lack the antigen(s) against which the recipient
is immunized and those from cross-reacting
groups (Fig 20-1).147
Antibodies provoked by or directed at a
wide variety of drugs can lead to thrombocyto-
penia.148 These drug-dependent platelet anti-
bodies (DDPAs) can lead to mild or profound
thrombocytopenia and to refractoriness to
transfused platelets.149 Although often thought
of as rare, DDPAs can be caused by many
drugs and are present in many patients. For
example, in one study, approximately 10% of
patients receiving gentamicin had a drop in
their platelet counts, and almost half of these
patients (5.9% overall) had an antibody that
interacted with platelets in the presence of
gentamicin.150 When refractoriness to platelet
transfusion cannot be explained by the pa-
tient’s condition or alloimmunization to HLA
or platelet antigens, consideration should be
given to the presence of DDPAs.
Dealing with Platelet Refractoriness
Responses to platelet transfusion are most of-
ten quantitated using the CCI 1 hour after
transfusion (Table 20-5).151 However, a sample
collected 10 minutes after transfusion yields
similar information and may be easier to ob-
tain routinely.152 The CCI calculation is based
on the count increment (count increment =
posttransfusion count – pretransfusion count),
platelet content of the unit (expressed as × 10-11),
and size of the patient [expressed as body sur-
face area (BSA) in m2]. For an adult patient
with a BSA of 2.0 m2 whose platelet count rose
from 5,000/µL to 25,000/µL after a platelet
transfusion containing 4.0 × 1011 platelets, the
CCI would be:
(count increment  BSA)/unit content =
(20,000/µL × 2.0 m2)/4.0 = 10,000
Units (m2/µL) are usually omitted when
reporting the result. A CCI above 7500 is con-
sidered evidence of a successful transfusion;
two transfusions with CCIs below 7500 within
an hour after transfusion are evidence of re-
fractoriness. A similar approach is used to
compare the recovery rate of platelets to the
expected rate.108
Occasionally, despite diligent efforts, no
compatible platelets can be found to transfuse
to a patient with platelet refractoriness. Alter-
native measures can be tried to stem or fore-
stall hemorrhage, but these are of unpredict-
able benefit. Administration of antifibrinolytic
agents, such as -aminocaproic acid (Amicar),
either intravenously or, in the case of gingival
bleeding, as a mouthwash may allow the clot
that is formed to be sustained.108
Curiously, administration of intravenous
Ig (IVIG), an effective therapy for autoimmune
thrombocytopenia, appears to be of little ben-
efit in alloimmune refractoriness. IVIG can
yield increased increments shortly after trans-
fusion, but the durability of the response is
limited and, by 24 hours after transfusion, the
patient usually returns to his or her baseline
level.108 The claim that platelets are accumu-
lating at sites of bleeding even though they are
not detected in circulation via a platelet count
 CHAPTER 20
increment has not been substantiated and is
unlikely to be true.108,153-155 Some experts have
advocated administration of platelets by slow
drip, perhaps after making aliquots of a thera-
peutic dose and administering it over 4 to 12
hours. This approach has the benefit of allow-
ing the clinician to feel that “something” is be-
ing done while minimizing the demand on the
transfusion service inventory; however, the
predicted benefit to the patient is based pri-
marily on anecdotal experience.
Contraindications to Platelet
Transfusion
Idiopathic (autoimmune) thrombocytopenia
(ITP) can cause profoundly low platelet counts,
but patients with autoimmune ITP (particular-
ly children) rarely suffer hemorrhagic conse-
quences.156,157 The transfusion of platelets in
the stable, nonbleeding patient with ITP offers
no benefit because the platelets are rapidly
cleared by the circulating antibodies. In situa-
tions where the patient is bleeding, clinicians
may feel compelled to take some action,
including using antifibrinolytic agents and
platelet transfusion. Success in stemming
hemorrhage with transfusion is not universal,
but bleeding may slow down or cease after
transfusion in perhaps half or two-thirds of at-
tempts; this proportion is high enough to war-
rant trying transfusion, particularly when the
bleeding is serious.158
Thrombotic thrombocytopenic purpura
(TTP) has traditionally been regarded as a
contraindication to platelet transfusion.159,160
Thrombocytopenia occurs in TTP as platelets
are activated and consumed in thromboses
triggered by abnormally large multimers of
vWF that are capable of inciting inappropriate
platelet activation without other cofactors.161
The resulting thrombocytopenia may be pro-
tective, then, in slowing the formation of addi-
tional pathologic thromboses. Case reports
describing the development of coma in close
temporal relationship to platelet transfusion
led to the idea that platelets may “add fuel to
the fire” and prompt further thromboses in
critical sites. A recent review of the literature
accompanied by a prospective observational
Hemotherapy Decisions 䡲 517
study of 54 patients with TTP found no in-
creased frequency of neurologic events or
death in patients who received platelet trans-
fusions.154 Other, well-designed trials are need-
ed to refute or confirm the belief that platelet
transfusion is contraindicated in TTP.
Platelet transfusion is also usually avoid-
ed in patients with heparin-induced thrombo-
cytopenia (HIT), especially the immunologic
(Type II) form, to forestall the development of
limb- and life-threatening thromboses.162
Other Uses of Platelets
Autologous or allogeneic platelets may also be
applied topically to an area of surgical recon-
struction. The presence of platelet-derived
growth factor through applications of PRP or
platelet gels is thought to stimulate angiogene-
sis and promote more rapid tissue repair.163,164
A recent systematic review of RCTs on PRP
concluded that, although the use of PRP may
reduce the volume of blood components
transfused for an average savings of 247 mL
per patient, the use of PRP in adult elective
surgery cannot be justified due to the risk of
adverse events, such as hypotension, and the
fact that other blood-sparing techniques (eg,
use of a lower transfusion threshold or antifi-
brinolytic agents) are supported by stronger
evidence.165
PL ASMA TR ANSFUSION
Indications for Plasma
The current data needed to support evidence-
based guidelines for plasma transfusion are
surprisingly weak. A systematic review of the
available data for subsequent development of
guidelines by an expert panel found the data
to be sparse and of low quality.166 The panel
developed a guidance document after taking
into account the potential benefits and harms
in the available data.167 Indications for plasma
transfusion with reasonable support included
massive transfusion and reversal of warfarin
anticoagulation in patients with intracranial
hemorrhage. In other clinical scenarios, such
as surgery without massive transfusion or
518 䡲 AABB TECHNICAL MANUAL
warfarin reversal in the absence of intracranial
hemorrhage, the panel did not develop recom-
mendations due to insufficient data. The panel
recommended against plasma infusion in situ-
ations that might result in prophylactic plasma
infusion, such as acute pancreatitis or critical-
ly ill nonsurgical noncardiac patients. In these
types of patients where coagulopathy or
bleeding is absent, the risk of lung injury and
mortality outweigh any perceived benefit.165 A
more recent review echoes the conclusion that
prophylactic plasma infusion may not provide
benefit.168
Common Clinical Scenarios and the
Limitations of Laboratory Tests to
Support Transfusion Decisions
Many studies have shown that abnormal coag-
ulation test results do not predict an increased
risk of hemorrhage.169 Standard coagulation
screening tests are poor predictors of hemor-
rhage risk, in part because they are inordinate-
ly sensitive to mild deficiencies of multiple
procoagulants.170 As a result, the test results
become abnormal at factor levels that are not
clinically associated with bleeding. Additional
confusion arises from the fact that the critical
thresholds in these studies, 1.3 times the up-
per limit of normal or 1.5 times the midpoint
of the reference range (usually almost the
same number), usually fall at about an inter-
national normalized ratio (INR) of 1.5 to 2.0.
Because the usual target for oral anticoag-
ulation therapy is an INR of 2.0 to 3.0, some cli-
nicians assume that patients with an INR close
to 2.0 require correction before surgery. How-
ever, although reducing coagulation in pa-
tients with an INR of 2.0 prevents their con-
genital risk factor for hypercoagulability (eg,
Factor V Leiden) or acquired abnormality (eg,
prosthetic cardiac valve) from triggering un-
wanted clotting, this does not mean that nor-
mal, physiologic triggers of the clotting sys-
tem will be unable to cause an appropriate
thrombotic sequence. This distinction is im-
portant to make when deciding whether a pa-
tient requires correction of a slightly abnor-
mal coagulation level. The guidelines for
plasma administration of several professional
associations, including the American Society
of Anesthesiologists and the College of Ameri-
can Pathologists, recommend correction of the
overcoagulation by plasma transfusion prior
to surgery or to facilitate thrombosis only for
an INR of approximately 1.5 to 2.0.171,172
The British Committee for Standards in
Haematology noted, similarly, the limited
utility of attempting to correct mild overcoag-
ulation.173 Their guidelines for correcting
excessive anticoagulation and those of the
American College of Chest Physicians174 (Ta-
ble 20-7) notably avoid calling for the use of
plasma in lieu of administration of vitamin K
until very abnormal INRs and bleeding are en-
countered. Administration of vitamin K can re-
verse the effects of warfarin promptly (within
6-24 hours) without complicating the reestab-
lishment of healthy levels of anticoagula-
tion.175
Prothrombin complex concentrates (PCCs)
can also be used to rapidly correct the effects
of warfarin.176 Success has been reported from
combining the transfusion of 2 units of plasma
with three-factor PCCs, or by using a four-fac-
tor PCC.177 This is discussed more fully in the
“PCC” section below.
With the approval of new anticoagulants
that act at different points of the coagulation
system, questions about reversal have arisen
when emergent surgery is required or bleeding
occurs. The direct thrombin inhibitors and
Factor Xa inhibitors function as their names
indicate and offer the benefits of short half-
lives and a return to baseline coagulation lev-
els in approximately 24 hours with adequate
renal function upon cessation of the drug.
However, there is currently no approved rever-
sal agent. Case reports indicate that attempted
reversal with plasma in bleeding patients was
not successful, and more evidence on the utili-
ty of PCCs and prohemostatic therapies is nec-
essary.178 Laboratory tests to determine the
amount of drug circulating are still in develop-
ment.
Patients with cirrhosis commonly have
coagulation abnormalities due to their syn-
thetic difficulties. They also often experience
gastrointestinal bleeding due to increased por-
tal vein pressure. However, much more fre-
 CHAPTER 20
TABLE 20-7. Guidelines for Correction of Excessive Oral Anticoagulation
Clinical Situation
INR > therapeutic but < 5, no significant
bleeding
INR > 5 but < 9, no significant bleeding
INR > 9, no significant bleeding
Serious bleeding at any elevation
of INR
Life-threatening hemorrhage
INR = international normalized ratio.
Adapted from guidelines developed by the American College of Chest Physicians.174
quent hemorrhage and an inability to clot
might be expected in these patients based on
their prothrombin times (PTs) only. The reason
why people with cirrhosis do not bleed abnor-
mally—and why intricate pro- and antithrom-
botic balances are inherent in their clotting
system—was recently elucidated by demon-
stration that thrombin generated in vitro in
plasma from healthy individuals and people
with cirrhosis was the same, but only after the
addition of thrombomodulin, which activates
protein C, to the test system.179
As the liver’s synthetic production de-
clines, the amount of procoagulants circulat-
ing in plasma decreases, but so does the level
of certain anticoagulants, such as protein C.
Hemotherapy Decisions 䡲 519
Guideline
Lower anticoagulant dosage.
Temporarily discontinue drug if necessary.
Omit 1-2 doses; monitor INR. Resume oral anticoagulation when
INR is in therapeutic range or, if patient is at increased risk of
hemorrhage, omit a dose and give 1-2.5 mg vitamin K1 orally.
For rapid reversal before urgent surgery: 2-4 mg vitamin K1 orally;
repeat dose with 1-2 mg at 24 hours if INR remains elevated.
Omit warfarin; give 5-10 mg vitamin K1 orally.
Closely monitor INR; give additional vitamin K1 if necessary.
Resume warfarin at lower dose when INR is within therapeutic
range.
Omit warfarin.
Give 10 mg vitamin K1 by slow intravenous infusion.
Supplement with plasma or prothrombin complex concentrate
depending on urgency of correction.
Vitamin K1 infusions can be repeated every 12 hours.
Omit warfarin.
Give prothrombin complex concentrate with 10 mg vitamin K1 by
slow intravenous infusion.
Repeat as necessary, depending on INR.
Less activity may be expected in the procoagu-
lant system of a patient with cirrhosis, but, at
the same time, there will be less of an
opposing force from the coagulation-control
system.179 The PT and partial thromboplastin
time do not reflect thrombin generation in
these patients in a way that predicts bleeding.
Until a more accurate predictive test becomes
available, one role of the transfusion medicine
physician will continue to be to help other cli-
nicians understand why patients with severe
liver disease often do not require heroic efforts
(or heroic amounts of plasma) to completely
normalize their coagulation system.
What happens if an attempt is made to
correct the patient’s PT before a procedure?
520 䡲 AABB TECHNICAL MANUAL
Often, the answer is “surprisingly little.” The
authors of one study noted that the mean re-
duction in INR was only 0.03 per unit of plas-
ma transfused.180 Another study showed that
the PT decreased to the normal range in less
than 1% of plasma recipients who had mildly
abnormal PTs before transfusion, and the dif-
ference in the upper limit of normal was re-
duced by half in only 14.5%.181 The mean de-
crease in PT was only 0.2 second, and there
was no correlation between PT abnormality
and subsequent RBC transfusion. This study
also revealed that only 1 in 10 patients receiv-
ing plasma had their PT rechecked within 8
hours—despite the fact that the ordering clini-
cian thought that the correction was clinically
important and the expected shortening of PT
occurred infrequently. The small corrections
in PT/INR may reflect the fact that these re-
ductions have an exponential relationship,
rather than a linear relationship, to the pro-
portion of coagulation factors in circulation
(Fig 20-2).
In cases of rapid bleeding, a more proac-
tive approach may be beneficial. The altera-
tions that occur in a massive transfusion
situation are a combination of dilutional coag-
ulopathy, injury-driven factor consumption,
and activation of fibrinolysis.182 Correction of a
true coagulopathy after it becomes clinically
7
6
5
4
INR
3
2
1
0
0 20 40
% Factor Levels
FIGURE 20-2. Exponential relationship of international normalized ratio (INR) to % factor levels.
(Used with permission from Wayne L. Chandler, MD, Houston Methodist Hospital.)
manifest can be much more difficult, and in-
creased transfusion of other components may
be needed. Although the early use of non-RBC
components reduces mortality risk,16 this ap-
proach fails to take into consideration each
patient’s situation; direct involvement of a
transfusion medicine specialist can best guide
hemotherapy in these complex, rapidly evolv-
ing situations. Such consultation also takes
into account other important factors, such as
reduced patient temperature and the presence
of acidosis that decrease the in-vivo activity of
the coagulation system significantly.183
Rapid whole-blood testing techniques
have become integrated into many centers’
transfusion protocols for massively bleeding
patients. Common viscoelastic coagulation
testing (VCT) platforms include thromboelas-
tography and rotational thromboelastometry.
These tests measure the speed and strength of
clot formation. The benefits of these tests
include that they generate initial results within
15-20 minutes and can be used to assess fibri-
nolysis. Current challenges, however, involve
lack of standardization of results, correlation
with standard coagulation tests, or training in
interpreting the results.184
A recent meta-analysis of RCTs on TEG-
based treatment in massive transfusion in-
cluded nine studies of cardiac surgery and one
60 80 100 120
 CHAPTER 20 Hemotherapy Decisions 䡲 521

liver transplant study in 776 patients.185 The
authors found no difference in mortality be-
tween TEG-guided therapy and conventional
practice. There were moderate decreases in
bleeding and numbers of patients transfused
with both FFP and platelets in patients under-
going TEG-guided treatment; however, the
heterogeneity of the studies limited the ability
to draw definitive conclusions. VCT use in
trauma appears to predict the need for mas-
sive transfusion and risk of mortality; however,
most data come from observational studies,
and this issue would benefit from additional
RCT evidence.186
Dose and Timing of Plasma
Transfusion
Although the level of coagulation factors nor-
mally varies widely between 50% and 150% of
the activity of circulating clotting factors, nu-
merous publications show that people have
the ability to form clots with significantly low-
er levels of clotting factors.187 For single factor
deficiencies, such as deficiencies of Factors
VIII or IX, 30% activity is often needed for he-
mostasis. In patients with multiple factor defi-
ciencies, such as after trauma, factor levels
closer to 40% may be needed for hemostasis
(see Fig 20-2 and Table 20-8). In addition, the
further the patient’s procoagulant activity is
from the normal range, the easier it is to effect
a change in the PT because the relationship
between factor activity and PT is more expo-
nential than linear.179,188
A usual dose of plasma is 10-20 mL/kg.
This dose is expected to increase the level of
coagulation factors by 20% immediately after
infusion. Precise prediction of the amount of
plasma needed to be transfused to correct a
particular coagulopathy is not currently possi-
ble. Thus, posttransfusion repetition of the co-
agulation test that prompted the transfusion is
warranted.179,181
When attempting to correct a coagulopa-
thy with plasma transfusion, the biological
half-life of procoagulants must also be consid-
ered. Factor VII has the shortest half-life in
vivo (approximately 5 hours). If a transfusion
raises the patient’s activity of this factor from
30% to 45% 5 hours later, for example, the
activity level will be halfway back to the
steady-state level for that patient (ie, to 37%).
Additional correction attempts must now
change the factor concentration in an en-
larged plasma volume, and multiple plasma
transfusions can produce pulmonary edema
through fluid overload. In addition, if correc-
tion is truly required before a hemostatic chal-
lenge, such as major surgery, the plasma
should be infused shortly before the procedure
for the benefit to be incurred at the time of the
hemostatic challenge.

TABLE 20-8. Coagulation Factor Half-Lives

Factor In-Vivo Half-Life % Needed for Hemostasis

I 3-6 days 12-50

II 2-5 days 10-25
V 5-36 hours 10-30
VII 2-5 hours >10
VIII 8-12 hours 30-40
IX 18-24 hours 15-40
X 20-42 hours 10-40
XI 40-80 hours 20-30
XIII 12 days <5
522 䡲 AABB TECHNICAL MANUAL
Types of Plasma
Several types of plasma may be available for
transfusion, and, for most situations, they can
be used interchangeably. To be called “FFP,”
the unit must be frozen within 8 hours of col-
lection and transfused within 24 hours of
thawing, thus optimizing the levels of the most
labile procoagulants, Factors V and VIII. How-
ever, congenital Factor V deficiency is rare.
Most patients requiring plasma transfusion al-
ready have an elevated Factor VIII level be-
cause it is an acute phase reactant. Therefore,
Plasma Frozen within 24 Hours After Phlebot-
omy (PF24) has procoagulant contents that are
as capable as FFP of correcting most clinical
coagulopathies.189,190
Thawed Plasma made from PF24 or FFP
and kept refrigerated after thawing can be ef-
fectively used throughout its 5-day shelf life
because most coagulation factors remain at
hemostatic levels.191,192 Thawed Plasma can be
part of an effective strategy aimed at reducing
wastage of plasma in combination with the
implementation of appropriate ordering prac-
tices, as described above.
Transfusion of Plasma for Other
Indications
There are several other clinical situations
where the transfusion of plasma is indicated
based on slightly different rationales. Thera-
peutic plasmapheresis may call for partial or
complete replacement of the removed volume
of patient plasma. This may occasionally be
necessitated by frequent (ie, daily) procedures
that can deplete procoagulant levels more
quickly than they can be replenished. Howev-
er, most patients do not require such an ag-
gressive plasmapheresis schedule, and the
need to use plasma for factor replenishment is
very unusual. TTP presents a different situa-
tion, however, because the plasma’s content of
ADAMTS13 is therapeutic, replenishing the
patient’s supply that has been reduced by au-
toantibody or congenital deficiency, for exam-
ple.161
Congenital deficiencies of procoagulants
may require prophylactic or therapeutic re-
plenishment via plasma transfusion, but pa-
tients with these deficiencies are rare. C1 es-
terase inhibitor deficiency causes hereditary
angioedema due to inappropriate activation of
complement. Its management may include
administration of a C1 inhibitor derived from
human plasma.191 Factor XI deficiency, al-
though not a cause of spontaneous bleeding,
often results in excessive postoperative bleed-
ing.191 In emergent situations where specific
concentrates are not available, transfused
plasma can be used as a source of the deficient
factor. 193,194
C RYO P R E C I PI TATE
TRANSF USION
The discovery that certain proteins critical to
the coagulation system could be concentrated
by thawing FFP at 4 C and centrifugally sepa-
rating the plasma from the supernatant pro-
vided the first truly effective treatment for pa-
tients with hemophilia A, the congenital
deficiency of Factor VIII. Cryoprecipitated an-
tihemophilic factor, known as “cryoprecipi-
tate,” is rarely if ever used today for its original
purpose, having been supplanted by other
simpler and safer means of providing the defi-
cient factor. Nevertheless, cryoprecipitate is an
essential component in modern hemotherapy.
Although the US regulatory requirements
for the content of cryoprecipitate are based on
Factor VIII content (minimum = 80 U/unit), it
is for its fibrinogen content that cryoprecipi-
tate is most commonly used. When fibrinogen
consumption (eg, in DIC) and/or loss (eg, due
to massive hemorrhage) occur, exogenous re-
placement may be necessary to maintain plas-
ma’s coagulation potential. The fibrinogen
concentration necessary to maintain hemo-
stasis is in the range of 50-100 mg/dL. Howev-
er, coagulation test results that are used to fol-
low patients with such conditions usually
become abnormal due to hypofibrinogen-
emia when the fibrinogen concentration drops
below 100 mg/dL. Therefore, maintaining the
fibrinogen concentration above this level aids
both the patient and those attempting to un-
derstand the situation through laboratory test-
ing. When fibrinogen levels drop as a result of
 CHAPTER 20
DIC or ongoing, high-volume hemorrhage, it
may be wise to initiate cryoprecipitate transfu-
sion (or at least prepare the component) as the
critical point of 100 mg/dL is approached (eg,
at approximately 120 mg/dL).
Although the dosage of cryoprecipitate is
often stated in tens of units (eg, 10, 20, or 30
units), the dosage required to achieve the de-
sired effect is readily calculated. This calcula-
tion is based on the difference between the
current and desired (usually 200 mg/dL) con-
centration of fibrinogen, a projection of the
patient’s plasma volume [as (1 – hematocrit) ×
0.7 dL/kg × body mass, or 30 dL if the patient’s
weight is unknown], and the usual fibrinogen
content of cryoprecipitate (250 mg/unit):
Dose (units) = [desired fibrinogen increment
(mg/dL) × plasma volume]/250 mg/unit
Although the small volume (5-15 mL) of
each cryoprecipitate unit facilitates rapid
thawing, the pooling of units (usually includ-
ing rinsing the bags to maximize recovery) can
be time consuming. Planning ahead for rapid
use later in case a patient needs a massive
transfusion, for example, can certainly facili-
tate patient care. Some facilities have chosen
to produce pools of cryoprecipitate using ster-
ile techniques before freezing the component.
This product has a higher fibrinogen content
and a shorter time to issue when needed.
Dysfibrinogenemias are rare congenital
abnormalities that result in dysfunctional fi-
brinogen molecules. These molecules can lead
to an increased risk of thrombosis, bleeding, or
both, although they can also have no clinical
manifestations.195 Patients may require admin-
istration of cryoprecipitate to correct the
deficiency. Some degree of dysfunctional fi-
brinogen is also produced in damaged or re-
generating livers and in neonates, but this
rarely results in the need to transfuse normal
fibrinogen.196
States of localized fibrinolysis can be in-
duced during surgeries that disrupt the pros-
tatic bed, urothelium, or salivary gland tissue
because plasminogen activators are produced
by these tissues. In these cases, cryoprecipitate
infusions may be warranted to stop bleeding
Hemotherapy Decisions 䡲 523
with or without the use of antifibrinolytic ther-
apy. Note that bleeding from the urinary tract
is a relative contraindication to antifibrinolytic
therapy. States of systemic fibrinolysis can oc-
cur with medical conditions such as wide-
spread amyloidosis or as a result of chemo-
therapy with L-asparaginase, typically used to
treat acute lymphoblastic leukemia.
Although no longer considered for rou-
tine use due to the availability of concentrated
plasma derivatives of copurified Factor VIII
and vWF, cryoprecipitate can be used as a
source of vWF for patients with von Wille-
brand disease (vWD). The most common form
of vWD is Type 1, which results in a deficiency
of a normal protein. Type 1 vWD most often
can be managed with administration of des-
mopressin. Patients with Types 2 and 3 vWD
need to have their bleeding managed with an
exogenous source of vWF. See Table 20-9 for
general factor replacement guidelines for the
treatment of vWD.
Cryoprecipitate can also be applied topi-
cally on surfaces where suturing is ineffective
to achieve hemostasis and that are slow to
generate clot, such as the raw surface of trau-
matically injured liver.199 When applied simul-
taneously with calcium and thrombin (usually
using two syringes), a thick, gelatinous mass
quickly forms over the area and quells the
bleeding. This approach can also be used to
bind together structures, such as in surgery in
the inner ear. Cryoprecipitate is not typically
requested from the transfusion service in cur-
rent practice because products that have both
lyophilized plasma and thrombin are avail-
able commercially.200 Similarly, virus-inactivat-
ed fibrinogen concentrates are available that
are more readily stored (lyophilized) and pack-
aged for simple use (discussed below).201
GRANULOCY T E TRANSFUSION
The development of apheresis technology sev-
eral decades ago allowed the collection of
granulocytes (neutrophils) to transfuse to pa-
tients with neutropenia and serious infections.
Most of these patients were not able to mount
an appropriate response because of marrow
hypoplasia (eg, due to chemotherapy). The
524 䡲 AABB TECHNICAL MANUAL

TABLE 20-9. General Factor Replacement Guidelines for Treatment of Hemophilia A and von
Willebrand Disease

Minimum Desired Factor VIII Dose Factor IX Duration

Indication Factor Level (%) (IU/kg) Dose (IU/kg) (days)

Hemophilia*
Severe epistaxis, oral mucosal 20-30 10-15 20-30 1-2
bleeding†
Hemarthrosis, hematoma, persistent 30-50 15-25 30-50 1-3
hematuria,‡ gastrointestinal bleed-
ing, retroperitoneal bleeding
Trauma without signs of bleeding, 40-50 20-25 40-50 2-4
tongue/retropharyngeal bleeding†
Trauma with bleeding, surgery, 100 50 100 10-14
intracranial bleeding§
von Willebrand Disease¶
Major surgery 50 40-60 daily
Minor surgery 30 30-50 daily or every other day
Dental extractions 30 20-30, single dose 12 hours
Spontaneous bleeding 30 20-30, single dose
*Data from US Pharmacopeia.197 Dosing intervals based on a half-life of Factor VIII over 8-12 hours (2-3 doses/day) and
half-life of Factor IX over 18-24 hours (1-2 doses/day). Maintenance doses of one-half the initial dose (as shown) may be
given at these intervals. The dosing frequency depends on the severity of bleeding, with more frequent dosing used for seri-
ous bleeding.
†
In addition to antifibrinolytics.
‡
Painless spontaneous hematuria usually requires no treatment. Increased oral or intravenous fluids are necessary to main-
tain renal output.
§
Factor may be administered continuously. Following the initial loading dose, a continuous infusion at a dose of 3 IU/kg per
hour is given. Subsequent doses are adjusted according to measured plasma factor levels.
¶
Concentrates labeled in terms of the ratio of von Willebrand factor to ristocetin cofactor. The recommended doses for
adults, number of infusions, and target plasma levels are the same as those for Factor VIII.198

therapy was also administered to patients with
congenital neutrophil dysfunction [eg, chronic
granulomatous disease (CGD)]. Not all pa-
tients benefited from this approach, however,
and only modest improvements in outcome
were noted in a majority of—but not all—
trials.202-206 The published literature offers re-
views of this and other topics related to granu-
locyte transfusion therapy.207,208
With the development of more potent an-
timicrobial drugs and the availability of cyto-
kines that promote more rapid marrow recov-
ery of granulocyte production, differentiation,
and function [such as granulocyte colony-
stimulating factor (G-CSF) and granulocyte/
macrophage colony-stimulating factor], the
frequency of granulocyte transfusion fell to
very low levels.209,210 This low utilization was
also prompted by the recognition that the
amount of granulocytes that could be collect-
ed from a donor, even one mobilized with pri-
or administration of a corticosteroid, on the
order of 1 × 1010, was only 1% to 10% of what a
healthy marrow would produce each day in re-
 CHAPTER 20
sponse to a serious infection. Trials whose au-
thors reported success in using granulocyte
transfusion therapy generally transfused high-
er doses of granulocytes, but these higher
yields were very difficult to obtain.
More recently, the concept of granulocyte
transfusion therapy using components with
higher, “more therapeutic” content has gener-
ated renewed attention to this component.
Despite the availability of newer antimicrobial
agents, infection remains a common problem
in patients undergoing rigorous chemothera-
py regimens, especially those culminating in
hematopoietic stem cell transplantation, and
more rigorous T-cell depletion schemes in al-
logeneic hematopoietic transplantation that
lead to an increased frequency of serious and
fatal infections, usually due to yeasts and
fungi.211,212 Case reports of success in treating
patients with CGD with fungal infections dur-
ing transplantation have also rekindled inter-
est in granulocyte transfusion.213,214
With the administration to donors of 8 mg
dexamethasone orally and 5 µg/kg G-CSF sub-
cutaneously 8 to 16 hours before apheresis,
yields of up to 1011 granulocytes (or more) have
been reported.215 A clinical trial to document
the effectiveness of these more potent compo-
nents is under way through the Transfusion
Medicine Hemostasis Clinical Trials Network
of the National Heart, Lung and Blood Insti-
tute. This use of G-CSF is not approved by the
FDA, and donor informed consent should be
obtained before using this approach. In addi-
tion, the suggestion that corticosteroid admin-
istration may lead to posterior subcapsular
cataracts has prompted some clinicians to
consider avoiding the dexamethasone admin-
istration to donors even though this decreases
yield by one-quarter.216 The potential utility of
prophylactic granulocyte transfusions remains
unclear at present, again owing to dosage con-
siderations.217
Neonates born preterm or after pro-
longed premature rupture of membranes are
at increased risk of bacterial sepsis. Their neu-
tropenia is related to a temporary limitation in
the production of neutrophils from a marrow
primed to produce red cells. Some of these pa-
tients have been treated with transfusions of
Hemotherapy Decisions 䡲 525
granulocytes generated either through an
apheresis procedure or from whole-blood-
derived buffy coats. The latter can provide
only a limited number of cells and has not
been found to be as clinically efficacious as
apheresis.218 Today, potent antibiotics are used
much more commonly than granulocyte
transfusions in neonates. IVIG may also be giv-
en because premature infants may have hypo-
gammaglobulinemia,219 although IVIG may in-
crease these infants’ susceptibility to other
potentially fatal infections.220
Patients with severe neutropenia (abso-
lute neutrophil count <500/µL) are consid-
ered for granulocyte therapy if the infection
cannot be controlled with appropriate bacteri-
cidal antimicrobials and the neutropenia is
temporary, meaning that recovery of endoge-
nous production in a few days is likely. Patients
with CGD are usually considered for granulo-
cyte transfusions if they have deep-seated ab-
scesses and/or fungal infections that are not
responding to antimicrobial therapy.
Components must be ABO compatible if
the granulocyte collection contains 2 mL or
more of red cells, as is often the case. If the pa-
tient requires CMV-reduced-risk transfusions,
the donor should be CMV seronegative be-
cause leukocyte reduction cannot be used on
these components. If the recipient is alloim-
munized to HLA antigens, HLA-compatible
donors need to be found or the transfused
cells could be cleared rapidly and possibly
cause untoward reactions without achieving
their intended purpose. If patients are at risk
of transfusion-associated graft-vs-host dis-
ease, as is usually the case for patients with
neutropenia, units may be gamma irradiated.
Donors often undergo infectious disease
testing when they present for predonation
stimulation to facilitate rapid release of the
collected component following documenta-
tion of ABO/Rh type. Granulocyte units should
be stored for the shortest period possible at
room temperature without agitation and ad-
ministered through regular blood component
filters (eg, 170 microns). Drugs known to inter-
act with granulocytes, such as amphotericin,
are best given at times remote from the granu-
locyte transfusions (ie, 12 hours before or
526 䡲 AABB TECHNICAL MANUAL
after). Transfusions are usually given daily, and
sometimes more frequently if yields are low,
until the patient recovers from the infection
and/or neutrophil production returns.
PL AS MA-D ER IVATIVE
TR ANSF USI O N
Plasma proteins are derived from both source
plasma, collected for the purpose of manufac-
turing plasma derivatives, and recovered plas-
ma that has been separated from whole-blood
donations. In the United States, approximately
6 million liters of source plasma and 2.5 mil-
lion units of recovered plasma are produced
each year. The final products are subjected to
one or more virus-inactivation techniques.
Additional testing (eg, for hepatitis A virus or
parvovirus) may be performed by nucleic acid
amplification techniques to add an additional
margin of safety.
The prions associated with variant
Creutzfeldt-Jakob disease (vCJD) appear to be
successively removed through multiple steps
in the separation process to levels that are be-
yond the limit of detection. Neither classical
CJD nor vCJD cases have reportedly been at-
tributed to the transfusion of plasma deriva-
tives.221
The pathogen-inactivation techniques
described above have an excellent record of
not transmitting infectious diseases. However,
most patients with hemophilia in the United
States prefer transfusions of Factor VIII or Fac-
tor IX concentrates produced via recombinant
DNA technology and, whenever possible, that
have not been manufactured using any human
proteins.
Immunoglobin products have often been
regarded as free of infectious risks because of
their production method and/or neutralizing
antibodies. However, multiple examples of
hepatitis C transmission have raised doubts
regarding this belief, although the units in-
volved in these cases were usually prepared
through nonstandard donor-selection or pro-
duction methods.222-225
Albumin
Albumin was the first of the modern plasma
proteins to be developed for transfusion. It
serves as a colloid, expanding plasma volume
by approximately the volume administered at
the 5% concentration with a half-life of ap-
proximately 16 hours.226 Unless the container
has been damaged, albumin’s pasteurization
at 60 C for 10 hours prevents the transmission
of viruses or bacteria.
There are several obvious and accepted
applications for albumin, including volume
replacement in patients who are unresponsive
to crystalloids, postparacentesis volume man-
agement in patients who are unresponsive to
colloids, volume replacement in patients with
severe necrotizing pancreatitis, patients with
diarrhea (>2 L/d) or hypoalbuminemia (<2.0
d/dL) on enteral feedings who do not respond
to short-chain peptide supplementation, and
plasmapheresis.227 Use of albumin infusions
simply to raise the albumin concentration in
patients with cachexia or other chronic hypo-
albuminemia is ineffective and inappropriate
but may be helpful in treating the complica-
tion of acute hypoalbuminemia if a concentra-
tion of at least 3.0 g/dL can be achieved.228
In most cases of hypovolemia, crystal-
loids are used as the primary means of volume
replacement because of their effectiveness,
availability, and cost. A meta-analysis of recent
RCTs found no decrease in mortality associat-
ed with the use of colloids compared to crys-
talloids in critically ill patients, with no benefit
from albumin vs crystalloid specifically.229
When colloid support is required, other, non-
human sources, such as hydroxyethyl starch
and dextran, may also be useful for both clini-
cal use and apheresis, although the volume
that can be administered in a day without af-
fecting coagulation function may be limited.230
Synthetic substitutes may prompt an anaphy-
lactic reaction in rare cases and have been as-
sociated with a syndrome of pruritus begin-
ning several weeks after administration.231
Clotting Factor Concentrates
A complete discussion of the treatment of he-
mophilia A (Factor VIII deficiency) and hemo-
 CHAPTER 20
philia B (Factor IX) deficiency is beyond the
scope of this text. Although the first effective
treatment for hemophilia A was cryoprecipi-
tate transfusion, patients with moderate (1%-
5% Factor VIII activity) or severe (<1% activity)
disease are now treated exclusively with a
lyophilized concentrate. The same treatment
has evolved for patients deficient in Factor IX,
also an X-linked recessive condition.
For both conditions, the dose of the factor
to be administered can be easily calculated
based on the patient’s plasma volume [body
mass  70 mL/kg  (1 – hematocrit)] multi-
plied by the desired increase in activity level.
Thus, a 70-kg male (hematocrit = 40%) with 1%
activity who needs his activity level raised by
99% (0.99 IU/mL) to 100% (1 IU/mL) would re-
quire approximately 2900 IU of Factor VIII. Be-
cause the half-life of Factor VIII is 8 to 12
hours, repeat doses will be required to ensure
hemostasis (ie, >30% activity) through surgery
or a bleeding episode (often a period of at least
10 days); see Table 20-9.
Because recovery of activity may vary by
product and patient, close follow-up of pa-
tients using repetitive assays of factor activity
is advised. Patients may also be maintained on
regular prophylactic doses of Factor VIII to re-
duce the risk of spontaneous hemorrhage and
preserve joint function, although the value of
this approach has been difficult to prove be-
cause of the small sizes of most trials.232,233
Nevertheless, prophylaxis is a widely recom-
mended course of action.
About one-third of patients with severe
hemophilia A develop antibodies against Fac-
tor VIII because they do not produce it or they
produce a defective form. These patients can
be very difficult to support because it is im-
practical to overwhelm the inhibitor (anti-
body) with higher and higher doses of Factor
VIII to stem hemorrhage. A variety of treat-
ments have been tried, including a high-dose
desensitization program, immunosuppres-
sion, and, when rapid reduction in inhibitor ti-
ters is needed, plasmapheresis. The advent of
recombinant Factor VIIa to bypass the inhibi-
tion of Factor VIII activity has improved the
management of bleeding in this dreaded com-
plication of hemophilia therapy.
Hemotherapy Decisions 䡲 527
Prothrombin Complex Concentrates
Procoagulants that require vitamin K for ap-
propriate carboxylation and normal activity—
Factors II (prothrombin), VII, IX and X—are
called the “vitamin K-dependent factors.”
They have long been known to be separable
from plasma by simple chemical means and
have been available as Factor IX complex con-
centrate or PCC. This product should not be
confused with a Factor IX concentrate used to
treat hemophilia B. The latter, now also avail-
able as a recombinant product, contains only
Factor IX.
The first technique to produce PCC re-
sulted in activation of a substantial propor-
tion of some procoagulants and sometimes led
to unwanted thromboses. This complication
precluded its routine use, but the product was
beneficial in bypassing the effect of Factor VIII
inhibitors in some patients with hemophilia A.
This activated form of PCC concentrate was
known for its Factor VIII inhibitor bypassing
activity. Subsequent development of PCC with
minimal amounts of activated factors has al-
lowed its reconsideration for broader use, par-
ticularly as an antidote to warfarin overdos-
age.175
Four-factor PCCs have been available in
Europe for several years and have demonstrat-
ed rapid correction of coagulation status. In
2013, a four-factor PCC was approved by the
FDA for reversal of acute major bleeding for
patients taking vitamin K antagonists.234 Be-
cause the product (Kcentra, CSL Behring,
Kankakee, IL) contains adequate concentra-
tions of coagulation Factors II, VII, IX, and X,
plasma infusion should not be necessary.
Experience with this product is limited at the
time of this writing and its value for reversal of
direct thrombin inhibitors or anti-Xa inhibi-
tors is not established.
The administration of PCCs in other off-
label settings (eg, massive hemorrhage in the
absence of warfarin or intracranial pressure
monitor placement in fulminant liver failure)
should be weighed against the risk of throm-
bosis from giving, in the case of PCCs, a non-
activated but supraphysiologic concentration
of clotting factors. The clinical trials preceding
528 䡲 AABB TECHNICAL MANUAL
FDA approval did not include patients who
had experienced acute thromboembolic
events, myocardial infarction, DIC, stroke, un-
stable angina, or severe peripheral vascular
disease within 3 months before administra-
tion. For this reason, the package insert indi-
cates that the product may not be suitable in
patients who have experienced thromboem-
bolic events in the prior 3 months. In patients
with a history of HIT who are subsequently
shown to be negative for HIT on enzyme-
linked immunosorbent assay, exposure is like-
ly to be safe because the heparin will be gone
by the time the HIT antibody is recalled. How-
ever, use is contraindicated in patients with
known HIT. The reduced capacity of a cirrhotic
liver to clear the circulating byproducts of the
coagulation cascade, such as D-dimers, may
lead to DIC or other derangements of the co-
agulation system. Therefore, the use of PCC in
patients with liver disease remains risky.173
Recombinant Factor VIIa
The production of the activated form of coagu-
lation Factor VII via recombinant Factor VIIa
(rFVIIa) has led to the exploration of this drug
as a means of addressing a wide variety of
hemorrhagic conditions. The drug was devel-
oped and is approved for use in bypassing an-
tibodies against Factor VIII in patients with he-
mophilia A and achieving hemostasis without
needing to achieve hemostatic levels of Factor
VIII in the face of potent antibodies. It can also
be used to address congenital deficiency of
Factor VII.235
Beyond these applications, rFVIIa been
considered for use in a wide variety of condi-
tions that are not related to hemophilia or Fac-
tor VII deficiency in which bleeding is difficult
to control or is threatening the patient’s life.
For example, rFVIIa has been used to counter-
act the effect of warfarin by replacing the inac-
tive factor (produced in the presence of the vi-
tamin K antagonist) with active rFVIIa that can
immediately stimulate the downstream proco-
agulants and produce fibrin.236 The use of
rFVIIa has also received much attention in
trauma treatment, liver transplantation, and
massive transfusion, where correction of pro-
coagulant deficiency and achievement of he-
mostasis can be challenging.237,238 However,
rFVIIa is less effective in patients with signifi-
cant acidosis or hypothermia.
In a registry that collected reports of ad-
verse events after use of rFVIIa, the drug was
listed as a contributing cause of death for a
large proportion of patients who died after its
administration.239 However, in a review of 35
placebo-control trials involving 4468 people, a
significant increase in arterial thrombosis, but
not venous thrombosis, was attributed to
rFVIIa use.240 A recent Cochrane analysis iden-
tified 29 RCTs with 4290 patients who were
treated off label with rFVIIa in a variety of clin-
ical situations. The meta-analysis included
separate analyses of 16 studies of prophylactic
use and 13 of therapeutic use in addition to an
analysis of all of the trials. The authors found
no effect on mortality in the prophylactic
group and a trend toward decreased mortality
in the therapeutic group. Bleeding and trans-
fusion rate outcomes favored rFVIIa use, but
these data were only reported in the smaller
studies, so these findings are of uncertain val-
ue. Of concern was a trend toward increased
thromboembolic events in the therapeutic
group. When all studies were analyzed togeth-
er, a statistically significant increase in arterial
thromboembolic events was found (RR 1.45;
95% CI 1.02 to 2.05).241 Given the open ques-
tion of whether any adverse events are related
to unwanted clotting and the exclusion of pa-
tients with a propensity to clot, including
those with atherosclerotic disease, from many
of the trials, it may be prudent to refrain from
employing rFVIIa in patients who might be at
increased risk of unwanted thrombosis.
A substantial stumbling block in the use
of rFVIIa has been its cost. If it is effective in
substantially reducing hemorrhage or morbid-
ity, then its use might be cost-effective. Anoth-
er approach is to adopt a standardized rather
than weight-based dosing practice, which re-
duces consumption of the drug and appears to
provide similar clinical results.242 Clinical con-
sultation by a transfusion medicine specialist
may be very helpful in guiding the use of this
product.243
 CHAPTER 20
Fibrinogen Concentrate
Originally, fibrinogen concentrate, which de-
livers a small volume of purified plasma-de-
rived factor, was approved by the FDA for
treatment of dysfibrinogenemia or deficiency.
As the pathogenesis of coagulopathy of trau-
ma and surgery and the importance of fibrino-
lysis in these situations is better understood,
the use of fibrinogen concentrate in these situ-
ations is being explored.182 Although cryopre-
cipitate can also replace fibrinogen, fibrinogen
concentrate can be prepared in advance with-
out the storage and thawing issues that arise
with cryoprecipitate.
A systematic review of the use of fibrino-
gen concentrate and plasma in patients at risk
of bleeding found 1) decreased bleeding, 2) re-
duced transfusion requirements, and 3) a low
rate of adverse events in patients treated with
fibrinogen concentrate. Results for bleeding
and transfusion requirements were inconsis-
tent in plasma-treated patients.201 The hetero-
geneity among the studies precluded any true
meta-analysis, but the overall trend favored
use of fibrinogen concentrate early in care.
Intravenous Immune Globulin
Concentrates of plasma immune globulins
were developed to treat congenital immuno-
deficiencies and treat or provide prophylaxis
against certain viral exposures. Because im-
mune globulins tend to polymerize during
Cohn fractionation and purification, initial
preparations were given intramuscularly to
avoid severe hypotensive and/or anaphylac-
toid reactions. Subsequent development of a
variety of chemical modifications has allowed
the development of IVIG preparations that
have low proportions of aggregates. These
products allow the administration of larger
quantities over shorter intervals to achieve
more pronounced clinical effects.
Although the initial intended use of IVIG
was to reduce infections in immunodeficient
patients, the optimal dose for this indication
remains under discussion. The clinical appli-
cation of IVIG has extended far beyond this in-
dication (Table 20-10).244 For a thorough re-
view of the indications for IVIG, see the report
Hemotherapy Decisions 䡲 529
by Durabi and colleagues.245 When used for the
treatment of hypogammaglobulinemia, an
IVIG dose of 600 mg/kg in adults or 800 mg/kg
in children every 4 weeks rather than the usual
300 mg/kg in adults or 400 mg/kg in children
slightly reduces the frequency and duration of
infections in patients with primary immune
deficiencies.246 However, the cost-effective-
ness of increased doses may be poor, and
these doses accelerate usage rates.
The ever-increasing off-label use of IVIG
poses an additional challenge to the adequacy
of the supply for treating the 50,000 patients
with congenital hypogammaglobulinemia in
the United States. The FDA-approved indica-
tions for IVIG account for only 30% of its us-
age. The other situations in which IVIG is
administered may have greater or lesser scien-
tific support, but the usage rate in developed
countries appears to be increasing at a rate of
approximately 10% per year. The wide vari-
ability of doses used (from < 0.03 g per person
in Australia to more than 0.06 g per person in
the United States and Canada) without con-
comitant variations in outcomes, however,
raises the question of the true utility of some of
these applications.
The administration of IVIG can be associ-
ated with a wide variety of adverse events (Ta-
ble 20-11). Some of these effects appear to
result from the use of idiosyncratic combina-
tions of a particular product with a particular
patient. Therefore, advantage should be taken
of the wide variety of IVIG products in the
marketplace to find the form of the medica-
tion that is well tolerated by a given patient.247
Slow rates of infusion and pretreatment with
antihistamines and/or steroids usually pre-
vent reactions. Clinically dramatic reactions
usually occur only in patients with agamma-
globulinemia who have not previously been
treated and who have an infection. IgA may be
present in sufficient concentration in some
preparations to prompt an anti-IgA reaction in
sensitized patients.
Although the antibody specificities in
IVIG products represent the range and relative
concentrations of the donors’ immune globu-
lin responses and, thus, may convey varying
degrees of protection against particular patho-
530 䡲 AABB TECHNICAL MANUAL

TABLE 20-10. Applications of Intravenous Immune Globulin

Primary Immune Deficiencies

Hypo/agammaglobulinemia
Selective antibody deficiency
Class/subclass deficiency with recurrent infection
Secondary (Acquired) Immune Deficiencies
Chronic lymphocytic leukemia
Multiple myeloma
Prevention of cytomegalovirus pneumonitis after hematopoietic stem cell transplantation
Reduction of bacterial infection in children with acquired immunodeficiency syndrome
Immune Cytopenias
(Auto)immune thrombocytopenic purpura [idiopathic thrombocytopenia (ITP)]*
Pure red cell aplasia
Other cytopenias: neonatal alloimmune thrombocytopenia, posttransfusion purpura, and warm autoimmune
hemolytic anemia
Human immunodeficiency virus-related idiopathic thrombocytopenia
(Presumed) Autoimmune Disorders
Kawasaki disease
Guillain-Barré syndrome
Multiple sclerosis
Myasthenia gravis
Dermatomyositis
Systemic vasculitides
Factor VIII inhibitor deficiency (congenital/acquired hemophilia)
Prevention/treatment of infections
Post-hematopoietic stem cell transplantation
Post-solid-organ transplantation and immunosuppression
Parvovirus infection
Neonatal sepsis
Other (Presumed) Immunologic Conditions
Recurrent spontaneous abortion
Graft-vs-host disease
Asthma
Myocarditis
 CHAPTER 20
TABLE 20-10. Applications of Intravenous Immune Globulin (Continued)
Inflammatory bowel disease
Stevens-Johnson syndrome
Other Conditions
Alzheimer’s disease
Predisposition to atherosclerosis
Autism
Chronic fatigue syndrome
Multifocal motor neuropathy
Rh prophylaxis (if patient cannot receive intramuscular product)
Approved indications shown in bold. Commonly accepted indications shown in italics.
*Usually used only in chronic ITP because most acute ITP cases are self-limited. (Many other agents potentially effective in
ITP are being developed.244
gens, the large pool sizes used to manufacture
these products reduces this variability. Hyper-
immune globulins are manufactured from
samples provided by donors chosen for their
higher titers of activity against selected agents,
such as hepatitis A or B virus, tetanus, rabies,
varicella-zoster virus, or CMV, but are usually
available only in the intramuscular form.
The products may, on occasion, convey
sufficient isoagglutinins or red cell alloanti-
bodies of a particular specificity (usually anti-
D) to cause a positive direct antiglobulin test
result in the recipient, but clinically detectable
or significant hemolysis following use of these
products is rare.248 Administration of hyperim-
mune anti-D to an Rh-positive patient for
treatment of ITP may induce a life-threatening
or fatal acute hemolysis, and the product car-
ries a black-box warning to remind physicians
of this potential outcome.
Antithrombin
Antithrombin circulates in plasma as a serine
protease inhibitor that inactivates the serine
proteases of the coagulation system, including
thrombin and Factors IXa, Xa, XIa, and XIIa, by
covalent binding at their serine active site.249
The ability of antithrombin to accomplish this
Hemotherapy Decisions 䡲 531
inhibition is greatly accelerated by heparin,
which induces a change in the polypeptide’s
conformation; this is why antithrombin is
known as “heparin cofactor.”
Patients who are congenitally deficient in
antithrombin have an increased risk of devel-
oping thrombosis.250 Acquired deficiency of
antithrombin can also occur due to reduced
antithrombin synthesis (as in hepatic dis-
ease), increased antithrombin loss (as in a ne-
phrotic syndrome), increased antithrombin
breakdown (eg, due to L-asparaginase treat-
ment), or increased antithrombin consump-
tion (eg, in DIC, trauma, or surgery). The ad-
ministration of heparin also accelerates the
metabolism of antithrombin and can lead to a
relative resistance to heparin.250
Although antithrombin is stable in frozen
or thawed plasma, antithrombin deficiencies
are usually addressed through the infusion of
antithrombin concentrate. Congenital anti-
thrombin deficiency is rare. However, anti-
thrombin has been used in the treatment of
sepsis and DIC to stem thrombotic complica-
tions and in patients being heparinized for
extracorporeal circulation who do not
experience the expected effects of heparin
treatment.
532 䡲 AABB TECHNICAL MANUAL

TABLE 20-11. Adverse Events Associated with Activated Protein C

Intravenous Immune Globulin Use Protein C is a serine protease that acts with
Infusion Associated protein S to hydrolyze and thus inactivate Fac-
tors Va and VIIIa. It therefore serves an anti-
Fever Chest tightness thrombotic regulatory function. Protein C is
normally activated along the prothrombotic
Chills Dyspnea
cascade, which leads to an innate regulation of
Facial flushing Wheezing the clotting system. When given as an infusion
over 96 hours to patients with sepsis, recombi-
Tachycardia Hypotension
nant activated protein C concentrate reduced
Palpitations Anxiety mortality by 19%, although the risk of serious
bleeding almost doubled.251
Abdominal pain Nausea

Headache Vomiting 1-Antitrypsin

Lumbar pain
Other Possible Adverse
Events
Volume overload
Arterial thrombosis
(myocardial infarction,
stroke)
Venous thromboembolism
Disseminated intravascular
coagulation
Hemolysis (due to alloanti-
bodies/isoagglutinins)
Nephrotoxicity
Neutropenia
Urticarial rash -Antitrypsin (also known as 1-proteinase
inhibitor) is a natural inhibitor of the elastase
elaborated by activated polymorphonuclear
leukocytes. If the activity of elastase is left un-
checked, as in congenital deficiencies of 1-
antitrypsin, excessive damage to pulmonary
parenchyma occurs after even minor infec-
tions, leading to development of fatal emphy-
sema at an early age in the 100,000 patients in
the United States with this deficiency. Hepatic
cirrhosis can also occur.
Until the development of several plasma-
derived replacements,252 congenitally deficient
patients could stem the advance of tissue de-
struction only by attempting to avoid and rap-
idly treat respiratory infections. Periodic (usu-
ally weekly) infusions of 1-antitrypsin can be
administered prophylactically to prevent addi-
tional lung damage in these patients.

KEY POINTS

1. Transfusion in the presence of an autoantibody that precludes a negative crossmatch is safe

and could be life-saving, provided that alloantibodies can be excluded.
2. The data needed to support evidence-based guidelines for RBC transfusion have become
stronger over the past several years. The data needed to support plasma transfusion remain
surprisingly weak.
3. Compared to amounts of circulating platelets and coagulation factors in healthy people, the
amount required to achieve hemostasis is surprisingly small. When component therapy is
used, the aim is to achieve hemostatic levels rather than normal amounts.
4. The laboratory test results that are currently available to aid in component therapy deci-
sions do not correlate with an individual patient’s physiologic and hemostatic state.
 CHAPTER 20 Hemotherapy Decisions 䡲 533

5. Data on using blood components to reverse the effects of newer antiplatelet agents and an-
ticoagulants are very limited, as are the data on using clotting factor concentrates.
6. In the setting of hemorrhagic shock, the current data support the transfusion of platelets,
plasma, and cryoprecipitate early in the resuscitation effort. An increased ratio of these
products to RBCs, often referred to as a 1:1:1 protocol, has been shown to decrease mortality
in one large prospective cohort study and a series of retrospective studies.
7. For patients without underlying cardiac disease, the data support using an RBC transfusion
threshold of 7 to 8 g/dL of hemoglobin. For patients with underlying cardiac disease, cur-
rent guidelines recommend a threshold of 8 g/dL of hemoglobin or less. Data are lacking on
the appropriate transfusion threshold in patients with acute coronary syndrome; therefore,
a transfusion trigger cannot be established in this setting at this time. The decision to trans-
fuse RBCs should also take into account the patient’s clinical situation and response to
anemia.
8. Although prophylactic platelet transfusions during aplasia are common practice, it is uncer-
tain whether a prophylactic vs a therapeutic transfusion approach is optimal.
9. The most common prophylactic transfusion threshold is 10,000 platelets/µL. The current
standard prophylactic dose of platelets of 3 to 4 × 1011 for an adult may be halved without
risk of bleeding.
10. When platelets bearing ABO antigens that are foreign to the recipient are transfused, the ef-
fect on the platelet count may be blunted. When plasma in platelet units contains antibod-
ies against A and/or B antigens expressed on the recipient red cells, hemolysis may occur,
with a 1:3000 to 1:10,000 risk from apheresis platelets. Steps are often taken to limit the
amount of incompatible plasma transfused or to avoid high-titer units, especially for pedi-
atric patients, when time permits.
11. Alloimmunized platelet refractory patients may be supported by transfusion of platelets
from donors lacking the targeted epitopes by either phenotyping the donor (unit) or provid-
ing HLA-matched units to patients with HLA alloimmunization.
12. There are very limited data to suggest a benefit in transfusing plasma in settings other than
intracranial hemorrhage after anticoagulation with vitamin K antagonists or massive trans-
fusion.

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 C h a p t e r 2 1

Administration of Blood
Components

Kim Maynard, BSN, RN, OCN

T H E S A F E A D M I N I S T R A T I O N of
blood and its components requires mul-
tidisciplinary collaboration among clinical
and ancillary services and clinicians. Policies
and procedures should be developed with in-
put from transfusionists, the transfusion ser-
vice, surgeons, anesthesiology care providers,
primary-care physicians, and transport per-
sonnel. The transfusionist typically provides
the last line of defense in the detection of er-
rors before the transfusion commences. All
personnel involved in the chain of preparing,
delivering, and administering a transfusion
should be given appropriate training to ensure
the provision of the safest transfusion possible
for patients.
EV E N TS A ND CO N S I D E R AT I O N S
BEFORE DISPENSING
CO MPO N EN TS
Before a transfusion begins, thoughtful con-
sideration, planning, and preparation are re-
quired.
Recipient Consent
The AABB Standards for Blood Banks and
Transfusion Services states, “The blood bank or
transfusion service medical director shall par-
ticipate in the development of policies, pro-
cesses, and procedures regarding recipient
consent for transfusion.”1(p44) Recipient in-
formed consent should address indications
for; risks, benefits, and possible side effects of;
and alternatives to transfusions of allogeneic
blood components. Some state laws require
certain additional elements in the patient con-
sent.
The patient has the right to choose or re-
fuse a transfusion and must have an opportu-
nity to ask questions of a learned professional
before providing consent. Documentation of
the consent process must be entered into the
patient’s medical record. Some facilities re-
quire an institution-approved signed consent
form to document that the consent process
has occurred and the risks/benefits of transfu-
sion were discussed with the patient or legal

Kim Maynard, BSN, RN, OCN, Breast Coordinator (former Transfusion Safety Officer, Transfusion Medicine
Service), Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire
The author has disclosed no conflicts of interest.

545
546 䡲 AABB TECHNICAL MANUAL
representative. Each institution needs to have
a process for recording a patient’s refusal to re-
ceive blood or blood components in the pa-
tient’s medical record. Institutional policies
should identify health-care providers who are
allowed to obtain consent and the length of
time for which that consent remains valid.
Consent for transfusion must be ob-
tained from patients who have the requisite
capacity to make such decisions. If a patient is
unable to give consent, a legally authorized
representative or surrogate may do so (de-
pending on local and state laws). If no one is
available to provide consent and the need for
transfusion is considered a medical emergen-
cy, the blood component may be administered
based on the doctrine of implied consent. In-
dividual state and local laws governing re-
quirements for implied consent may vary, but
the emergent need for transfusion should be
carefully documented in the medical record.2
Patient Education and History
The transfusionist should educate the patient
about reporting any symptoms that may be in-
dicative of a reaction and how long the trans-
fusion will take. The patient’s questions should
be answered before the transfusion is started.
It is important to collect a history from
the patient before the component is ordered to
assess whether the patient is at increased risk
of a transfusion reaction. This history includes
previous transfusions and any adverse reac-
tions. If the patient has had previous reactions
to transfusions, the medical team should de-
termine whether the patient needs to receive
medications before the transfusion or wheth-
er special processing of the component is indi-
cated to mitigate the risk of an adverse reac-
tion.
Baseline Assessment of Recipient
A baseline physical assessment should include
vital signs. Many institutions also routinely
measure oxygen saturation using oximetry.
The assessment should include pretransfusion
symptoms, such as shortness of breath, rash,
pruritus, wheezing, and chills, as a basis for
comparison after the transfusion starts. A pa-
tient with renal or cardiopulmonary disease
may require a slower infusion rate to prevent
fluid overload, for example.
A patient with an elevated temperature
may destroy cellular components at an in-
creased rate.3 Moreover, if the patient presents
with an elevated temperature before the trans-
fusion, it may be difficult to determine later
whether this was caused by a transfusion reac-
tion. Administration of an antipyretic should
be considered in such cases.
Medical Order
Blood Component
A licensed provider writes two orders for the
components to be administered. The first or-
der requests that compatible crossmatch (if
appropriate) unit(s) be prepared for the pa-
tient and notes special processing require-
ments. The second order explains to the trans-
fusionist how to administer the component,
including the transfusion rate. Both of these
orders should specify:
䡲 Patient name and other independent iden-
tifier (eg, date of birth or medical record
number).
䡲 Component [eg, Red Blood Cells (RBCs) or
apheresis platelets] to prepare or adminis-
ter.
䡲 Special processing required (eg, leukocyte
reduction, irradiation, or washing).
䡲 Number of units or volume to administer.
䡲 Date and time for the infusion.
䡲 Flow rate or period for administering the
component.
Note: It is appropriate for the rate and du-
ration of the transfusion (eg, not to exceed 4
hours from the time that the container is en-
tered until completion)4 to be described in a
policy approved by the hospital’s medical staff.
Laboratory Testing
After receiving an order from a licensed pro-
vider, the transfusion service ensures that
compatible components are provided. To issue
blood components for a specific patient (ex-
 CHAPTER 21 Administration of Blood Components
cept in the case of an emergency transfusion),
the laboratory must collect a pretransfusion
blood sample. Typically, the sample is ob-
tained within 3 days of transfusion, with the
draw date considered to be day 0.1(p36) Institu-
tional policies may vary regarding the sample
outdate. If the patient has not had a transfu-
sion or been pregnant in the prior 3 months,
the sample may be acceptable for longer than
3 days for testing purposes.
According to The Joint Commission, con-
tainers used for blood specimens must be la-
beled in the presence of the patient.5 The sam-
ple must be labeled at the patient’s side with at
least two unique identifiers (eg, the patient’s
name, date of birth, or identification number).
The identification of the person collecting the
sample and the date on which the sample was
collected must be traceable.1(p35)
Pretransfusion testing of the recipient’s
blood sample, including for unexpected anti-
bodies to red cell antigens, is described in de-
tail in Chapters 15 and 16. The time interval
from sample receipt to availability of the re-
quested component can vary greatly. One
reason is that availability depends on the in-
ventory of components maintained by the
transfusing facility, and some components
may need to be obtained from an external sup-
plier. Furthermore, if the test results from the
pretransfusion sample show that the patient
has clinically significant red cell antibodies,
obtaining corresponding antigen-negative or
crossmatch-compatible units may require ad-
ditional time. Moreover, some components re-
quire thawing, pooling, relabeling, or other
preparation before release. All of these factors
necessitate timely communication between
laboratory and transfusing personnel. For ex-
ample, components that are pooled or require
thawing may have a shortened shelf life after
being prepared (4-24 hours); transfusionists
should be aware of the decreased time avail-
able to complete a transfusion of such compo-
nents.1(pp51-59)
Venous Access
The transfusionist must determine whether
the patient’s venous access [with a central line,
䡲 547
peripheral intravenous line, implanted port, or
peripherally inserted central catheter (PICC)]
is acceptable for the infusion of blood compo-
nents.
Acceptable intravenous catheter sizes for
use in transfusing cellular blood components
range from 22 to 14 gauge.6 A 20- to 18-gauge
intravenous catheter is suitable for the general
adult population and provides adequate flow
rates without excessive discomfort to the pa-
tient. When an infant or a toddler is trans-
fused, a 24- to 22-gauge intravenous catheter
may be suitable but requires infusion through
a syringe (see Chapter 23).7 The flow rate may
need to be adjusted depending on the catheter
size.
There is an increased possibility of hemo-
lysis when red cells are transfused rapidly us-
ing handheld syringes through 23-gauge or
smaller needles.8 With the use of smaller cath-
eters, blood dilution and a pump are helpful to
administer the unit to prevent slow flow rates
that lead to clogging of the intravenous cathe-
ter. Units suspended in a preservative solution,
such as Adsol, usually do not require addition-
al dilution. It is important that the infusion
line be patent before the component is re-
ceived at the patient’s bedside.
Prophylactic Medications Given
Before Transfusion
Although antipyretics (eg, acetaminophen)
were commonly ordered in the past to reduce
the risk of febrile nonhemolytic transfusion re-
actions, indications for their use are contro-
versial.9 Some providers use antipyretics in a
prophylactic manner, some wait to use them
until the patient has experienced at least one
febrile transfusion reaction, and others believe
that prophylactic use of antipyretics may mask
the elevated temperature that results from a
transfusion reaction. Evidence supporting any
of these approaches is limited.9-12 Some experts
recommend that “in the absence of definitive
evidence-based studies, pretransfusion medi-
cation to prevent transfusion reactions should
not be encouraged.”13
In a Cochrane review14 of studies on pre-
medication to prevent allergic reactions and
548 䡲 AABB TECHNICAL MANUAL
febrile nonhemolytic transfusion reactions
(FNHTRs), the authors stated that current evi-
dence from three randomized controlled trials
(RCTs) involving 462 patients indicate that no
pretransfusion medication regimen reduces
the risk of allergic reaction or FNHTR. They
further state that no evidence shows that pre-
transfusion medications prevent NHTR. How-
ever, the conclusion is based on the evidence
from a review of three trials with a moderate
risk of bias and low to moderate quality. A bet-
ter-powered RCT is necessary to evaluate the
role of pretransfusion medication in the pre-
vention of NHTR.
Antihistamines (diphenhydramine and/
or H2 blockers) may be ordered as premedica-
tion for individuals who have had allergic reac-
tions to transfusions in the past. Meperidine or
corticosteroids are occasionally ordered for
patients who have experienced severe rigors
during transfusion.15 Corticosteroids have also
been used to premedicate patients who have
had prior anaphylactoid reactions or recurrent
febrile nonhemolytic transfusion reactions.
The onset of corticosteroid immunosuppres-
sion takes a long time. The efficacy of premed-
ication with corticosteroids has not been ade-
quately assessed, and the optimal timing and
efficacy of corticosteroids have not been es-
tablished in the transfusion setting. However,
corticosteroids have been used to prevent ana-
phylactoid reactions to iodinated contrast me-
dia in the radiology setting in patients who
have had prior reactions, a situation that is
somewhat analogous to that of transfusion.
The authors of a number of publications rec-
ommend repeat corticosteroid dosing in the
radiology setting, often in combination with
H1 and H2 blockers over 13 to 24 hours before
the procedure.16,17
If premedication is required, it should be
administered in advance of the component’s
arrival. If the premedication is given orally, the
transfusionist should wait 30 minutes before
initiating the transfusion. If the premedica-
tion is given intravenously, a 10-minute wait-
ing period before initiating the transfusion is
suggested.
Equipment
Blood Warmers
Infusions of cold components can cause hypo-
thermia and cardiac complications, increasing
morbidity and mortality.18 The likelihood of
clinically important hypothermia is increased
when blood is transfused through a central ve-
nous device directly into the right atrium.
Blood warmers are rarely needed during
routine transfusions. However, they are used
when rapid transfusion of components is re-
quired, especially in trauma or surgery set-
tings. Blood warmers are also advantageous
during transfusions to neonates, where hypo-
thermia can cause serious adverse effects.
Opinions vary on the utility of blood warmers
in patients with cold agglutinins.19,20
AABB Standards1(p6) states that warming
devices shall be equipped with a temperature-
sensing device and a warning system to detect
malfunctions and prevent hemolysis or other
damage to blood or blood components.
Warming blood to temperatures >42 C may
cause hemolysis.21 The transfusion service
should work with departments that use blood
warmers to make sure that the devices are ap-
proved by the Food and Drug Administration
(FDA) for infusion of components. Warming
devices should be validated and maintenance
and testing of alarms should be performed ac-
cording to the manufacturer’s suggestions.
Blood components should not be warmed by
placing them in a microwave, on a heat source,
or in hot water, or by using devices that are not
approved by the FDA specifically for blood
warming.
Infusion Systems
Infusion pumps or systems are used to admin-
ister fluids, medications, blood, and blood
components through clinically accepted routes
of administration. These devices allow for a
controlled infusion rate and, thus, controlled
administration over a desired period; they also
provide an alarm system to notify clinicians of
problems with the infusion. Consequently, the
use of infusion pumps or systems may be pre-
ferred over simple gravity administration.
 CHAPTER 21 Administration of Blood Components
However, there is a potential for hemolysis of
the cellular components infused through these
pumps. The manufacturer of the pump should
be consulted to determine whether the pump
is approved for the infusion of blood compo-
nents. If it is not approved, the institution
should establish a validation plan to confirm
that the pump will not damage components
before their use. Many of the electromechani-
cal pump devices that are approved for blood
administration require use of administration
sets with standard in-line filters.
Using infusion pumps to deliver transfu-
sions via PICC lines may lead to pressure in the
catheter that exceeds the allowed pounds per
square inch. The institution should ensure
that the infusion pump used does not generate
pressure that exceeds the recommended limits
of the catheter.22
Syringe Infusion Pumps
A syringe infusion pump may be used for
small-volume transfusions to neonatal or pe-
diatric patients. Using this pump requires
drawing the blood component through a filter
into the syringe. For more details, see Chapter
23.
Pressure Devices
The use of an externally applied pneumatic
pressure device may achieve flow rates of 70 to
300 mL per minute, depending on the pressure
applied. The device should have a gauge to
monitor the pressure, which should be applied
evenly over the entire bag. Pressure in excess
of 300 mmHg may cause the seams of the
blood component bag to leak or rupture.
When a pressure device is used, a large-gauge
cannula should be employed to prevent he-
molysis.
The application of an external pressure
device to the blood bag to expedite the trans-
fusion of RBC units causes minimal damage to
the red cells and is a safe practice in the major-
ity of patients.23 However, the use of pressure
devices has been reported to provide only a
small increase in component flow rates. When
rapid infusion is desired, an increase in cannu-
la size typically provides better results.
䡲 549
Availability of Emergency Equipment
The transfusionist should be prepared to ob-
tain and administer emergency interventions
when needed. Items used to respond to a
transfusion reaction include the following:
䡲 A 0.9% sodium chloride intravenous (IV)
solution and administration set to keep an
IV line open.
䡲 Medications to treat a reaction along with a
mechanism to obtain emergency medica-
tions prescribed to treat the sequelae of
transfusion reactions.
䡲 A mechanism to activate emergency resus-
citation measures in the event of a severe
reaction.
䡲 Ventilatory assistance and an oxygen
source.
B LO OD CO M P O N E NT
TRANSP ORTATION AND
D I S PE N S I N G
Delivery of Components to the Patient
Area
Each institution must have policies and proce-
dures for issuing components and ensuring
that they are delivered in a timely manner to
the receiving transfusionist. Components
should not leave the controlled environment
until the patient has been properly prepared,
including insertion of a patent intravenous
catheter, and the transfusionist is ready to be-
gin the infusion.
Transfusion service personnel should in-
spect the unit before issuing it for abnormal
appearance (significant color change, cloudi-
ness, clots, clumps, or loss of bag integrity).
The component must not be used if any of
these are noted.4 There must be a mechanism
to correctly identify the intended recipient and
component at the time of the request to issue
the component. Institutions should train staff
to request components so that they arrive in
an expedited manner and are not left outside a
controlled environment.
550 䡲 AABB TECHNICAL MANUAL
Institutions may use dedicated personnel
or automated delivery systems (eg, pneumatic
tube systems, automated blood delivery ro-
bots, or blood dispensing kiosks in remote
sites) to facilitate the delivery of components
to their final destination. Provision of RBCs via
automated blood vending machines [remote,
automated, computerized controlled blood
storage and dispensing refrigerators, such as
BloodTrack HemoSafe (Neoteric Technology,
Vancouver, BC, Canada)] at the point of care
may help circumvent delays in transportation.
This solution employs an electronic issuance
process, requiring infrastructure and bidirec-
tional informatics connectivity with the cen-
tral blood bank to safely issue and return
blood units to/from remote sites.24 Staff educa-
tion in the electronic issuance process em-
ployed by vending machines is required to use
these devices. The use of automated and re-
mote delivery systems requires validation pri-
or to implementation to ensure a safe and ef-
fective system for blood delivery and remote
blood issue.25
Transfusion services generally allow the
issue of only 1 unit at a time unless it is an
emergent or large-volume transfusion.
1. At the time a unit is issued, AABB Standards
requires a final clerical check of transfusion
service records with each unit or compo-
nent. Verification must include1(p42) the
intended recipient’s two independent iden-
tifiers (name, date of birth, or patient iden-
tification number and/or unique identifier
given at the time the crossmatch sample is
drawn), ABO group, and Rh type.
2. The donation identification number, donor
ABO group, and, if required, donor Rh type.
3. The interpretation of results of crossmatch
tests, if performed.
4. Special transfusion requirements.
5. The expiration date and, if applicable, time.
6. The date and time of issue.
AABB Standards requires that there be a
process to confirm agreement among identify-
ing information, records, the blood or blood
component, and the request. Discrepancies
must be resolved before a unit is issued.1(p42)
Delays in Starting Transfusion
If an unexpected occurrence does not allow for
the transfusion to start immediately, the blood
component unit should be promptly returned
to the transfusion service for proper storage.
The transfusion service may set limits,
based on validation, on the amount of time
that a unit may be out of controlled storage be-
fore it is considered unsuitable for reissue.
Components may be suitable for reissue if the
appropriate temperature has been main-
tained.1(p40) They should never be stored or
held in a patient care unit unless there is a
controlled and monitored environment for
storing components. Trauma areas and surgi-
cal units may have arrangements with the
transfusion service to provide controlled stor-
age of units. If the temperature of a transport-
ed component falls outside of 1-10 C, reissue is
not permissible.1(p47)
If a unit has been entered (spiked for
transfusion), it may not be returned to the
transfusion service for reissue. It must either
be infused within 4 hours of the time it was
spiked, or it must be discarded.
EV E N TS A ND CO N SI D E R AT I O NS
B E F O R E COM P O N E N T
ADMINISTRATION
Identification of the Recipient and
Correct Component
Once the unit is received, a qualified transfu-
sionist who will administer the blood or blood
component to the patient verifies the compo-
nent at the patient’s side with another health-
care provider who is qualified in performing
identification verification, as determined by
the hospital.5 Alternatively, a one-person veri-
fication process using automated identifica-
tion technology, such as bar coding, may be
used.
The following items should be checked
immediately before transfusion:
䡲 Appearance of the unit. Units should be re-
turned to the transfusion service if there is
discoloration, abnormal cloudiness, pres-
 CHAPTER 21 Administration of Blood Components
ence of clots or clumps, or loss of bag integ-
rity.
䡲 Identification of patient and unit. The pa-
tient’s two independent identifiers (eg,
name and identification number) must
match the information on both the label on
the unit or attached tag and the medical or-
der. The requirements of the institution for
patient identification must be satisfied. In-
stitutions often require, for example, an ad-
ditional check to verify that the unit was se-
lected for the patient whose sample was
collected for crossmatch.
䡲 Medical order. The transfusionist should
verify that the component matches the unit
called for in the medical order and that any
special processing in the order was per-
formed. In particular, the transfusionist
should ensure that leukocyte reduction or
irradiation was performed if ordered.
䡲 Blood type. The patient’s ABO group (and
Rh type if required) should be compatible
with that of the unit. Interpretation of
crossmatch tests (if performed) is also veri-
fied.
䡲 Donation identification number.
䡲 Expiration date (and time, if applicable).
The unit should not be transfused if the ex-
piration date or time has passed.
The transfusion should be withheld if any
discrepancy or abnormality is found.
Proper bedside identification of the recip-
ient is the final step to prevent the administra-
tion of an incorrect blood component to a pa-
tient. Although individuals often worry about
the possibility of exposure to infectious agents
from transfusion, equal concern should focus
on the inadvertent transfusion of incompati-
ble blood. Approximately 1 in every 19,000
units of RBCs is transfused to the wrong pa-
tient each year; 1 in 76,000 transfusions results
in an acute hemolytic transfusion reaction,
and 1 in 1.8 million units of transfused RBCs
results in death from an acute hemolytic trans-
fusion reaction.26
To prevent the potentially fatal conse-
quences of misidentification, specific systems
have been developed and marketed, including
identification bracelets with bar codes and/or
䡲 551
radiofrequency identification devices, biomet-
ric scanning, mechanical or electronic locks
that prevent access to bags assigned to other
patients, and handheld computers suitable for
transferring blood request and administration
data from the patient’s bedside to the transfu-
sion service information system in real time.
Each system provides a method to bring staff
toward self-correction during the proce-
dure.27,28 Studies show that rates of positive re-
cipient identification can be increased by such
systems. However, none of these systems ne-
gates the need for good quality management,
such as adequate standard operating proce-
dures, regular training, periodic competency
assessment, and system monitoring.
Infusion Sets
Components must be administered through
special IV tubing with a filter designed to re-
move blood clots and particles that are poten-
tially harmful to the patient. Standard blood
administration tubing typically has a 170- to
260-micron (macroaggregate) filter, but this
micron size is not mandated or required. The
tubing can be primed with either 0.9% sodium
chloride or the component itself. The manu-
facturer’s instructions should be reviewed for
proper use. The IV setup should have a mecha-
nism that allows bypass of the blood IV admin-
istration tubing to start 0.9% sodium chloride
in the event of a reaction. A suggested mecha-
nism is to have a “Y” port or three-way stop-
cock close to the infusion site that allows for
the administration of 0.9% sodium chloride.
Microaggregate Filters
Microaggregate filters are not used for routine
blood administration. These second-genera-
tion filters were originally developed to re-
move leukocytes and to complement or
replace the clot screen in the 1970s.29 They
have since been replaced by more efficient
leukocyte reduction filters.30 Microaggregate
filters have a screen filter depth of 20 to 40
microns and retain fibrin strands and clumps
of dead cells. Red cells, which are 8 microns in
diameter, can flow through the filters. Micro-
aggregate filters are typically used for the
552 䡲 AABB TECHNICAL MANUAL
reinfusion of shed autologous blood collected
during or after surgery.
Leukocyte Reduction Filters
Leukocyte reduction filters are designed to re-
duce the number of leukocytes to less than 5 ×
106 per RBC unit (so that more than 99.9% of
the leukocytes are removed from the unit).
Leukocyte reduction decreases the incidence
of febrile transfusion reactions, risk of HLA al-
loimmunization, and transmission of cyto-
megalovirus by cellular blood components
(see Chapter 6).29,30 These filters are provided
by various manufacturers for prestorage use
shortly after collection of the units or for
poststorage use at the patient’s bedside.
Prestorage leukocyte reduction is usually
preferred for a number of reasons, one of
which is that it allows monitoring of leukocyte
reduction efficacy. Furthermore, the use of
bedside leukocyte reduction filters has been
associated with dramatic hypotension in some
individuals, often in the absence of other
symptoms. This happens more frequently with
patients taking angiotensin-converting en-
zyme inhibitors. The use of components that
were filtered in the blood center or transfu-
sion service before storage decreases the inci-
dence of such reactions.31 If a precipitous drop
in blood pressure is noted, the transfusion
should be stopped immediately.
It is important to verify that the filter is in-
tended for use with the component being
transfused (RBCs or platelets) and to note the
maximum number of units that can be admin-
istered through one filter. Filters designed for
RBCs or platelets may not be used inter-
changeably. The manufacturer’s instructions
should be followed for priming and adminis-
tering blood components through the filter.
Otherwise, leukocyte removal may be ineffec-
tive or an air lock may develop, preventing
passage of the component through the filter.
Leukocyte filters should never be used to ad-
minister granulocytes or hematopoietic pro-
genitor cells.
Compatible IV Solutions
No medications or solutions other than 0.9%
sodium chloride injection, USP, should be ad-
ministered with blood components through
the same tubing. Solutions containing dex-
trose alone may cause red cells to swell and
lyse. Lactated Ringer’s solution or other solu-
tions containing high levels of calcium may
overcome the buffering capacity of the citrate
anticoagulant in the blood preservative solu-
tion and cause clotting of the component.32
AABB Standards allows exceptions to the
above restrictions when 1) the drug or solution
has been approved by the FDA for use with
blood administration, or 2) there is documen-
tation available to show that the addition is
safe and does not adversely affect the blood or
component.1(p45) Acceptable solutions accord-
ing to these criteria include ABO-compatible
plasma, 5% albumin, or plasma protein frac-
tion. Certain solutions are compatible with
blood or blood components as noted in the
package inserts reviewed by the FDA, includ-
ing Normosol-R pH 7.4 (Hospira, Lake Forest,
IL), Plasma-Lyte-A injection pH 7.4 (Baxter
Healthcare, Deerfield, IL), and Plasma-Lyte
148 injection (Multiple Electrolytes Injection,
Type 1, USP, Baxter Healthcare). There are sev-
eral formulations of Plasma-Lyte that are not
isotonic or that contain calcium; package in-
serts must be checked to confirm their com-
patibility with components.
MANUAL ADMINISTRATION
Starting the Transfusion
Once the identification of the unit and the re-
cipient is verified, the unit is spiked using an
aseptic technique. At institutions that use Joint
Commission hospital accreditation, Joint
Commission requirements for the transfusion-
ist (HR.01.02.01) apply: “If blood transfusions
and intravenous medications are adminis-
tered by staff other than doctors of medicine
or osteopathy, the staff members must have
special training for this duty.”33
 CHAPTER 21 Administration of Blood Components
The blood administration tubing should
be filled with either 0.9% sodium chloride or
the contents of the blood component. If any
solution or medication other than 0.9% sodi-
um chloride is infused before component ad-
ministration, the tubing should be flushed
with 0.9% sodium chloride immediately before
the blood infusion.
The infusion should start slowly, at ap-
proximately 2 mL per minute, for the first 15
minutes while the transfusionist remains near
the patient. Severe reactions may occur after
as little as 10 mL has been transfused. Poten-
tially life-threatening reactions most com-
monly occur within 10 to 15 minutes of the
start of a transfusion.34
The rate of transfusion should be in-
creased after 15 minutes to ensure the unit is
administered within the 4-hour window. The
advantages of using relatively rapid transfu-
sion rates (eg, 240 mL/hour) include correc-
tion of deficiency as rapidly as possible as well
as reduced patient and nursing time dedicated
to transfusion. Disadvantages include the po-
tential to cause reactions (eg, volume over-
load) or to make reactions more severe (eg,
FNHTR, septic reactions, or allergic reac-
tions). More rapid infusion of leukocytes (with
use of nonleukoreduced cellular components)
can result in a more rapid rise in body temper-
ature, a FNHTR, or the transmission of bacte-
ria and/or allergenic substances.35 Many
FNHTRs as well as septic, allergic, and even
some hemolytic reactions may not be evident
within the first 15 minutes.
If there is no sign of a reaction after the
first 15 minutes, the flow rate can be increased
to the designated infusion rate, taking into
consideration the patient’s size, blood vol-
ume, and hemodynamic condition in deter-
mining the flow rate (see Table 21-1). Careful
attention should be paid to avoid transfusing
the unit too rapidly in relation to the patient’s
cardiac and/or and respiratory status.
Monitoring the Transfusion
The unit identifiers should never be removed
during the transfusion.
䡲 553
The transfusionist should continue to
monitor the patient throughout the infusion
and check the IV site and flow rate. If the IV
rate has slowed down, the transfusionist
should take one or more of the following ac-
tions: 1) check to make sure that the IV is pat-
ent and there is no swelling at the site; 2) at-
tempt to administer the component through
an infusion pump; 3) raise or elevate the unit;
4) examine the filter for air, excessive debris, or
clots; or 5) consider the addition of 0.9% sodi-
um chloride as a diluent if the unit is too vis-
cous. Frequent patient monitoring during the
infusion helps alert the transfusionist to a pos-
sible transfusion reaction and allows for early
interdiction.
Vital signs should be taken within 5 to 15
minutes of beginning the transfusion and then
according to institutional policy. There is little
evidence to support a best practice related to
the frequency of vital-sign monitoring other
than at baseline, soon after the start of the
transfusion, and after transfusion.36 AABB
Standards requires that the medical record
must include pre- and posttransfusion vital
signs.1(p45) Vital signs should be taken at once if
there is a suspected transfusion reaction.
The transfusionist should be knowledge-
able about signs and symptoms indicative of
an adverse reaction and able to act quickly
(see Chapter 27). Visual observation and pa-
tient reporting of any changes should be uti-
lized to determine that a reaction has occurred
because the patient may experience symp-
toms before changes occur in vital signs. If a
transfusion reaction is suspected, the transfu-
sion should be stopped and 0.9% sodium chlo-
ride administered. The 0.9% sodium chloride
should be infused near the IV insertion site to
avoid flushing the tubing with the residual
component. The unit identification informa-
tion should be rechecked. The transfusionist
should notify the patient’s care provider of any
suspected transfusion reaction and obtain
emergency medication orders as needed to
treat the suspected reaction. It is helpful for in-
stitutions to summarize and have readily avail-
able to the transfusionist descriptions of com-
mon reactions and immediate steps to be
taken in the event of certain symptoms.
 554

TABLE 21-1. Blood Component Transfusions in Nonemergency Settings

Suggested Adult Flow Rate

䡲

Component First 15 Minutes After 15 Minutes Special Considerations ABO Compatibility Filter

Red Blood Cells 1-2 mL/min As rapidly as tolerated; Infusion duration should Whole blood: ABO identical In-line (170-260 micron)
(RBCs) (60-120 mL/hour) approximately 4 mL/ not exceed 4 hours. RBCs: ABO compatible with Leukocyte reduction if
minute or 240 mL/hour For patients at risk of fluid recipient’s plasma indicated
overload, may adjust flow Crossmatch required
rate to as low as 1 mL/kg/
hour.
Platelets 2-5 mL/min 300 mL/hour or as toler- Usually given over Crossmatch not required In-line (170-260 micron)
(120-300 mL/hour) ated 1-2 hours ABO/Rh compatibility pref- Leukocyte reduction if
For patients at risk of fluid erable but not required indicated
overload, use slower flow
rate (see under RBCs) May be HLA matched

Plasma 2-5 mL/min As rapidly as tolerated; Thaw time may be needed Crossmatch not required In-line (170-260 micron)
AABB TECHNICAL MANUAL

(120-300 mL/hour) approximately 300 mL/ before issue
hour For patients at risk of fluid
overload, use slower flow
rate (see under RBCs)
Granulocytes 1-2 mL/min 120-150 mL/hour or as Over approximately 2
(60-120 mL/hour) tolerated hours
Infuse as soon as possi-
ble after collection/release
of product; irradiate
Cryoprecipitated AHF As rapidly as tolerated Infuse as soon as possi-
ble after thawing; pooling
is preferred.
ABO compatibility with
recipient red cells
Crossmatch required In-line (170-260 micron)
ABO/Rh compatibility No leukocyte reduction fil-
required ter or depth-type micro
May be HLA matched aggregate filters
Crossmatch and ABO com- In-line (170-260 micron)
patibility not required
 CHAPTER 21 Administration of Blood Components
Once the patient is stable, the transfusion
service should be notified of a suspected
transfusion reaction, and institutional policy
should be followed for returning the compo-
nent bag and/or to order the laboratory stud-
ies needed to evaluate the reaction.
Completing the Transfusion
The patient is assessed at the completion of
the transfusion, and his or her vital signs are
obtained. The bag and tubing are discarded in
a biohazard container if the transfusion was
uneventful.
Because patients can experience transfu-
sion reactions several hours to days after the
transfusion is complete, clinical staff should
continue to monitor the patient periodically
for 4 to 6 hours after the end of the transfusion
to detect febrile or pulmonary reactions that
may be associated with blood administration.
If the patient is not under direct clinical super-
vision after a transfusion, clinical staff should
provide written instructions to the patient and
caregiver regarding signs and symptoms to re-
port and a phone number to call or a person to
contact should a reaction occur later.
The transfusion should be documented in
the patient’s medical record. At a minimum,
AABB Standards requires documentation of
the following1(p45):
1. Transfusion order.
2. Recipient consent.
3. Component name.
4. Donation identification number.
5. Date and time of transfusion.
6. Pre- and posttransfusion vital signs.
7. Volume transfused.
8. Identification of the transfusionist.
9. Transfusion-related adverse events.
If additional units are transfused, the in-
stitution’s guidelines and/or manufacturer’s
recommendations should be consulted to de-
termine whether the same blood administra-
tion tubing may be used for subsequent units.
If there are no contraindications from the
manufacturer, institutions frequently allow the
tubing to be reused as long as subsequent
䡲 555
units are transfused within 4 hours of the start
of the initial transfusion. Therefore, if more
than 1 unit can be infused in 4 hours, blood
administration tubing sets may be used for
more than one component.
UNIQUE TRANSFUSION
S ET T IN G S
See Chapter 23 for information about transfu-
sion in pediatric and neonatal patients.
Operating Room and Trauma: Rapid
Infusions
If components need to be administered rapid-
ly, the use of pressure infusion, large-bore ad-
ministration tubing, and 8-Fr intravenous
catheters can decrease the infusion time with-
out inducing hemolysis.37,38 Specific tubing
sets are designed for rapid blood administra-
tion with appropriate filters. Flow rates as fast
as 10 to 25 mL/second (600-1500 mL/minute)
have been reported with such tubing. Howev-
er, at such rapid infusion rates, steps should be
taken to avoid hypothermia. Furthermore,
when multiple units are infused through the
same tubing, the flow rate may decrease ap-
preciably.
Hypocalcemia has been noted with rapid
transfusions. This is usually transient and de-
pendent on the amount and rate of citrate in-
fused. Calcium replacement may be adminis-
tered based on the patient’s ionized serum
calcium level and the rate of citrate adminis-
tration.39
Transfusion-associated hyperkalemic car-
diac arrest has been reported with rapid ad-
ministration of RBCs. It may develop with rap-
id RBC administration even with a modest
transfusion volume of between 1 (in a neo-
nate) and 54 units. Contributing factors are ac-
idosis, hypoglycemia, hypocalcemia, and hy-
pothermia at the time of cardiac arrest.40
If components are urgently needed and a
delay in transfusion could be detrimental to
the patient, the transfusion service should
have a process to provide components before
all pretransfusion compatibility testing is
completed. In such cases, uncrossmatched
556 䡲 AABB TECHNICAL MANUAL

units are released with a signed statement
from the requesting physician indicating that
the clinical situation requires urgent release
before the completion of testing. If compo-
nents in the transfusion service inventory are
not immediately accessible to a trauma unit or
operating room, a supply of group O red cells
may be maintained at these sites. The transfu-
sion service must ensure proper storage of
components at these satellite storage sites.
Out-of-Hospital Transfusion
Transfusing blood in a nonhospital setting re-
quires a well-planned program that incorpo-
rates all the relevant aspects of the hospital
setting and emphasizes safety consider-
ations.41,42 An outpatient surgery center, oncol-
ogy clinic, or dialysis center is likely to be able
to provide medical assistance in a timely man-
ner in the event of an adverse reaction. Medi-
cal staff availability, medications, and equip-
ment to handle adverse reactions must be
arranged for in advance. Staff should be com-
petent in performing blood administration
procedures and patient monitoring. Blood ad-
ministration outside the hospital should be
performed by personnel with substantial ex-
perience in blood administration in this set-
ting.
Transfusion in the home generally allows
close monitoring of the transfusion event be-
cause the personnel-to-patient ratio is one to
one. The disadvantage is that there is no
trained assistant available in the event of a se-
vere adverse reaction. Issues to consider when
preparing for a transfusion in the home in-
clude the following:
䡲 Availability of a competent adult in the
home to assist in patient identification and
to summon medical assistance if needed.
䡲 A mechanism to obtain immediate physi-
cian consultation.
䡲 A telephone to contact emergency person-
nel and easy access for emergency vehicles.
䡲 Documentation of prior transfusions unac-
companied by adverse reactions.
䡲 The ability to properly dispose of medical
waste.
CO N C LUS IO N S
Transfusion of blood components and the cre-
ation of blood administration procedures and
policies should be patient centric. Following
these policies helps the transfusionist quickly
recognize and report suspected transfusion re-
actions. Close monitoring and early interven-
tion can make a critical difference in patient
outcomes. The transfusion service should pe-
riodically audit the blood administration pro-
cess to identify instances of nonconformance,
analyze their causes, and institute corrective
actions.

KEY POINTS

1. Blood administration involves the process of informed consent, preparation of the recipi-
ent, administration of the appropriate product to the correct recipient, and careful observa-
tion of the recipient during and after the transfusion for any adverse reaction. All steps must
be appropriately documented in the patient’s medical record.
2. A licensed care provider should initiate requests for blood administration with an order for
the appropriate blood component and an order for the administration of the component(s).
3. The recipient should be informed of and educated about the upcoming administration of
the blood component so that he or she can give informed consent for the transfusion.
4. A baseline assessment of the recipient should be performed for subsequent comparison.
5. Just before the planned administration, the transfusionist must verify appropriate venous
access, administration of any prophylactic medications required, and availability of re-
quired equipment (eg, blood warmer, infusion pump, pressure devices, and emergency
equipment).
 CHAPTER 21 Administration of Blood Components 䡲 557

6. Institutions should identify appropriate blood and blood component issue and delivery
mechanisms to ensure that the transfusionist receives the components in a timely manner.
7. Transfusion services should ensure that other departments are aware of the requirement for
returning components if a transfusion is delayed.
8. Before a transfusion is initiated, verification of recipient and component identification
should be performed. The following items should be verified: 1) the appearance of the unit,
2) identity of the recipient and unit, 3) medical order, 4) blood type, and 5) expiration date/
time of the component.
9. Components must be administered through the appropriate infusion sets and filter if indi-
cated. No solution other than 0.9% sodium chloride injection, USP, should be administered
through the same tubing unless the tubing has been flushed with 0.9% sodium chloride,
USP, immediately before and after the transfusion.
10. The infusion should start slowly at approximately 2 mL per minute for the first 15 minutes.
During this time, the transfusionist should remain near the patient. If no sign of reaction
appears, the infusion rate can be increased. The transfusionist monitors the patient
throughout the infusion and stops the infusion in the event of an adverse reaction.
11. Infusions must be completed within 4 hours. After completion, the transfusionist takes the
patient’s vital signs. If the patient will not be under direct clinical supervision after the
transfusion, the patient and caregiver should receive instructions regarding signs and
symptoms to report and whom to report these reactions to.
12. The following information, at a minimum, about the transfusion must be documented in
the patient’s medical record: 1) the transfusion order, 2) patient consent for transfusion, 3)
name of component, 4) donation identification number, 5) date and time of infusion, 6)
pre- and posttransfusion vital signs, 7) volume transfused, 8) identity of the transfusionist,
and 9) any adverse reaction.

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1. Levitt J, ed. Standards for blood banks and
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AABB, 2014.
2. Stowell CP, Sazama, K, eds. Informed consent
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Patients, donors, and research subjects.
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8. Miller MA, Schlueter AJ. Transfusions via
hand-held syringes and small-gauge needles
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9. Kennedy LD, Case LD, Hurd DD, et al. A pro-
spective, randomized, double-blind controlled
trial of acetaminophen and diphenhydramine
pretransfusion medication versus placebo for
the prevention of transfusion reactions. Trans-
fusion 2008;48:2285-91.
10. Ezidiegwu CN, Lauenstein KJ, Rosales LG, et
al. Febrile nonhemolytic transfusion reac-
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implications in adult patients. Arch Pathol Lab
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11. Geiger TL, Howard SC. Acetaminophen and
diphenhydramine premedication for allergic
and febrile nonhemolytic transfusion reac-
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Transfus Med Rev 2007;21:1-12.
12. Wang SE, Lara PN Jr, Lee-Ow A, et al. Acet-
aminophen and diphenhydramine as premed-
ication for platelet transfusions: A prospective
randomized double-blind placebo-controlled
trial. Am J Hematol 2002;70:191-4.
13. Tobian AR, King K, Ness PM. Prevention of fe-
brile nonhemolytic and allergic transfusion re-
actions with pretransfusion medication: Is this
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48:2274-6.
14. Marti-Carvajal AJ, Sola I, Gonzalez LE, et al.
Pharmacological interventions for the preven-
tion of allergic and febrile non-haemolytic
transfusion reactions. Cochrane Database Syst
Rev 2010;(6):CD007539.
15. Patterson BJ, Freedman J, Blanchette V, et al.
Effect of premedication guidelines and leuko-
reduction on the rate of febrile nonhaemolytic
platelet transfusion reactions. Transfus Med
2000;10:199-206.
16. Goss JE, Chambers CE, Heupler FA, et al. Sys-
temic anaphylactoid reactions to iodinated
contrast media during cardiac catheterization
procedures: Guidelines for prevention, diag-
nosis, and treatment. Cath Cardiovasc Diagn
1995;34:99-104.
17. Tramer MR, von Elm E, Loubeyre P, Hauser C.
Pharmacological prevention of serious ana-
phylactic reactions due to iodinated contrast
media: Systematic review. Br Med J 2006;333:
675-81.
18. Boyan CP, Howland WS. Cardiac arrest and
temperature of bank blood. JAMA 1963;183:
58-60.
19. Donham JA, Denning V. Cold agglutinin syn-
drome: Nursing management. Heart Lung
1985;14:59-67.
20. Iserson KV, Huestis DW. Blood warming: Cur-
rent applications and techniques. Transfusion
1991;31:558-71.
21. Hirsch J, Menzebach A, Welters ID, et al. Indi-
cators of erythrocyte damage after microwave
warming of packed red blood cells. Clin Chem
2003;49:792-9.
22. Houck D, Whiteford J. Improving patient out-
comes: Transfusion with infusion pump for
peripherally inserted central catheters and
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2007;30:341-4.
23. Frelich R, Ellis MH. The effect of external pres-
sure, catheter gauge, and storage time on he-
molysis in RBC transfusion. Transfusion
2001;41:799-802.
24. Wong KF. Virtual blood bank. J Pathol Inform
2011;2:6.
25. Lum G, D’Amarino MJ. Use of Laboratory ro-
bots for transport and delivery of blood prod-
ucts. Lab Med 2009;40:517-22.
26. Vamvakas EC, Blajchman MA. Transfusion re-
lated mortality: The ongoing risks of allogene-
ic blood transfusion and the available strate-
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17.
27. Pagliaro P, Rebulla P. Transfusion recipient
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28. Koshy R. Navigating the information technolo-
gy highway: Computer solutions to reduce er-
rors and enhance patient safety. Transfusion
2005;45(Suppl 4):189S-205S.
29. Wortham ST, Ortolano GA, Wenz B. A brief his-
tory of blood filtration: Clot screens, microag-
gregate removal, and leukocyte reduction.
Transfus Med Rev 2003;17:216-22.
30. Lane TA. Leukocyte reduction of cellular blood
components: Effectiveness, benefits, quality
control, and costs. Arch Pathol Lab Med 1994;
118:392-404.
31. Zoon KC, Jacobson ED, Woodcock J. Hypoten-
sion and bedside leukocyte reduction filters.
Int J Trauma Nurs 1999;5:121-2.
32. Dickson DN, Gregory MA. Compatibility of
blood with solutions containing calcium. S Afr
Med J 1980;57:785-7.
33. The Joint Commission. Comprehensive ac-
creditation manual for hospitals. Oakbrook
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34. Bradbury M, Cruickshank JP. Blood transfu-
sion: Crucial steps in maintaining safe prac-
tice. Br J Nurs 2000;9:134-8.
35. Perkins HA, Payne R, Ferguson J, Wood M.
Nonhemolytic febrile transfusion reactions:
Quantitative effects of blood components with
emphasis on isoantigenic incompatibility of
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36. Oldham J, Sinclair L, Hendry C. Right patient,
right blood, right care: Safe transfusion prac-
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37. Iserson KV, Knauf MA. Confirmation of high
blood flow rates through 150 micron filter/
highflow tubing. J Emerg Med 1990;8:689-91.
 CHAPTER 21 Administration of Blood Components
38. Floccare DJ, Kelen GD, Altman RS, et al. Rapid
infusion of additive red blood cells: Alternative
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Emerg Med 1990;19:129-33.
39. Denlinger JK, Nahrwold ML, Gibbs PS, Lecky
JH. Hypocalcaemia during rapid blood trans-
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1976;48:995-1000.
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40. Smith HM, Farrow SJ, Ackerman JD, et al. Car-
diac arrests associated with hyperkalemia dur-
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41. Fridey JL. Practical aspects of out-of-hospital
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42. Evans CS. Out-of-hospital transfusion. Trans-
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 C h a p t e r 2 2

Perinatal Issues in Transfusion

Practice

Melanie S. Kennedy, MD; Meghan Delaney, DO, MPH;

and Scott Scrape, MD

H E M O L Y T I C D I S E A S E O F the fetus ing red cell antigen, causing the antibody-

and newborn (HDFN), fetal/neonatal al-
loimmune thrombocytopenia (FNAIT), and
immune thrombocytopenia (ITP; previously
known as “immune thrombocytopenic purpu-
ra”) affect pregnant women and their fetuses
and newborns. The blood bank and transfu-
sion service play critical roles in supporting
the diagnosis and treatment of these condi-
tions, including the appropriate provision of
Rh Immune Globulin (RhIG).
HD FN
HDFN is the destruction of fetal and newborn
red cells by maternal red cell alloantibodies
that are specific for inherited paternal red cell
alloantigen(s). The maternal IgG antibody is
transported across the placenta into the fetal
circulation, where it binds to the correspond-
coated red cells to be destroyed by macro-
phages in the fetal spleen. The fetal hemato-
poietic tissue initially responds by increasing
erythropoiesis and releasing many of the new-
ly produced red cells into the circulation pre-
maturely as nucleated precursors, a condition
known as “erythroblastosis fetalis.” With wors-
ening anemia, excess erythropoiesis occurs in
the liver and spleen, causing organ enlarge-
ment and portal hypertension. A resulting de-
crease in liver production of albumin leads to
reduced plasma colloid osmotic pressure, gen-
eralized edema, ascites, and effusions known
as “hydrops fetalis.” Untreated, hydrops feta-
lis, with its associated high-output cardiovas-
cular failure, can lead to fetal death. Severe
disease can occur as early as 18 to 20 weeks’
gestation; severity usually increases in subse-
quent pregnancies.

Melanie S. Kennedy, MD, Clinical Associate Professor Emeritus, Attending Physician, Transfusion Medicine,
Wexner Medical Center, Department of Pathology, The Ohio State University, Columbus, Ohio; Meghan Del-
aney, DO, MPH, Medical Director, Red Cell Genomics, Puget Sound Blood Center, Medical Director, Blood
Bank, Seattle Children’s Hospital, Assistant Professor, University of Washington, Seattle, Washington; Scott
Scrape, MD, Assistant Professor, Director, Transfusion Medicine, Wexner Medical Center, Department of
Pathology, The Ohio State University, Columbus, Ohio
The authors have disclosed no conflicts of interest.

561
562 䡲 AABB TECHNICAL MANUAL
Maternal Alloimmunization
Females can be alloimmunized to red cell anti-
gens by previous transfusion or transplanta-
tion and/or previous or current pregnancy.
Fetomaternal hemorrhage (FMH) occurs
spontaneously during pregnancy and its likeli-
hood increases with gestational age (from 3%
in trimester I to 12% and 45% in trimesters II
and III, respectively).1,2 Because exposure to
fetal red cells and resulting maternal alloim-
munization typically occur late during preg-
nancy and at delivery, the fetus and newborn
of the first pregnancy are rarely affected. The
risk of FMH is higher in women who have ex-
perienced trauma or undergone amniocente-
sis, cordocentesis, abortion, or other proce-
dures.2
Complex factors influence the ability of
individuals to immunologically respond to red
cell antigens. Rh(D) is the most potent immu-
nogen, in that a 200-mL transfusion of Red
Blood Cells (RBCs) stimulates anti-D in about
85% of D-negative individuals, except those
who are immunosuppressed. About half of the
remaining 15% will never become immunized,
even after repeated challenges with D-positive
red cells. Although as little as 0.1 to 1 mL of D-
positive red cells can stimulate antibody pro-
duction, the volumes of FMH are generally
small, which contributes to relatively low
alloimmunization rates in pregnancy.3 Before
Rh(D) immunoprophylaxis, 16% of ABO-com-
patible, D-negative mothers with D-positive
infants became immunized; the rate was 2%
in ABO-incompatible, D-negative mothers.3-5
Antibodies targeted to blood group anti-
gens in addition to RhD can cause HDFN. The
most common of these antigens are K and c.6
Unlike HDFN caused by anti-D, HDFN caused
by anti-K uniquely results in destruction of fe-
tal erythropoietic precursors in addition to he-
molysis.7,8(p527) Other antibodies that have been
less commonly reported to cause moderate or
severe disease include E, k, Kpa, Kpb, Ku, Jsa, Jsb,
Jka, Fya, Fyb, S, s, and U.8 In rare cases, anti-Ge
and anti-M have been reported to cause de-
struction of fetal erythropoietic precursors.
Pathophysiology of HDFN
Hemolysis occurs when the maternal antibody
binds to a fetal red cell antigen, causing at-
tachment to the Fc receptor of macrophages in
the spleen of the fetus. The rate of hemolysis
and severity of disease are determined by the
IgG subclass, amount of antibody, and number
of antigenic sites on the red cells.9 The sub-
classes IgG1 and IgG3 are more efficient in
causing hemolysis than IgG2 or IgG4. Trans-
portation of IgG1 and IgG3 across the placenta
is mediated by the Fc receptor beginning in
the second trimester and continuing until
birth.10 Because IgG1 is transported across the
placenta earlier and in larger amounts than
IgG3, IgG1 is associated with more severe dis-
ease. After delivery, antibody persistence can
cause continuing destruction of red cells, pro-
gressive anemia, and hyperbilirubinemia.
However, the amount of maternal antibody
present continues to decrease over the next 12
weeks, with a half-life of about 25 days.
During gestation, fetal red cell destruc-
tion releases hemoglobin. The hemoglobin is
broken down into bilirubin, which passes into
the maternal circulation and is conjugated by
the maternal liver, preventing the bilirubin’s
return to the fetus. Birth severs the connection
with the mother, causing the bilirubin (result-
ing from red cell destruction) to remain in the
neonatal circulation. Because of the infant’s
immature liver enzymatic pathways, the
amount of unconjugated bilirubin can in-
crease to dangerous levels and cause perma-
nent damage to the brain, known as “kernic-
terus.”11
Diagnosis
The diagnosis and management of HDFN in-
volve the close cooperation of the patient, ob-
stetrician, and blood bank/laboratory person-
nel. During the first trimester, the patient’s
obstetric and transfusion history should be
obtained. A previously affected pregnancy
alerts the obstetrician to possible problems in
the current pregnancy.12
 CHAPTER 22 Perinatal Issues in Transfusion Practice
Laboratory Testing
During the first prenatal visit, maternal ABO
and Rh should be typed and an antibody
screen should be performed. If an RhD-nega-
tive woman has an initial negative antibody
screening result, she is a candidate for RhIG
administration (see “RhIG” section below).
The antibody screen should be conducted us-
ing techniques that detect IgG antibodies that
are reactive at 37 C and in which separate
screening cells represent all clinically impor-
tant specificities. A positive antibody screen-
ing result requires antibody identification. An-
tibodies such as anti-I, -P1, -Lea, and -Leb,
whether IgM or IgG, may be ignored because
these antigens are poorly developed at birth.
Treatment of the mother’s plasma with dithio-
threitol (DTT) can help distinguish IgG from
IgM antibodies.13
After identifying an antibody known to
cause HDFN, the next step for fetal risk stratifi-
cation is to test the father for the presence of
the corresponding red cell antigen. If the fa-
ther is homozygous for the corresponding an-
tigen, 100% of the fathers’ offspring will be at
risk of HDFN. If the father is heterozygous,
50% of the father’s offspring will be at risk.
For pregnancies sensitized with anti-D,
no serologic methods can determine paternal
zygosity. In these situations, paternal DNA
testing is indicated.14 Using fetal amniocytes,
direct testing of the fetal red cell genotype can
predict the red cell phenotype. The reported
sensitivity and specificity of DNA testing by
polymerase chain reaction are 98.7% and
100%, respectively, with a low false-negative
rate (1-3%).14 Molecular typing of fetal DNA
can also be performed on maternal plasma
early in the second trimester.15
During a sensitized pregnancy, antibody
titers can assist with management decisions.
The AABB-recommended method is the use of
saline antihuman globulin (AHG) incubated
for 60-minutes at 37 C (Method 5-3). Other
methods, such as using albumin AHG or gel,
may result in higher titers than the recom-
mended method and should be validated with
clinical findings and laboratory data to ensure
appropriate interpretation by the obstetrician
䡲 563
and avoid inappropriate referral of patients for
high-risk obstetric care. The critical titer for
anti-D (the level below which HDFN and hy-
drops fetalis are unlikely and no invasive pro-
cedures are needed) is 16 in the AHG phase. As
long as the titer is 8 or lower, except in the case
of anti-K, the pregnancy can be followed by
titers.
Kell-system antigens are present on early
red cell precursors, so even a maternal anti-K
titer that is relatively low can cause erythropoi-
etic failure and severe anemia. A critical titer of
8 is generally accepted for Kell system antibod-
ies.7 Due to the poor reproducibility of titers,
individual blood banks should validate their
testing internally and keep previous speci-
mens for subsequent comparison. Flow
cytometric quantitation of antibody mass has
proven to be more precise than antibody
titers.16
Pregnancy Monitoring
Obstetric care usually involves monitoring af-
fected fetuses with a combination of maternal
antibody titers and fetal ultrasound of the
middle cerebral artery to assess fetal anemia.12
When a critical antibody titer is reached at 16
weeks of gestation, ultrasound and color Dop-
pler ultrasonography are performed to estab-
lish disease severity. Decisions about how and
when to treat an affected fetus are based on
the degree of fetal anemia and gestational age.
Treatment
In cases of severe fetal anemia from hemolytic
disease, fetal transfusion is indicated to treat
the anemia and suppress fetal erythropoiesis.
Intrauterine transfusion (IUT) is performed by
inserting a needle into the umbilical vein using
color Doppler enhancement of high-resolu-
tion sonography to guide the procedure. A pre-
transfusion sample of fetal blood is obtained
at the same time to determine the fetus’s blood
type, direct antiglobulin test (DAT) result, anti-
gen type, hemoglobin level, hematocrit, plate-
let count, and bilirubin level. DNA typing may
be performed as well. Cordocentesis is associ-
ated with a 1% to 2% risk of fetal infection,
bleeding, bradycardia, and/or premature
564 䡲 AABB TECHNICAL MANUAL
membrane rupture. Because cordocentesis
may cause FMH, RhIG immunoprophylaxis
should be administered after the procedure to
D-negative women who do not have anti-D.
Blood Product Selection and
Administration
For IUT, the blood should be 1) irradiated to
prevent transfusion-associated graft-vs-host
disease in the immunologically immature fe-
tus, 2) cytomegalovirus (CMV) reduced-risk
(through leukocyte reduction and/or by being
CMV seronegative), and 3) known to lack he-
moglobin S to prevent sickling under low oxy-
gen tension. Donor group O RBCs that are an-
tigen negative for the mother’s corresponding
antibodies and crossmatch compatible with
maternal plasma are selected. Generally, RBC
units that were collected within 7 days before
transfusion are preferred if they are available.
Centers have reported that using RBCs that are
antigen matched to the mother decreases the
risk of further maternal sensitization from
IUT.17
In rare cases, the mother’s antibody is di-
rected at a high-prevalence antigen and no
compatible blood is available. In these situa-
tions, the mother’s washed or frozen and de-
glycerolized RBCs can be used for fetal IUT.
Testing the mother’s siblings or searching rare
donor registries may provide additional sourc-
es of compatible units.
The volume of blood to be transfused can
be calculated, as shown in the following exam-
ple, by 1) determining the fetal and placental
total blood volume by multiplying the ultra-
sound-estimated fetal weight in grams (eg,
1000 g) by 0.14 mL/g, 2) multiplying this
amount (eg, 140 mL) by the difference in post-
transfusion (desired) and pretransfusion he-
matocrit (eg, 0.40 – 0.15 = 0.25), and 3) dividing
the resulting amount by the hematocrit of the
RBC unit (eg, 0.85). In this example, the result
is 41.2 mL.
Example:
[(1000 g) × (0.14 mL/g) × (0.40 – 0.15)]/85 =
41.2 mL
The blood volume transfused by cordo-
centesis and the rate of transfusion should be
adjusted to accommodate the clinical status of
the fetus.18 Transfusion is repeated if needed
according to the severity of the disease or
based on an estimated decline in hematocrit
of 1% per day to maintain the fetal hematocrit
at about 30%.
Other Treatments
Maternal plasma exchange and the adminis-
tration of intravenous immune globulin (IVIG)
have been used early in gestation before IUT
can be accomplished or as alternatives to in-
trauterine transfusion.4 Plasma exchange can
temporarily reduce antibody levels by as much
as 75%. Because its efficacy was established
before the use of IUT was widespread, its use
today is reserved for treatment failures on a
case-by-case basis. The American Society for
Apheresis classifies plasma exchange as a Cat-
egory II treatment based on weak (Grade 2C)
evidence for this indication.19
IVIG infusion has been shown to stabilize
anti-D titers, and results were best when the
procedure was started before 28 weeks’ gesta-
tion in a case series of 24 patients.20
Neonatal Management
During the first days after birth, close monitor-
ing of the bilirubin level is necessary because
of the threat of kernicterus, especially in pre-
mature neonates.21 The infant may require
blue-green light therapy, which oxidizes the el-
evated unconjugated bilirubin, allowing the
oxidation products to be excreted in the urine.
In addition, IVIG may be given to the infant to
help control hemolysis and, thus, elevated bili-
rubin. In neonates who are unresponsive to
phototherapy and IVIG, a double-volume-
exchange transfusion removes approximately
90% of the fetal red cells and 50% of the biliru-
bin. Exchange transfusion is generally unnec-
essary if the infant received IUTs. See Chapter
23 for a discussion of exchange transfusion in
the newborn.
 CHAPTER 22 Perinatal Issues in Transfusion Practice
RhIG
RhIG is available in 300-µg and 50-µg doses to
prevent alloimmunization to the D antigen.
The risk of a D-negative mother becoming im-
munized by a D-positive fetus can be reduced
from about 16% to less than 0.1% by the ap-
propriate administration of RhIG.3,4
Screening and Dosing for RhIG
Antepartum Administration
When a pregnant mother is D negative and the
father is D positive, the fetus may be D positive
and the mother may be at risk of D alloimmu-
nization. Such women are candidates for RhIG
prophylaxis to prevent alloimmunization. D-
negative females whose infants are D negative,
D-negative females who have been previously
immunized to D, and D-positive females are
not candidates for RhIG. Whether women with
variants of D should be considered to be D
positive is controversial. Maternal red cell ge-
notyping can assist with RhIG treatment deci-
sions because some D variants have been
found to make anti-D.22,23
The American College of Obstetricians
and Gynecologists (ACOG) recommends RhIG
administration at 28 weeks’ gestation because
92% of women who develop anti-D during
pregnancy do so at or after 28 weeks.3,24 Ante-
natal RhIG administration reduces alloimmu-
nization to 0.1% compared to 1.5% with post-
partum administration only. In addition, RhIG
is indicated after amniocentesis, cordocente-
sis, version, abortion, or abdominal trauma.
Postpartum Administration
D-negative mothers without anti-D should
receive RhIG after delivery of a D-positive in-
fant. A postpartum blood sample should be
screened for FMH to determine the RhIG dose.
The rosette test is 99.5% sensitive to an FMH of
10 mL or more. After incubation with anti-D,
indicator D-positive red cells form aggluti-
nates (rosettes) with the fetal D-positive red
cells. Weak D phenotypes in the mother or fe-
tus can cause false-positive or -negative test
results. Therefore, D-negative mothers with
䡲 565
positive results on a test for fetal blood in the
maternal circulation should undergo a sero-
logic weak D test or molecular RHD testing. If
the rosette test result is negative, a dose of 300
µg RhIG (100 µg in the United Kingdom) is giv-
en, which is sufficient to prevent immuniza-
tion by 15 mL of red cells (5 mL in the United
Kingdom) or 30 mL of whole blood. The pres-
ence of residual anti-D from antepartum RhIG
does not indicate ongoing protection.
A positive rosette test result indicates a
large FMH. About 0.3% of deliveries have an
FMH larger than 30 mL. When a rosette test re-
sult is positive, a quantitative test, such as the
Kleihauer-Betke (acid/elution) test, or flow cy-
tometry is needed to calculate the dose of
RhIG. Flow cytometry can precisely measure
fetal hemoglobin and/ or D-positive red cells.
The use of both markers avoids false-positive
results from maternal red cells containing fetal
hemoglobin.25,26
The Kleihauer-Betke test has been shown
to be far less precise than flow cytometry be-
cause of its technical difficulty. The Kleihauer-
Betke test is based on the resistance of fetal he-
moglobin to acid treatment (Method 5-2). A
thin smear of maternal blood is placed on a
slide, treated with acid, rinsed, counter-
stained, and read microscopically by counting
2000 cells. The maternal cells appear as ghosts,
and the fetal cells are pink. The formula below
is used to calculate the fetal bleeding:
(Fetal cells/total cell counted) × maternal
blood volume (mL) = fetal hemorrhage (mL)
Example:
(6 cells/2000 cells) × 5000 mL =
15 mL fetal whole blood
According to the results for this example,
a 300-µg vial of RhIG will suppress alloimmu-
nization by 30 mL of fetal whole blood. Fetal
hemorrhage is 15 mL, so the number of RhIG
vials is 15 mL/30 mL/vial = 0.5 vial. Because of
the inherently wide estimate generated by the
test, if the calculated dose to the right of the
decimal point is 0.5 vial, it should be rounded
up to the next whole number plus one vial; if
566 䡲 AABB TECHNICAL MANUAL

the calculated dose to the right of the decimal
point is <0.5 of a vial, it should be rounded
down to the next whole number plus one vial
(Table 22-1). In the above example, the dose to
be given is two vials. Additional examples are
as follows:
Examples:
1.6 vials calculated =
2 (round up) + 1 (add 1) = 3
1.4 vials calculated =
1 (round down) + 1 (add 1) = 2
Postpartum RhIG should be given to the
mother within 72 hours of delivery. If prophy-
laxis is delayed, the ACOG recommends that
treatment still be administered. If the D type of
the newborn is unknown or undetermined (eg,
for a stillborn infant), RhIG should be admin-
istered.
Depending on the preparation, RhIG can
be given by intramuscular (IM) or intravenous
(IV) injection. If IM preparations are used,
multiple doses are given in different sites or at
different times within 72 hours. Multiple doses
of the IV preparation may be administered ac-
cording to the instructions in the package in-
sert.
Serology and Mechanism
Administration of RhIG during pregnancy may
produce a positive antibody screening result in
the mother, but the titer is rarely greater than 4
and thus poses no risk to the fetus. Occasion-
ally, the DAT result may be positive in a new-
born with no evidence of hemolysis. About
10% of the 28-week gestation dose will be pres-
ent at delivery (the half-life of IgG is 25 days).
This anti-D is not active immunization, so
postpartum RhIG should be given if the new-
born is D positive.
RhIG is entirely IgG, whereas active im-
munization has an IgM component. Thus, new
anti-D produced by the mother can often be
detected in the saline phase and can be com-
pletely or partially inactivated by 2-mercapto-
ethanol or DTT treatment, whereas RhIG can-
not. In addition, passively acquired anti-D
rarely achieves a titer above 4. Antibody titers
do not correlate with the effectiveness of the
RhIG or the amount of FMH.
The mechanism of action of RhIG has not
been completely elucidated. Current evidence
shows that D-positive red cells are opsonized
by RhIG and removed by macrophages, which
release cytokines that result in immunomodu-
lation.27 The number of IgG molecules known
to prevent immunization is much smaller than
the D antigen sites on red cells.
ABO HEM O LY TIC DISEASE
Because of the use of RhIG, ABO incompatibil-
ity is now the most common cause of HDFN.
HDFN is triggered when naturally occurring

TABLE 22-1. Amount of RhIG to Administer Based on Amount of Fetomaternal Hemorrhage

Dose

% Fetal Cells Vials to Inject g (mcg) IU

0.3-0.8 2 600 3000

0.9-1.4 3 900 4500
1.5-2.0 4 1200 6000
2.1-2.6 5 1500 7500

Notes:
1. Based on a maternal blood volume of 5000 mL.
2. 1 vial of 300 g (1500 IU) is needed for each 15 mL of fetal red cells or 30 mL of fetal whole blood.
 CHAPTER 22 Perinatal Issues in Transfusion Practice
IgG anti-A,B in a group O mother is transport-
ed across the placenta and bound to fetal red
cells expressing A or B antigens. Destruction of
fetal red cells rarely leads to severe anemia be-
cause fetal ABO antigens are poorly developed
and antibody is neutralized by tissue and solu-
ble antigens. Also, ABO HDFN has no comple-
ment-mediated hemolytic mechanism. If an
umbilical cord DAT result is negative, ABO
HDFN is unlikely even if the mother has the
corresponding ABO antibody(ies). After birth,
hyperbilirubinemia can be successfully treat-
ed with phototherapy in most cases; in rare sit-
uations, exchange transfusions may be re-
quired.
Group A and B infants of group O mothers
are more severely affected by ABO HDFN. In
populations of European or Asian ancestry,
group A infants are most commonly affected;
in populations of African ancestry, group B in-
fants are most likely to be affected. The overall
incidence of ABO HDFN is higher in people of
African than European ancestry.8(p529) In pa-
tients with severe disease, the DAT result is
nearly always positive.28
If ABO HDFN is ruled out, antibodies
against low-prevalence red-cell antigens in-
herited from the father should be suspected.
Testing the eluate from cord blood or maternal
serum (if ABO compatible) against the father’s
red cells with an antiglobulin technique is of-
ten diagnostic.
IMM U NE T HRO M B O C Y T OP E N I A
Maternal IgG antibodies to platelets can cross
the placenta and cause severe thrombocyto-
penia. Two categories of immune thrombocy-
topenia are recognized: FNAIT and ITP. The
diagnostic distinction between them is impor-
tant for therapy selection.
FNAIT
FNAIT is caused by antibodies that are specific
for platelet antigens inherited from the father
that are are absent in the mother. Platelet anti-
gens represent specific polymorphisms in
platelet membrane glycoproteins. Approxi-
mately 80% of FNAIT cases are caused by hu-
䡲 567
man platelet antigen HPA-1a, which is present
in about 98% of the US population.29 About
10% of cases are caused by anti-HPA-5b, 4% by
anti-HPA-1b, 2% by anti-HPA-3a, and 6% by
other antibodies. The incidence of affected
pregnancies is approximately 1 per 1500 to
2000.30
In about 25% of FNAIT cases, the platelet
antibody develops during the first pregnancy
and that fetus is affected. The maternal anti-
body has been detected as early as 17 weeks’
gestation and the fetus may develop thrombo-
cytopenia as early as 20 weeks’ gestation. How-
ever, the disease is often not discovered until
birth, when the newborn presents with pete-
chiae, ecchymoses, or intracranial hemor-
rhage. Intracranial hemorrhage occurs in 10%
to 30% of infants and 50% of fetuses with
FNAIT.29 The greatest risk of hemorrhage oc-
curs when the fetal platelet count is less than
50,000/µL. The response of the fetal hemato-
poietic system to FNAIT is variable and may
include compensatory extramedullary hema-
topoiesis. In rare cases, hydrops fetalis devel-
ops. Fetal anemia without red cell incompati-
bility can also occur.
A history of giving birth to a newborn with
thrombocytopenia can alert the obstetrician
to a potentially affected pregnancy. The preg-
nant woman and the father should be typed
for platelet antigens, and the woman should
be screened for the alloantibody. DNA testing
of the father can determine the zygosity of the
antigen involved.31
The DNA genotype of the fetus can be de-
termined as early as 11 to 13 weeks’ gestation.
Assessment of the fetus should begin at or be-
fore 20 weeks’ gestation, when severe throm-
bocytopenia and hemorrhage can occur.
When cordocentesis is used to determine the
platelet count, irradiated, CMV-reduced-risk,
antigen-negative platelets should be used.
If needed, platelet transfusion should be
given to the fetus to treat thrombocytopenia
and avoid hemorrhage. Many blood suppliers
have identified donors who are negative for
human platelet alloantigen 1a, the most
widely implicated platelet antibody, and can
prepare suitable platelets for IUT. The mother
is negative for the implicated alloantigen and
568 䡲 AABB TECHNICAL MANUAL

can be a donor if her platelets are washed.
When platelet transfusion is needed urgently
and maternal platelets are unavailable, un-
selected platelets may be used.32
The mother is given IVIG after the fetus is
determined to be affected. The dose is usually
1 g/kg each week. In a retrospective review,
IVIG treatment alone appeared to be as safe
and effective as cordocentesis with platelet
transfusion.33 However, IVIG treatment failures
may occur and can necessitate fetal platelet
count monitoring by cordocentesis and re-
quire platelet transfusions.34 The goal of both
transfusion and IVIG treatment is to avoid
hemorrhage. Ultrasound monitoring of the fe-
tus to detect hemorrhage is not recommend-
ed because intracranial hemorrhage usually
indicates permanent brain damage. Before
vaginal delivery, the fetal platelet count should
be >50,000/µL.
After birth, the infant’s platelet count may
decrease in the first few hours to days. In 2 to 3
weeks, when the antibody has been cleared,
the infant’s platelet count returns to normal.
The presence or absence of severe thrombocy-
topenia and intracranial hemorrhage in the
firstborn correlates with the outcomes of sub-
sequent pregnancies.35
ITP
Autoantibodies against platelets, which are re-
active with the mother’s own platelets as well
as donor or fetal platelets, are another cause of
fetal and neonatal thrombocytopenia. Throm-
bocytopenia in a pregnant woman is common
and is rarely associated with ITP. However, ITP
may cause thrombocytopenia in both the
mother and fetus. Fortunately, only about 10%
of newborns with ITP have platelet counts
<50,000/µL, and only 1% to 2% have a high risk
of hemorrhage.36
A pregnant woman with thrombocytope-
nia or a preexisting diagnosis of ITP should be
tested for serum platelet autoantibody. A neg-
ative result generally indicates that the throm-
bocytopenia was caused by other conditions
and suggests that the fetus or neonate is not at
risk. In contrast, a pregnant woman with sig-
nificant thrombocytopenia and petechiae or
other evidence of hemorrhage caused by auto-
antibody should undergo similar treatment to
that used in nonpregnant women with ITP.
Prednisone at a dose of 1 to 2 mg/kg is
usually effective. However, with persistent ma-
ternal thrombocytopenia, IVIG 1 g/kg/day for
2 to 5 days is indicated. Although the maternal
platelet count is often monitored in these cas-
es, it does not correlate with the newborn’s
platelet count.36 Fetal blood sampling to deter-
mine platelet count is not usually recom-
mended because the risk of morbidity and
mortality from cordocentesis is greater than or
equal to the risk of severe bleeding in utero or
at delivery. However, if fetal sampling is per-
formed, a platelet transfusion should be ad-
ministered at the same time. Platelet transfu-
sions may be needed in about 15% of
newborns.36

KE Y POI N T S

1. HDFN is caused by maternal antibodies that are specific to a paternal red cell antigen. The
maternal IgG antibody is transported across the placenta, where it destroys the fetal red cells.
2. Some antibodies, such as anti-I, -P1, -Lea and -Leb, can be ignored. The most common clini-
cally significant antibodies are anti-D, -K, and -c.
3. Molecular typing of fetal DNA can be performed on maternal plasma early in the second tri-
mester.
4. The recommended titer method is saline AHG with 60-minute incubation at 37 C. Other
methods, such as those using albumin AHG or gel, may result in higher titers that may lead
to misinterpretation by the obstetrician.
5. For IUT, the blood should be irradiated, CMV reduced-risk, hemoglobin S negative, group O
(in most cases), and less than 7 days old.
 CHAPTER 22 Perinatal Issues in Transfusion Practice 䡲 569

6. The rosette test is a sensitive method for detecting fetomaternal hemorrhage of ap-
proximately 10 mL or more. Flow cytometry can precisely measure hemoglobin F and/
or D-positive red cells.
7. The calculated RhIG dose should be rounded up if the number to the right of the decimal
point is 0.5 or rounded down if the number is <0.5. In either case, a vial should be added to
the result.
8. Despite the prevalence of ABO HDFN, severe anemia rarely occurs. After birth, hyperbiliru-
binemia can usually be treated with phototherapy alone.
9. In fetal/neonatal alloimmune thrombocytopenia, the platelet antibody may develop at
around 17 weeks of gestation in the first pregnancy and fetal thrombocytopenia may devel-
op as early as 20 weeks. Irradiated, CMV-reduced-risk, antigen-negative platelets should be
given to treat thrombocytopenia and avoid hemorrhage.

R E F E R EN C E S

1. Bowman JM, Pollock JM, Penston LE. Fetoma-
ternal transplacental hemorrhage during
pregnancy and after delivery. Vox Sang 1986;
51:117-21.
2. Sebring ES, Polesky HF. Fetomaternal hemor-
rhage: Incidence, risk factors, time of occur-
rence, and clinical effects. Transfusion 1990;
30:344-57.
3. Bowman JM. The prevention of Rh immuniza-
tion. Transfus Med Rev 1988;2:129-50.
4. Bowman JM. Controversies in Rh prophylaxis:
Who needs Rh immune globulin and when
should it be given? Am J Obstet Gynecol 1985;
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5. Klein HG, Anstee DJ. The Rh blood group sys-
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medicine. 12th ed. Oxford: Wiley-Blackwell,
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6. van der Schoot CE, Martine Tax GH, et al. Pre-
natal typing of Rh and Kell blood group system
antigens: The edge of a watershed. Transfus
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7. Vaughan JI, Manning M, Warwick RM, et al. In-
hibition of erythroid progenitor cells by anti-
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8. Klein HG, Anstee DJ. Haemolytic disease of the
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fusion in clinical medicine. 12th ed. Oxford:
Wiley-Blackwell, 2014:499-548.
9. Pollock JM, Bowman JM. Anti-Rh(D) subclass-
es and severity of Rh hemolytic disease of the
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10. Firan M, Rawdon R, Radu C, et al. The MHC
class I-related receptor, FcRn, plays an essen-
tial role in the maternal transfer of gamma-
globulin in humans. Int Immunol 2001;13:993-
1002.
11. Dennery PA, Seidman DS, Stevenson DK. Neo-
natal hyperbilirubinemia. N Engl J Med 2001;
344:581-90.
12. Moise KJ Jr. Management of rhesus alloimmu-
nization in pregnancy. Obstet Gynecol 2008;
112:164-76.
13. Kanra T, Erdem G, Tekinalp G, et al. Further
hemolytic disease of the newborn caused by
anti-M. Am J Hematol 1996;53:280-1.
14. Wagner FF, Flegel WA. RHD deletion occurred
in the Rhesus box. Blood 2000;95:3662-8.
15. Akolekar K, Finning K, Kuppusamy R, et al, Fe-
tal RHD genotyping in maternal plasma at 11-
13 weeks of gestation. Fetal Diagn Ther 2011;
29:301-6.
16. Hilden JO, Backleman K, Nilsson J, Ernerudh J.
Flow-cytometric quantitation of anti-D anti-
bodies. Vox Sang 1997;72:172-6.
17. Schonewille H, Klumper FJCM, Watering
LMG, et al. High additional maternal red cell
alloimmunization after Rhesus- and K-
matched intrauterine intravascular transfu-
sions for hemolytic disease of the fetus. Am J
Obstet Gynecol 2007;196:143.e1-6.
18. Rodunovic N, Lockwood CJ, Alvarez M, et al.
The severely anemic and hydropic isoimmune
fetus: Changes in hematocrit associated with
intrauterine death. Obstet Gynecol 1992;79:
390-3.
19. Schwartz J, Winters JL, Padmanabhan A, et al.
Guidelines on the use of therapeutic apheresis
in clinical practice—Evidence-based ap-
proach from the Writing Committee of the
American Society for Apheresis. The Sixth Spe-
cial Issue. J Clin Apher 2013;28:145-284.
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20. Margulies M, Voto LS, Mathet E. High dose in-
travenous IgG for the treatment of severe Rhe-
sus alloimmunization. Vox Sang 1991;61:181-
9.
21. American Academy of Pediatrics Subcommit-
tee on Hyperbilirubinemia. Management of
hyperbilirubinemia in the newborn 35 or more
weeks gestation. Pediatrics 2004;114:297-316.
22. Domen RE. Policies and procedures related to
weak D phenotype testing and Rh immune
globulin administration. Arch Pathol Lab Med
2000;124:1118-21.
23. Flegel, WA. How I manage patients and donors
with weak D phenotype. Curr Opin Hematol
2006;13:476-83.
24. Prevention of Rh D alloimmunization. ACOG
practice bulletin #4. Washington, DC: Ameri-
can College of Obstetricians and Gynecolo-
gists, 1999.
25. Radel DJ, Penz CS, Dietz AB, Gastineau DA. A
combined flow cytometry-based method for
fetomaternal hemorrhage and maternal D.
Transfusion 2008;48:1886-91.
26. Sandler SG, Delaney M, Gottschall JL, College
of American Pathologists Transfusion Medi-
cine Resource Committee. Proficiency tests re-
veal the need to improve laboratory assays for
fetomaternal hemorrhage for Rh immunopro-
phylaxis. Transfusion 2013;53:2098-102.
27. Branch DR, Shabani F, Lund N, Denomme GA.
Antenatal administration of Rh-immune glob-
ulin causes significant increases in the immu-
nomodulary cytokines transforming growth
factor- and prostaglandin E2. Transfusion
2006;48:1316-22.
28. Herschel M, Karrison T, Wen M, et al. Isoim-
munization is unlikely to be the cause of he-
molysis in ABO-incompatible but direct anti-
globulin test-negative neonates. Pediatrics
2002;110:127-30.
29. Davoren A, Curtis BR, Aster RH, McFarland JG.
Human platelet antigen-specific alloantibod-
ies implicated in 1162 cases of neonatal allo-
immune thrombocytopenia. Transfusion
2004;44:1220-5.
30. Williamson LM, Hackett G, Rennie J, et al. The
natural history of fetomaternal alloimmuniza-
tion in the platelet-specific antigen HPA-1a
(PlA1, Zwa) as determined by antenatal screen-
ing. Blood 1998;92:2280-7.
31. Radder CM, Brand A, Kanhai HH. A less inva-
sive treatment strategy to prevent intracranial
hemorrhage in fetal and neonatal alloimmune
thrombocytopenia. Am J Obstet Gynecol 2001;
185:683-8.
32. Kiefel V, Bassler D, Kroll H, et al. Antigen-posi-
tive platelet transfusion in neonatal alloim-
mune thrombocytopenia (NAIT). Blood 2006;
107:3761-3.
33. Van den Akker ESA, Oepkes D, Lopriore E, et al.
Noninvasive antenatal management of fetal
and neonatal alloimmune thrombocytopenia:
Safe and effective. Br J Obstet Gynaecol 2007;
114:469-73.
34. Silver RM, Porter TF, Branch DW, et al. Neona-
tal alloimmune thrombocytopenia: Antenatal
management. Am J Obstet Gynecol 2000;182:
1233-8.
35. Birchall JE, Murphy MF, Kroll H. European col-
laborative study of the antenatal management
of feto-maternal alloimmune thrombocytope-
nia. Br J Haematol 2003;122:275-88.
36. Webert KE, Mittal R, Sigouin C, et al. A retro-
spective 11-year analysis of patients with idio-
pathic thrombocytopenic purpura. Blood
2003;102:4306-11.
 C h a p t e r 2 3

Neonatal and Pediatric

Transfusion Practice

Cassandra D. Josephson, MD, and Erin Meyer, DO, MPH

TR A N S F U S I O N P R A C T I C E I N n e o -
natal and pediatric patients differs from
that in adults.1 The differences are related to
physiologic changes occurring during the
transition from fetus to adolescent. Blood
volume, hematologic values, immune system
maturity, and physiologic responses to hypo-
volemia and hypoxia are variable in this het-
erogeneous population, contributing to the
complexity and intricacies of pediatric trans-
fusion practice.
This chapter discusses neonatal and pedi-
atric transfusion practice during two distinct
periods: infancy from birth to 4 months, and
infancy after 4 months and childhood. The pe-
diatric practices addressed include 1) small-
volume component preparation, 2) transfu-
sion indications for blood components, 3)
transfusion administration and exchange
transfusion, 4) transfusion support in specific
diseases, 5) rationale for special processing of
blood components, and 6) pediatric massive
transfusion protocols (MTPs).
T R A N SF U SI O N I N IN FAN TS
YO U N G E R TH A N 4 M ON TH S
Considerations for Component
Preparation and Therapy
The fact that patients younger than 4 months
have small blood/plasma volumes and imma-
ture organ system functions necessitates spe-
cial approaches to component therapy. This is
especially important for very-low-birthweight
(VLBW) infants (<1500 g) and extremely low-
birthweight infants (<1000 g).
Fetal and Neonatal Physiology Affecting
Transfusion Practice
Healthy full-term neonates have a mean cord
blood hemoglobin level of 16.9 ± 1.6 g/dL,
whereas that of preterm neonates is 15.9 ±
2.4 g/dL. The hemoglobin concentration nor-
mally declines during the first few weeks of
life, resulting in the physiologic anemia of in-
fancy in newborns and physiologic anemia of

Cassandra D. Josephson, MD, Director of Transfusion, Tissue, and Apheresis Services, Children’s Healthcare of
Atlanta, and Associate Professor, Pathology and Pediatrics, Emory University School of Medicine, and Erin
Meyer, DO, MPH, Associate Director of Transfusion, Tissue, and Apheresis Services, Children’s Healthcare of
Atlanta, and Assistant Professor of Pathology and Laboratory Medicine, Emory University School of Medicine,
Atlanta, Georgia
C. Josephson has disclosed financial relationships with Immucor and Octapharma. E. Meyer has disclosed no
conflicts of interest.

571
572 䡲 AABB TECHNICAL MANUAL
prematurity in preterm infants.2 Both anemias
are considered self-limited and are usually tol-
erated without harmful effects.
The rate of decline in hemoglobin levels is
a function of gestational age at birth. At 4 to 8
weeks after birth, hemoglobin decreases to as
low as 8.0 g/dL in preterm infants weighing
1000 to 1500 g and 7.0 g/dL in neonates weigh-
ing less than 1000 g at birth.3 The physiologic
decrease in hemoglobin concentration is due
to several factors: 1) a decrease in erythropoie-
tin (EPO) resulting in diminished red cell pro-
duction, 2) a decrease in survival of fetal red
cells, and 3) an increasing blood volume due to
rapid growth. Reduced EPO production re-
sults from increased oxygen delivery to tissues
because of increased pulmonary blood flow,
elevated arterial pO2 levels, and increased red
cell 2,3-diphosphoglycerate (2,3 DPG) and he-
moglobin A levels.
Clinical Considerations Related to
Infant Size and Blood Volume
Blood volumes of pediatric patients vary with
body weight. A full-term newborn has a blood
volume of approximately 85 mL/kg compared
to 100 mL/kg in a preterm newborn. Due to
the small total blood volumes of preterm in-
fants (100 mL or less), blood banks must be ca-
pable of providing appropriately sized blood
components.
Many factors, including iatrogenic blood
loss from repeated phlebotomy, lead to fre-
quent transfusions. Hypovolemia is not toler-
ated well in newborns because their left ven-
tricular stroke volume decreases without an
increase in heart rate when >10% of their
blood volume is lost. Thus, newborns must
physiologically increase their peripheral vas-
cular resistance with decreasing cardiac out-
put to maintain systemic blood pressure. Ulti-
mately, poor tissue perfusion and oxygenation
occur, resulting in metabolic acidosis.4
Although transfusion may be required, it
is neither necessary nor acceptable to replace
the amount of blood lost mL for mL; rather,
transfusions can be administered to maintain
a target hemoglobin level in certain clinical sit-
uations.5 When ill preterm and full-term neo-
nates receive multiple transfusions, they have
proportionately lower levels of circulating fetal
hemoglobin and an increase in adult hemo-
globin levels.
Erythropoietic Response
The EPO response in newborns differs from
that in adults and older children. In the latter
groups, oxygen sensors in the kidney recog-
nize decreases in oxygen delivery, resulting in
the release of EPO into the circulation. In con-
trast, in the fetus, this sensor is located in the
liver and is less sensitive, resulting in reduced
EPO production in the face of hypoxia (hypo-
responsiveness). This response likely occurs to
prevent polycythemia of the fetus in the hy-
poxic intrauterine environment.
Although EPO production eventually
shifts from the liver to the kidney, most prema-
ture infants produce the smallest amount of
EPO for any degree of anemia.6 As an alterna-
tive to transfusion, the use of recombinant hu-
man EPO has been shown to reduce donor ex-
posures in premature infants and minimize
the severity of anemia.7 As compliance with
strict transfusion threshold criteria has in-
creased, clinicians have decreased their phle-
botomy rates in VLBW infants, reducing the
rates of iatrogenically induced anemia and
numbers of transfusions. Thus, in most cases,
this approach achieves the same goal as EPO
therapy (ie, decreases the use of transfusions
and the number of donor exposures) without
expensive pharmacotherapy and its risk of ad-
verse effects.
Cold Stress
Hypothermia in the neonate can trigger or ex-
aggerate a number of responses, including 1)
an increase in metabolic rate; 2) hypoglyce-
mia; 3) metabolic acidosis; and 4) potential
apneic events that may lead to hypoxia, hypo-
tension, and cardiac arrest. In-line blood
warmers are required for all Red Blood Cell
(RBC) exchange transfusions to combat the ef-
fects of hypothermia. A radiant heater should
never be used to warm the blood being trans-
fused due to the risk of hemolysis. Further-
more, to prevent hemolysis in neonates under-
 CHAPTER 23 Neonatal and Pediatric Transfusion Practice
going phototherapy, the blood-administration
tubing should be positioned to minimize ex-
posure to phototherapy light.8
Immunologic Status
Their immature immune system predisposes
infants to infectious and noninfectious haz-
ards of transfusion. In fact, much of the special
processing of products for preterm infants and
neonates is directly related to their underde-
veloped immune function. Most of their hu-
moral immunity (antibody protection) is pro-
vided by the mother starting early in
pregnancy (approximately 12 weeks) through
placental transfer of immunoglobulins. Be-
tween 20 and 33 weeks of gestation, fetal IgG
levels rise significantly because of selective
transport system maturation in the placenta.
The breakdown of IgG occurs at a slower rate
in the fetus than in the mother, enabling con-
servation of the transplacental maternal anti-
body during the neonatal period. Unexpected
red cell alloantibodies of either IgM or IgG
class are rarely produced by the infant during
the neonatal period. This lack of red cell allo-
antibody production is not well understood
but has been postulated to be due to deficient
T-helper-cell function, enhanced T-suppres-
sor-cell activity, and poor antigen-presenting-
cell function.9
Cellular immune responses are also in-
completely developed during this period and
may make infants susceptible to transfusion-
associated graft-vs-host disease (TA-GVHD).
TA-GVHD has been reported most frequently
in newborns with confirmed or suspected
congenital immunodeficiency. The majority of
TA-GVHD cases reported in nonimmunocom-
promised infants have occurred after intra-
uterine transfusion and subsequent postnatal
exchange transfusion.10,11 There have also been
rare cases of TA-GVHD associated with ex-
treme prematurity, neonatal alloimmune
thrombocytopenia, or extracorporeal mem-
brane oxygenation (ECMO).10,12 Once an in-
fant develops TA-GVHD, the chance of associ-
ated mortality is higher than 90%. TA-GVHD
can be prevented by pretransfusion irradiation
of cellular blood components.12,13
䡲 573
Metabolic Problems
In infants younger than 4 months, large-vol-
ume transfusions of reconstituted whole blood
or plasma may result in acidosis and/or hypo-
calcemia because of the immature liver’s in-
ability to effectively metabolize citrate. The
immature kidneys also contribute to these
complications because they have lower glo-
merular filtration rates and concentrating abil-
ity than older infants and children, leading to
difficulties in excreting excess potassium, acid,
and/or calcium.
POTASSIUM. Small-volume, simple transfu-
sions administered slowly have been shown to
have little effect on serum potassium concen-
trations in infants younger than 4 months de-
spite the high potassium levels in the plasma
of stored RBCs. In calculating levels of infused
potassium, Strauss13,14 determined that an RBC
unit (80% hematocrit) stored in an extended
storage medium for 42 days would deliver
2 mL of plasma containing only 0.1 mmol/L of
potassium when transfused at 10 mL/kg. This
amount of potassium is much less than the
daily requirement of 2 to 3 mmol/L for a pa-
tient weighing 1 kg. It must be stressed that
this calculation does not apply to the transfu-
sion of large volumes of RBCs (>20 mL/kg). Se-
rum potassium can rise rapidly in these small
patients—particularly during surgery, exchange
transfusion, or ECMO—and is dependent on the
plasma potassium levels in the blood and ma-
nipulation of the blood components.15,16
The type of anticoagulant-preservative
solution used to store RBCs at collection deter-
mines the amount of potassium leakage. For
instance, a unit of RBCs preserved in an addi-
tive solution (AS), such as AS-1, AS-3, or AS-5,
delivers less extracellular potassium than
RBCs stored in citrate-phosphate-dextrose-
adenine (CPDA)-1.13,17 In addition, special
component processing, such as irradiation,
can potentiate potassium leaks. If such com-
ponents are stored for more than 24 hours,
washing may be required to remove the excess
potassium before transfusion.12 This washing
practice is supported by several reports of
infants who received via central line or intra-
574 䡲 AABB TECHNICAL MANUAL

cardiac line either older RBC units or units that Indications

had been irradiated (>1 day before transfu-
Several guidelines have been published over
sion) and had severe adverse effects, including
the past 15 years regarding the indications for
cardiac arrest and death.18,19 This washing
RBC transfusions in neonates.22-26 Most of the
practice is controversial, however, and several
recommendations are based on experience ac-
institutions accept AS units for low-volume
quired in clinical practice rather than pub-
neonatal transfusions without washing as long
lished evidence. To this end, a critical need ex-
as these units do not exceed a certain storage
ists for clinical studies in this area.27 Table 23-1
age or time after irradiation.20
lists the most recently published guide-
2,3-DPG. Levels of 2,3-DPG in red cells are lines.23,26
known to decline rapidly after 1 to 2 weeks of
storage. This deficit does not affect older chil- Compatibility Testing
dren and adult recipients negatively because
AABB Standards allows limited pretransfusion
of their ability to replenish the missing 2,3-
serologic testing for infants younger than 4
DPG in vivo and to compensate for hypoxia by
months.28(p39) Initial patient testing must in-
increasing their heart rate. Infants younger
clude ABO and D typing of the patients’ red
than 4 months are not able to do this as effec-
cells and screening for unexpected red cell an-
tively as a result of their low levels of intracel-
tibodies using either plasma or serum from
lular 2,3-DPG that reach even lower levels with
the infant or mother. During any hospitaliza-
respiratory distress syndrome or septic shock.
tion, crossmatch-compatibility testing and re-
Thus, if a large proportion of the neonate’s
peat ABO and D typing need not be conducted
blood volume is composed of transfused 2,3-
as long as all of the following criteria are met:
DPG-depleted blood, the resulting shift in the
1) the antibody screening result is negative; 2)
hemoglobin oxygen dissociation curve further
transfused RBCs are group O, ABO identical, or
increases oxygen affinity for hemoglobin and
ABO compatible; and 3) transfused cells are
reduces oxygen availability to the tissues.
either D negative or the same D type as the pa-
Therefore, the recommended therapy for
tient. Testing the infant’s reverse type for anti-A
newborns is an exchange transfusion with
and/or anti-B is not necessary. However, be-
RBCs that are usually less than 14 days old, al-
fore non-group O RBCs can be issued, testing
though this practice is variable and depen-
of the infant’s plasma or serum is required to
dent on institutional standard operating pro-
detect passively acquired maternal anti-A or
cedures. However, the medical need for fresh
anti-B and should include antiglobulin phase.
RBC units for small-volume transfusions has
If the antibody is present, ABO-compatible
not been established and these transfusions
RBCs must be transfused until the acquired
have even been characterized as unneces-
antibody is no longer detected.
sary.5,13,16 A prospective randomized controlled
If an unexpected non-ABO alloantibody is
trial to assess the outcomes of longer vs short-
detected in the infant’s or mother’s specimen,
er storage times of RBCs is necessary in this
the infant must be transfused with RBC units
population (see “RBC Age” section below).21
lacking the corresponding antigen(s) or units
that are compatible by antiglobulin cross-
RBC Transfusion Support
match. This regimen should continue until the
Ill neonates are more likely to receive RBC maternal antibody is no longer detected in the
transfusions than any other patient age group, infant’s plasma or serum. The policy of the
and RBCs are the component most often hospital transfusion service determines the
transfused during the neonatal period.5 RBC frequency for reevaluating the patient’s anti-
replacement is considered for sick neonates bodies. Once a negative antibody screening
when approximately 10% of blood volume has result is obtained, crossmatches and use of an-
been lost or they have symptomatic anemia. tigen-negative blood are no longer required in
 CHAPTER 23 Neonatal and Pediatric Transfusion Practice 䡲 575

TABLE 23-1. Transfusion Guidelines for RBCs in Infants Younger than 4 Months23,26

1. Hematocrit <20% with low reticulocyte count and symptomatic anemia (tachycardia, tachypnea, poor
feeding).

2. Hematocrit <30% and any of the following:

a. On <35% oxygen hood.

b. On oxygen by nasal cannula.

c. On continuous positive airway pressure and/or intermittent mandatory ventilation on mechanical

ventilation with mean airway pressure <6 cm of water.

d. With significant tachycardia or tachypnea (heart rate >180 beats/minute for 24 hours, respiratory
rate >80 beats/minute for 24 hours).

e. With significant apnea or bradycardia (>6 episodes in 12 hours or 2 episodes in 24 hours requir-
ing bag and mask ventilation while receiving therapeutic doses of methylxanthines).

f. With low weight gain (<10 g/day observed over 4 days while receiving  100 kcal/kg/day).

3. Hematocrit <35% and either of the following:

a. On >35% oxygen hood.

b. On continuous positive airway pressure/intermittent mandatory ventilation with mean airway

pressure  6-8 cm of water.

4. Hematocrit <45% and either of the following:

a. On extracorporeal membrane oxygenation.

b. With congenital cyanotic heart disease.

infants younger than 4 months because of
their immature immunologic status.
Multiple observational studies have
shown that alloimmunization to red cell anti-
gens is rare during the neonatal period.9,29,30
For this reason, repeated typing and screening,
which are required for adults and children old-
er than 4 months, is unnecessary in younger
infants and contributes to significant iatrogen-
ic blood loss. Also, the transfusion service
should avoid transfusing any components that
may passively transfer unexpected alloanti-
bodies or ABO-incompatible antibodies to re-
cipients.31
Components for Neonatal Transfusion
Advances in neonatology now permit the sur-
vival of extremely premature infants with sur-
factant therapy, nitric oxide therapy, high-
frequency ventilators, and compliance with
transfusion practice guidelines. These advanc-
es have substantially decreased the number of
RBC transfusions administered in this popula-
tion. Most neonatal transfusions are now given
to VLBW infants.32
Aliquoting for Small-Volume
Transfusion
The purpose of creating small-volume aliquots
is to limit donor exposures, prevent circulatory
overload, and potentially decrease donor-
related risks.33-37 Several technical approaches
can be used to accomplish these goals and
minimize blood wastage.38
Small-volume RBC transfusion aliquots
are commonly made with a multiple-pack
576 䡲 AABB TECHNICAL MANUAL
system.38,39 Quad packs, employed mostly by
blood centers, are produced from a single unit
of whole blood that is diverted into a primary
bag with three integrally attached smaller
bags. The plasma is then separated and divert-
ed into one bag during component prepara-
tion. The remaining red cells are drawn into
the smaller bags as needed for transfusion.
Each of the smaller units has the same expira-
tion date as the original unit because the sys-
tem’s original seal has remained intact and a
“closed system” is maintained.
A hospital transfusion service can then re-
move (either by heat sealer or metal clips) each
aliquot as needed. For hospital transfusion
services without a sterile connection device
(Fig 23-1), this method provides three aliquots
FIGURE 23-1. A sterile tubing welder (reproduced
with permission from Terumo Medical Corp.,
Somerset, NJ).
from a single unit.38 However, blood compo-
nents may still be wasted when used in ali-
quots that are larger than the dose selected for
each patient based on body weight.
Hospital transfusion services that have a
sterile connection device have multiple addi-
tional options to produce aliquots, such as
transfer packs [eg, PEDI-PAK system (Genesis
BPS, Hackensack, NJ) Fig 23-2], small-volume
bags, or tubing with integrally attached syring-
es.38 Syringe sets (Fig 23-3) offer the greatest
accuracy for obtaining the desired volume to
be transfused based on volume-per-weight
calculations.38 Some syringe sets have an at-
tached 150-micron in-line filter for use during
the aliquoting process so that, when issued by
the blood bank, the cells are ready to be placed
on a syringe pump without further manipula-
tion of the component at the bedside. This
process eliminates the need for the nurse to
transfer blood from the pack to a syringe at the
bedside for delivery by a syringe pump. Re-
moving this additional step reduces the risk of
contamination, mislabeling, or damage to the
unit that results in blood loss or spillage.38
Reducing donor exposures is more readily
accomplished by this technique, which en-
ables a recipient to receive multiple small-vol-
ume transfusions from a single unit until it
reaches its expiration date.40,41 Many hospital
transfusion services assign a single unit of
RBCs to one or more infants based on their
weight.34-40
Once an aliquot is produced at either the
blood center or hospital blood bank, it must be
labeled with the expiration date and the origin
and disposition of each smaller unit must be
recorded. Aliquot expiration dates vary from
institution to institution, and local standard
operating procedures should always be fol-
lowed.
RBC Additive Solutions
Historically, transfused RBCs for children con-
tained CPDA-1 anticoagulant-preservative so-
lution.40 However, as ASs (see Chapter 6)
evolved to extend the shelf life of RBCs, many
experts began to question their safety in neo-
nates. One concern is the large amount of ade-
 CHAPTER 23 Neonatal and Pediatric Transfusion Practice 䡲 577

FIGURE 23-2. Diagram of PEDI-PAK (reproduced with permission of Genesis BPS, Hackensack, NJ).

nine and mannitol in AS and its relation to re-
nal toxicity. Moreover, mannitol is a potent
diuretic with effects on fluid dynamics that
can result in fluctuations in the cerebral blood
flow of preterm infants.
Most evidence suggests that small-vol-
ume transfusions (5 to 15 mL/kg) containing
AS are safe for this patient population. Specifi-
cally, when AS-1 and AS-3 were compared, no
harmful effects were observed in neonates re-
ceiving small-volume, simple transfusions.40-42
These transfusions were as effective as CPDA-1
RBCs in increasing hemoglobin levels in recip-
ients. Luban and colleagues used theoretical
calculations in a variety of clinical settings to
demonstrate that red cells preserved in
extended-storage media present no major risk
when used for small-volume transfusions.43
However, for infants with renal or hepatic
insufficiency, it is recommended that AS solu-
tion be removed from RBC units, particularly if
multiple transfusions from the same unit are
expected. The safety of AS-preserved RBCs in
trauma-related massive transfusions, cardiac
surgery, or exchange transfusions is unknown
in this population. Therefore, AS-preserved
RBC units should be used with caution in
these settings.21,42-45
RBC Age
The age of RBC units and its impact on patient
outcomes has become a concern, although

FIGURE 23-3. Syringe with filter (reproduced with permission from Charter Medical, Ltd, Winston-Salem, NC).
578 䡲 AABB TECHNICAL MANUAL
clinical confirmation of the basis for this con-
cern is controversial. A randomized controlled
trial conducted in Canada, Age of Red Blood
Cells in Premature Infants (ARIPI), randomly
assigned low birthweight infants to be trans-
fused with RBCs that were 7 days old or less
(mean = 5.1 days, n = 188) or with standard-
issue RBCs divided into aliquots and stored for
2 to 42 days (mean = 14.6 days, n = 189).46 The
primary composite endpoints included necro-
tizing enterocolitis (NEC), intraventricular
hemorrhage (IVH), and bronchopulmonary
dysplasia. The ARIPI trial found no differences
in the primary endpoints between infants in
the two arms, suggesting that in the study pop-
ulation, the age of RBCs does not affect these
common morbidities of prematurity.
ARIPI’s external validity has been ques-
tioned because of the study's liberal transfu-
sion strategy, use of SAG-M units, and average
duration of blood storage. These practices do
not reflect the transfusion practices or storage
solution and ages of RBCs used in many cen-
ters in the United States.47 Thus, it has not
been firmly established that there is a causal
relationship between morbidities and transfu-
sion of older RBC units in premature infants.47
TABLE 23-2. Blood Components and Dosing of Small Volumes in Neonatal and Pediatric Patients49
Component Dose
Red Blood Cells 10-15 mL/kg
Fresh Frozen Plasma 10-15 mL/kg
Platelets [whole-blood-derived 5-10 mL/kg or
(WBD) or apheresis] 1 WBD unit/10 kg
(patients  10 kg)
Cryoprecipitated AHF 1-2 units/10 kg
*Dependent on anticoagulant-preservative solution: with 3 g/dL increment for CPD and CPDA-1 and 2 g/dL for AS-1, AS-3,
and AS-5.
†
Assumes  5.5 × 1010 platelets in 50 mL of plasma (whole-blood-derived) and  3.0 × 1011 platelets in 250-300 mL plasma
(apheresis).
CPD = citrate-phosphate-dextrose; CPDA-1 = citrate-phosphate-dextrose-adenine-1; AS = additive solution.
Specific Indications for RBCs
In neonates, symptomatic anemia is the major
indication for simple transfusion. Specifically,
a venous hemoglobin of <13 g/dL in the first 24
hours of life necessitates clinical consideration
of an RBC transfusion.48 RBC transfusions are
also considered when approximately 10% of a
sick neonate’s blood volume has been re-
moved or lost. When 10 mL/kg of RBCs with a
hematocrit of >80% are transfused, the expect-
ed increase in hemoglobin concentration in a
neonate is approximately 3 g/dL. A similar vol-
ume of RBCs with AS usually has a hematocrit
of 65%, and its transfusion results in a project-
ed posttransfusion hemoglobin increase of
<3 g/dL (see Table 23-2 for blood component
dosing recommendations and expected re-
sults).49
Two randomized controlled trials, the
Premature Infants in Need of Transfusion
(PINT) and its follow up study [PINT Follow-
up Outcomes Study (PINTOS)] as well as a
University of Iowa study compared the out-
comes of restrictive (Hb = 7 g/dL) vs liberal
RBC transfusion triggers (Hb = 10 g/dL) in
VLBW infants.50-52 The Iowa trial revealed a
lower rate of transfusion events (3.3 vs 5.2;
p=0.025) with the restrictive strategy com-
Expected Increment
Hemoglobin increase 2-3 g/dL*
15%-20% rise in factor levels (assuming 100%
recovery)
50,000/μL rise in platelet count (assuming 100%
recovery)†
60-100 mg/dL rise in fibrinogen (assuming 100%
recovery)
 CHAPTER 23 Neonatal and Pediatric Transfusion Practice
pared to the liberal strategy.52 However, rates of
periventricular leukomalacia and death were
higher in the restrictive arm. The PINT study
found no significant difference between the two
arms, which had the same thresholds as the
Iowa study, in the composite endpoint of death
or any of bronchopulmonary dysplasia, reti-
nopathy of prematurity (Stage >3), or brain in-
jury (periventricular leukomalacia, intracranial
hemorrhage Grade 4, or ventriculomegaly).50,52
The PINTOS study revealed that at 18 to 24
months after birth, infants in the PINT study’s
restrictive arm had more neurodevelopmental
impairments than those in the liberal arm.50,51
In summary, these studies indicated that
maintenance of higher hemoglobin levels in
low birthweight infants may provide long-
term neurologic protection.50-52 Therefore,
whether a restrictive or liberal RBC transfusion
strategy should be adopted in this population
is not clear and requires further prospective
randomized controlled trial data. The Transfu-
sion of Premature Infants Study is currently
being conducted in the United States.53
Exchange Transfusion for
Hyperbilirubinemia
Exchange transfusion in neonates involves re-
placement of one or two whole-blood vol-
umes. The primary purpose of this therapy is
to treat excessively high levels of unconjugated
bilirubin (hyperbilirubinemia). In high con-
centrations, bilirubin may cross the blood-
brain barrier; concentrate in the basal ganglia
and cerebellum of preterm and full-term in-
fants; and cause irreversible damage, known
as “kernicterus,” to the central nervous sys-
tem. Preterm and full-term infants are suscep-
tible to hyperbilirubinemia because their im-
mature liver conjugates bilirubin poorly and
their incompletely developed blood-brain bar-
rier allows bilirubin transit. Phototherapy (use
of fluorescent ultraviolet lights) is the current
treatment of choice for hyperbilirubinemia;
exchange transfusion is reserved for patients
who fail phototherapy.
Two critical objectives of exchange trans-
fusions are the removal of unconjugated bili-
rubin and maximization of albumin binding of
䡲 579
residual bilirubin. In addition, in antibody-
mediated hemolytic processes, exchange
therapy removes both free antibody and anti-
body-coated red cells, replacing them with
antigen-negative red cells.
Exchange transfusion needs to be per-
formed before the development of kernicterus.
In full-term infants, kernicterus rarely develops
at bilirubin levels less than 25 mg/dL. However,
in ill VLBW infants, kernicterus can occur at bil-
irubin levels as low as 8 to 12 mg/dL.54
A double-volume exchange transfusion
(two 85-mL/kg transfusions for full-term in-
fants and two 100-mL/kg transfusions for
VLBW infants) removes approximately 70% to
90% of the circulating red cells and approxi-
mately 50% of the total bilirubin.55 However,
after the first exchange transfusion, bilirubin
levels may rise again because of a re-equilibra-
tion of the extravascular tissue and plasma bil-
irubin, which may necessitate another ex-
change transfusion.56
Occasionally, exchange transfusion is
used to eliminate toxins, drugs, or chemicals
administered to the mother near the time of
delivery. Exchange transfusion is also used
when toxic doses have been administered to
the infant or accumulate at high levels in the
infant as a result of prematurity and/or an in-
born error of metabolism.57,58
Exchange Transfusion
COMPONENT CHOICE AND PHYSIOLOGIC
EFFECTS. Typically, RBCs are resuspended in
ABO-compatible thawed Fresh Frozen Plasma
(FFP) for an exchange transfusion. No single
method of combining components has been
shown to be superior to another. Most often,
RBCs <5 to 7 days old and stored in CPDA-1
are used to avoid high levels of potassium and
to maximize red cell survival.59 When using AS-
RBC units, some blood banks elect to remove
the additive-containing plasma to reduce the
volume transfused.
Most transfusion services provide RBC
units that are hemoglobin S negative, cyto-
megalovirus (CMV) reduced-risk (leukocyte
reduced and/or CMV seronegative), and irra-
diated. Irradiation should be performed just
580 䡲 AABB TECHNICAL MANUAL
before the exchange to prevent potentiation of
the potassium storage lesion. Some experts
recommend washing or removing the super-
natant of red cells that have been irradiated to
avoid the complications of hyperkalemic car-
diac arrhythmias.60
The glucose load administered during ex-
change transfusion can be high in some cases,
which stimulates the infant’s pancreas to re-
lease insulin and results in rebound hypogly-
cemia. Therefore, infant plasma glucose levels
should be monitored during the first few hours
following exchange transfusion.
VOLUME A ND HE MATOCRIT CONSIDER-
ATIONS. A double-volume exchange in neo-
nates rarely necessitates the infusion of more
than 1 RBC unit. The unit’s hematocrit should
be approximately 45% to 60%, and the unit
should have sufficient plasma (based on esti-
mated blood volume) to provide clotting fac-
tors.60 If the neonate’s condition requires a
higher postexchange transfusion hematocrit, a
small-volume RBC transfusion may be given
or a unit with a higher hematocrit can be used
for the initial exchange transfusion. The re-
constituted blood should be well mixed to sus-
tain the intended hematocrit throughout the
exchange. The infant’s hematocrit and biliru-
bin can be measured by removing the last ali-
quot of the exchange unit.
VA SCUL AR ACCESS. Umbilical venous cath-
eters are used for exchange transfusions in
preterm and full-term infants just after birth. If
umbilical venous catheters are not available,
small saphenous catheters may be used.
TECHNIQUES. Two exchange-transfusion
techniques are commonly employed: isovolu-
metric and manual push-pull. In isovolumet-
ric exchange transfusion, two catheters of
identical size provide vascular access. The
catheters allow simultaneous withdrawal and
infusion of blood and are regulated by a single
peristaltic pump. The umbilical vein is typical-
ly used for infusion, and the umbilical artery is
used for withdrawal. The manual push-pull
technique uses a single vascular access portal
with a three-way stopcock attached to the unit
of blood, the patient, and a graduated discard
container. A standard filter and in-line blood
warmer are recommended.
With both techniques, the absolute maxi-
mum volume of each withdrawal and infusion
depend on the infant’s body weight and hemo-
dynamic status. Usually, no more than 5 mL/
kg body weight or 5% of the infant’s blood vol-
ume is removed and replaced during a 3- to 5-
minute cycle.59 The exchange transfusion
should not be performed rapidly because sud-
den hemodynamic changes may affect cere-
bral blood flow and shift intracranial pressure,
contributing to IVH.61 A total double-volume
exchange transfusion typically takes 90 to 120
minutes.59
Platelet Transfusion Support
Mild-to-moderate thrombocytopenia (plate-
let count <150,000/µL) is the most common
hemostatic abnormality in ill preterm and full-
term infants, and it affects approximately 20%
of infants in neonatal intensive care units.62
The causes of thrombocytopenia include im-
paired platelet production, increased platelet
destruction, abnormal platelet distribution,
and/or platelet dilution secondary to massive
transfusion. The most common cause is in-
creased destruction of platelets that is general-
ly associated with a variety of self-limited con-
ditions.
Indications
Most platelet transfusions in preterm and full-
term infants are performed to treat platelet
counts less than 50,000/µL in the presence of
active bleeding.63
Prophylactic platelet transfusions in this
population are controversial (see Table 23-3
for transfusion indications and thresholds).27,64
Unlike adult patients who rarely have severe
bleeding complications until platelet counts
decline to less than 10,000/µL, preterm infants
with other complicating illnesses may bleed at
higher platelet counts. This increased risk may
be attributable to 1) lower concentrations of
plasma coagulation factors, 2) circulation of
an anticoagulant that potentiates thrombin
inhibition, 3) intrinsic or extrinsic platelet
 CHAPTER 23 Neonatal and Pediatric Transfusion Practice 䡲 581

TABLE 23-3. Transfusion Guidelines for Platelets in Neonates and Older Children23,26

With Thrombocytopenia
1. Platelet count 5,000 to 10,000/μL with failure of platelet production.
2. Platelet count <30,000/μL in neonate with failure of platelet production.
3. Platelet count <50,000/μL in stable premature infant:
a. With active bleeding, or
b. Before an invasive procedure, with failure of platelet production.
4. Platelet count <100,000/μL in sick premature infant:
a. With active bleeding, or
b. Before an invasive procedure in patient with DIC.
Without Thrombocytopenia
1. Active bleeding in association with qualitative platelet defect.
2. Unexplained excessive bleeding in a patient undergoing cardiopulmonary bypass.
3. Patient undergoing ECMO with:
a. A platelet count of <100,000/μL, or
b. Higher platelet counts and bleeding.
DIC = disseminated intravascular coagulation; ECMO = extracorporeal membrane oxygenation.

dysfunction/hyperreactivity, or 4) increased demonstrated to raise the platelet count of an

vascular fragility.63
A severe complication of prematurity is
IVH, which occurs in approximately 40% of
preterm neonates in the first 72 hours after
birth. Although prophylactic platelet transfu-
sions may increase platelet counts and short-
en bleeding times, this approach has not been
shown to reduce the incidence of IVH, and the
severity of thrombocytopenia appears to be
independent of the risk if IVH >Grade 2.62,65
Hence, the use of platelets in this situation and
the appropriate platelet dose remain contro-
versial.66 Posttransfusion platelet counts 15 to
60 minutes after transfusion can help when
platelet survival is evaluated; however, these
counts are not good predictors of hemostatic
efficacy.
Components and Dose
The use of whole-blood-derived platelets at
doses of 5 to 10 mL/kg body weight have been
average full-term newborn by 50,000 to
100,000/µL, depending on the concentration
of the platelet component used.14,62 A similar
dosing regimen is typically used with aphere-
sis platelets. For larger children (>10 kg), trans-
fusion of 1 platelet unit per 10 kg should in-
crease the platelet count by approximately
50,000 µL.14,67
When possible, the platelet component
should be ABO group specific/compatible and
should not contain clinically significant and
unexpected red cell antibodies. Transfusion of
ABO-incompatible plasma should be avoided
in children, and especially in infants, because
of their small blood and plasma volumes.31 If it
becomes necessary to administer ABO-incom-
patible platelets in an infant, plasma may be
removed either by volume reduction or wash-
ing (see Method 6-14). The platelets may then
be resuspended in saline or compatible plasma.
However, routine centrifugation to remove
plasma from platelets should be avoided
582 䡲 AABB TECHNICAL MANUAL

because it is unnecessary and harmful to the
platelets.62,63
In addition, when platelets are stored in a
syringe, the pH has been shown to decrease
rapidly, a potential problem for an already ill
and acidotic recipient.68-70 Therefore, when
volume reduction in an open system is used
and the product is placed in a syringe, the pro-
cessing should be done as close to the time of
issuance as possible and the product should
be infused within 4 hours of processing.
Plasma Transfusion Support to
Enhance Hemostasis
Infants must synthesize their own coagulation
factors because significant amounts are not
transplacentally transferred from the mother.
Furthermore, infants are unable to produce
normal levels of these proteins in the early
postnatal period.
Physiologically, low levels of vitamin K-
dependent factors (Factors II, VII, IX, and X)
and contact factors (Factor XI, Factor XII,
prekallikrein, and high-molecular-weight ki-
ninogen) contribute to altered coagulation test
results (see Table 23-4).71,72 Also, the naturally
occurring vitamin K-dependent anticoagu-
lants (proteins C and S) and the non-vitamin-
K-dependent antithrombin protein are at low
levels at birth. In spite of these issues, the pro-
coagulant and anticoagulant systems are usu-
ally in balance in healthy newborns, so spon-
taneous bleeding and thrombosis are rare (see
Table 23-4).72 However, the reserve capacity for
both systems is limited. Therefore, serious
bleeding may occur in sick premature infants
during the first week of life. Cryoprecipitate
and FFP can be transfused to treat bleeding or
clotting complications, such as disseminated
intravascular coagulation (DIC).73,74
FFP
FFP is frequently used to replace coagulation
factors in preterm and full-term infants, par-
ticularly if multiple factor deficiencies are
present, such as hemorrhagic disease of the
newborn or vitamin K deficiency (Table 23-5).
The usual dose of FFP is 10 to 15 mL/kg, which
is expected to increase all factor activity levels
by 15% to 20% unless there is a marked con-
sumptive coagulopathy.64,68
To limit donor exposure for each recipient
while minimizing plasma wastage, blood can
be collected into a system with multiple, inte-

TABLE 23-4. Screening Laboratory Tests for Hemostasis: Neonates vs Adults (reproduced with
permission)71

Preterm Neonates vs Neonates vs Older Approximate Age Adult

Full-Term Neonates Children/Adults Values are Reached

aPTT Longer Longer 16 years

Prothrombin time Longer Same or longer 16 years
INR Higher Same or higher 16 years
Thrombin time Longer Same or longer 5 years
Bleeding time Longer Shorter 1 months
PFA-100 Longer Shorter 1 months
ROTEM/TEG
Clotting time Same Shorter 3 months
Clot formation time Same Shorter 3 months
Maximal clot firmness Stronger Stronger 3 months
aPTT = activated partial thromboplastin time, INR = international normalized ratio.
 CHAPTER 23 Neonatal and Pediatric Transfusion Practice 䡲 583

TABLE 23-5. Transfusion Guidelines for Plasma Products in Neonates and Older Children23,26

Fresh Frozen Plasma (FFP)

1. Support during treatment of disseminated intravascular dissemination.
2. Replacement therapy:
a. When specific factor concentrates are not available, including, but not limited to, antithrombin; pro-
tein C or S deficiency; and Factor II, Factor V, Factor X, and Factor XI deficiencies.
b. During therapeutic plasma exchange when FFP is indicated (cryopoor plasma, plasma from which the
cryoprecipitate has been removed).
3. Reversal of warfarin in an emergency situation, such as before an invasive procedure with active bleeding.
Note: FFP is not indicated for volume expansion or enhancement of wound healing.
Cryoprecipitated AHF
1. Hypofibrinogenemia or dysfibrinogenemia with active bleeding.
2. Hypofibrinogenemia or dysfibrinogenemia while undergoing an invasive procedure.
3. Factor XIII deficiency with active bleeding or while undergoing an invasive procedure in the absence of
Factor XIII concentrate.
4. Limited directed-donor cryoprecipitate for bleeding episodes in small children with hemophilia A (when
recombinant and plasma-derived Factor VIII products are not available).
5. In the preparation of fibrin sealant.
6. von Willebrand disease with active bleeding, but only when both of the following are true:
a. Deamino-D-arginine vasopressin (DDAVP) is contraindicated, not available, or does not elicit
response.
b. Virus-inactivated plasma-derived Factor VIII concentrate (which contains von Willebrand factor) is
not available.

grally attached bags that create ready-to-
freeze aliquots.26 Once thawed, the aliquots
can be subdivided further for several patients
if they can be used within a 24-hour period.
FFP for infants must be ABO compatible
and free of clinically significant and unexpect-
ed antibodies. Transfused antibodies can
reach high concentrations in infants and chil-
dren with very small plasma volumes. A com-
mon practice at some institutions is to use
group AB FFP because a single unit can pro-
vide multiple small-volume doses for several
neonates.
Cryoprecipitated Antihemophilic Factor
Cryoprecipitate transfusions are primarily
used to treat conditions resulting from de-
creased or dysfunctional fibrinogen (congeni-
tal or acquired) or Factor XIII deficiency. Cryo-
precipitate is usually given in conjunction with
platelets and FFP to treat DIC in newborns.
Typically, 1 unit is sufficient to achieve hemo-
static levels in an infant.
ABO-compatible cryoprecipitate is pre-
ferred because transfusion of a large volume of
ABO-incompatible cryoprecipitate may result
in a positive DAT result and, in very rare cases,
a mild hemolysis.75,76
Cryoprecipitate transfusion is not recom-
mended for patients with Factor VIII deficien-
cy because the standard therapy is to treat this
condition with recombinant Factor VIII prod-
ucts or virus-inactivated, monoclonal-anti-
body-purified, plasma-derived products.73,77
584 䡲 AABB TECHNICAL MANUAL
Furthermore, cryoprecipitate should be used
only to treat von Willebrand disease if plasma-
derived, virus-inactivated concentrates con-
taining von Willebrand factor are not available.
See Table 23-5 for other guidelines regarding
cryoprecipitate use.64
Granulocyte Transfusion Support
Indications
The role of granulocyte transfusion for sepsis
in neonates is unclear, and this treatment is
rarely used. It is important to establish the fol-
lowing factors before the transfusion of granu-
locytes: 1) strong evidence of bacterial or fun-
gal septicemia; 2) absolute neutrophil count
less than 500/µL, chronic granulomatous dis-
ease, or leukocyte adhesion deficiency; and 3)
diminishing storage pool (such that 7% of nu-
cleated cells in the marrow are granulocytes
that are metamyelocytes or more ma-
ture).15,78,79 (See Table 23-6 for guidelines on
granulocyte transfusion support.)
Components and Dose
Granulocyte concentrates are produced by
standard apheresis techniques or by pooling
buffy coats from whole blood. A typical dose
for infants is 10 to 15 mL/kg body weight,
which is approximately 1 × 109 to 2 × 109 poly-
morphonuclear cells/kg.15,78 Treatment should
be administered daily until an adequate neu-
trophil count is achieved and/or the patient
shows clinical improvement.
According to AABB Standards, these com-
ponents must be irradiated when the patient is
TABLE 23-6. Transfusion Guidelines for
Granulocytes in Neonates and Older
Children23,26
1. Neonates or children with neutropenia or
granulocyte dysfunction with bacterial sepsis
and lack of responsiveness to standard ther-
apy.
2. Neutropenic neonates or children with fungal
disease not responsive to standard therapy.
at risk of TA-GVHD, and the components must
be obtained from CMV-seronegative donors to
prevent virus transmission if the recipient is
CMV seronegative.28(p40) Granulocytes must
also be ABO compatible with the red cells of
the recipient infant because of the significant
red cell content in these components.28(p37)
Many institutions also provide D-compatible
components to decrease Rh alloimmuniza-
tion.
Intravenous immune globulin (IVIG) in
the treatment of early neonatal sepsis has also
been studied, but there is a lack of agreement
on its routine application to this patient popu-
lation.78,80-85
Transfusion Administration
Vascular Access
Vascular access is the most difficult aspect of
transfusion administration in patients young-
er than 4 months, particularly in preterm in-
fants who require long-term or continuous in-
travenous infusions. The umbilical vein is
most frequently cannulated after birth to ad-
minister fluids and transfusions and to moni-
tor central venous pressure.86 Vascular cathe-
ters (24-gauge) and small needles (25-gauge)
generally can be safely used for RBC transfu-
sions without causing hemolysis if constant
flow rates are applied. The outcomes of trans-
fusions using smaller-gauge needles and cath-
eters have not been evaluated.
Pumps and Warming
When administered slowly, small-volume
transfusions typically do not require a blood
warmer; however, control of the rate and vol-
ume transfused is important. Electromechani-
cal syringe delivery pumps administer the
blood at a constant rate and are able to provide
adequate control. These pumps cause mini-
mal hemolysis and may even be used with in-
line leukocyte-reduction filters.87,88 Although
there are several devices that can be used to
transfuse blood components, it is important to
test and validate the mechanical system cho-
sen for blood component administration.
 CHAPTER 23 Neonatal and Pediatric Transfusion Practice
Filters and Transfusion Sets
All blood component transfusions require a
standard filter (150 to 260 microns), even if the
components have undergone leukocyte reduc-
tion before storage or at the bedside.75 Micro-
aggregate filters (20 to 40 microns) are occa-
sionally used for simple transfusions because
of their small priming volume. However, he-
molysis may occur when stored RBCs are ad-
ministered through these filters using negative
pressure.89
The plastic tubing in the administration
sets can add significant amounts of dead-
space volume to the transfusion and may need
to be accounted for when preparing a transfu-
sion dose. Pediatric infusion sets created for
platelets and other small-volume components
have less dead space than standard sets.
Administration Rates
A lack of clinical studies and evidence-based
practices related to blood transfusions in neo-
nates has led to variability both in rates of
transfusion and in devices used among insti-
tutions. The rate of RBC and other blood com-
ponent administration is dictated by the clini-
cal needs of the pediatric patient. Despite
concerns from neonatologists that rapid blood
infusion rates may adversely affect intravascu-
lar volume and electrolyte levels, an increased
risk of IVH in these small and fragile patients
has not been clearly demonstrated. Therefore,
administering a simple transfusion over 2 to 4
hours is adequate in nonemergent situations.
However, in states of shock or severe bleeding,
a rapid infusion is often required.
Unique Therapies and Situations in
Neonates
Polycythemia
Neonatal polycythemia is defined as a venous
hematocrit >65% or a hemoblogin >22 g/dL at
any time during the first week after birth. Ap-
proximately 5% of all newborns develop poly-
cythemia, and this risk may be higher in small-
for-gestational-age neonates and infants of
diabetic mothers. Once the hematocrit rises
䡲 585
above 65%, viscosity increases and oxygen
transport decreases. However, in neonates, the
exponential rise in viscosity can occur at a he-
matocrit as low as 40%.90 Congestive heart fail-
ure can result because infants have limited ca-
pability to increase their cardiac output to
compensate for hyperviscosity. Central ner-
vous system abnormalities, pulmonary and re-
nal failure, and NEC can occur from the resul-
tant decreased blood flow.
A partial exchange is used to normalize
the hematocrit to between 55% and 60% and
improve tissue perfusion while maintaining
blood volume. This exchange is accomplished
by removing whole blood and replacing it with
normal saline or other crystalloid solutions.
Plasma is not used to replace whole blood be-
cause NEC has been reported as a complica-
tion of plasma transfusion.91
The formula below can be used to ap-
proximate the volume of replacement fluid re-
quired and the volume of whole blood that
must be withdrawn for the partial exchange:
(Blood (Observed Hct –
Volume of volume) × Desired Hct)
replacement =
Observed Hct
fluid
ECMO
ECMO is a prolonged treatment where blood is
removed from the patient’s venous circulation,
circulated through a machine to remove CO2
and replenish O2, and then returned to the pa-
tient. ECMO has been successfully used since
the early 1980s to provide gas exchange inde-
pendently of a patient’s lungs. ECMO allows
patients to recover without exposure to aggres-
sive ventilator support that can cause baro-
trauma and permanent lung damage and to
maintain the circulation of oxygenated blood
during cardiac surgery or in patients with dis-
ease when other forms of treatment fail.92,93 In
neonates and children, ECMO has become a
lifesaving advance treatment of meconium as-
piration syndrome, persistent pulmonary hy-
pertension of the newborn, congenital dia-
phragmatic hernia, and respiratory failure due
586 䡲 AABB TECHNICAL MANUAL

to sepsis. It is also used for postoperative sup- TR A N SF U SI O N I N IN F A NT S

port following cardiac surgery.
Because standardized guidelines for
transfusion practice in ECMO have not been
established, centers typically establish their
own criteria. Table 23-7 provides some guide-
lines for ECMO.94 Bleeding complications are
frequent during ECMO treatment and may be
caused by 1) systemic heparinization, 2) plate-
let dysfunction, 3) thrombocytopenia, 4) other
coagulation defects, or 5) the nonendothelial
ECMO circuit. Hospital blood banks and trans-
fusion services must be in close communica-
tion with the ECMO staff and observe local
protocols to ensure safe, efficient, and consis-
tent care.
ECMO typically requires 1 to 2 units of
ABO- and Rh group-specific and crossmatch-
compatible RBCs for blood priming. In addi-
tion, 1 unit of group-specific FFP should be al-
located to the ECMO patient. RBC units are
usually negative for hemoglobin S, relatively
fresh (<5 to 7 days old), irradiated, and CMV
seronegative and/or leukocyte reduced.43 Be-
cause the ECMO circuit consumes platelets,
higher platelet counts are often maintained.
OLDER THA N 4 MO NTHS AND
C H I L D RE N
RBC Transfusion Support
RBC transfusions in infants older than 4
months and children are similar to transfu-
sions in adults. The most significant differenc-
es between this young group and adults are 1)
blood volume, 2) the ability to tolerate blood
loss, and 3) age-appropriate hemoglobin and
hematocrit levels. In these infants and chil-
dren, the most common indication for RBC
transfusion is to treat or prevent tissue hypoxia
caused by decreased red cell mass, typically
because of surgery, anemia of chronic disease,
or hematologic malignancies. Chronic RBC
transfusions are the treatment of choice for
children with sickle cell disease (SCD) or thal-
assemia. These transfusions are administered
to combat tissue hypoxia and suppress endog-
enous hemoglobin production. Table 23-8 can
help guide transfusion decisions in patients
older than 4 months.

TABLE 23-7. Blood Component Protocols for ECMO94

Clinical Scenario Urgency Components Blood Groups Storage

Cardiac arrest 5-10 min 2 units RBCs O-neg RBCs <14 days, AS

ECMO circuit disruption 5-10 min 2 units RBCs O-neg RBCs <14 days, AS

Progressive septic 30 min 2 units RBCs O-neg RBCs or <10 days,

shock (nonneonate) type specific any preservative

Neonate transferred for 1-2 hours 2 units RBCs O-neg RBCs <10 days,
ECMO 1 unit FFP AB plasma CPD or CPDA
1 unit platelets

Cardiac ICU 30-60 min 2 units RBCs Type specific <7 days, AS

Gradual respiratory or Hours to days 2 units RBCs Type specific <10 days, CPD
cardiac failure on con-
ventional support

ECMO = extracorporeal membrane oxygenation; RBCs = Red Blood Cells; AS = additive solution; FFP = Fresh
Frozen Plasma; CPD = citrate-phosphate-dextrose; CPDA = citrate-phosphate-dextrose-adenine; ICU = inten-
sive care unit.
 CHAPTER 23 Neonatal and Pediatric Transfusion Practice 䡲 587

TABLE 23-8. Transfusion Guidelines for RBCs in Patients Older than 4 Months23,26

1. Emergency surgical procedure in patient with significant postoperative anemia.

2. Preoperative anemia when other corrective therapy is not available.
3. Intraoperative blood loss >15% total blood volume.
4. Hematocrit <24% and:
a. In perioperative period, with signs and symptoms of anemia.
b. While on chemotherapy/radiotherapy.
c. Chronic congenital or acquired symptomatic anemia.
5. Acute blood loss with hypovolemia not responsive to other therapy.
6. Hematocrit <40% and:
a. With severe pulmonary disease.
b. On extracorporeal membrane oxygenation.
7. Sickle cell disease and:
a. Cerebrovascular accident.
b. Acute chest syndrome.
c. Splenic sequestration.
d. Aplastic crisis.
e. Recurrent priapism.
f. Preoperatively when general anesthesia is planned (target hemoglobin 10 mg/dL).
8. Chronic transfusion programs for disorders of red cell production (eg, -thalassemia major and Diamond-
Blackfan syndrome unresponsive to therapy).

Before receiving any RBC transfusion, all Simple or partial-exchange transfusions

pediatric patients older than 4 months require
ABO and Rh testing and screening for the pres-
ence of clinically significant antibodies. Com-
patibility testing should be performed accord-
ing to AABB Standards.28(p39)
SCD
Chronic transfusion therapy has been shown
to reduce the risk of stroke in patients with
SCD by decreasing the proportion of RBCs
containing hemoglobin S, reducing sickling,
and preventing blood viscosity increases.95-98
Chronic transfusions can reduce the risk of re-
current stroke to <10% if hemoglobin levels are
maintained at between 8 and 9 g/dL with a he-
moglobin S level <30%.
can be administered every 3 to 4 weeks. Eryth-
rocytapheresis has also been used to prevent
iron overload in patients with SCD. See Table
23-8 for a list of other complications of SCD
necessitating either simple or chronic RBC
transfusions.
Chronic transfusion therapy must be pro-
vided indefinitely because its cessation can
lead to a stroke.95,96 The Transcranial Dopplers
with Transfusions Changing to Hydroxyurea
trial is currently evaluating the role of hydroxy-
urea in stroke prevention compared to chronic
transfusions in patients with SCD.99 Of note,
RBCs for patients with SCD should ideally be
screened for hemoglobin S and leukocyte re-
duced to prevent HLA alloimmunization and
588 䡲 AABB TECHNICAL MANUAL
decrease platelet refractoriness in preparation
for possible stem cell transplantation.
RBC ALLOIMMUNIZ ATI ON IN SCD. Patients
with SCD have the highest rates of alloimmu-
nization of any patient group.100-102 These anti-
bodies are produced against common Rh, Kell,
Duffy, and Kidd system antigens. Many SCD
treatment centers perform thorough pheno-
type analysis of a patient’s red cells before be-
ginning transfusion therapy. This testing helps
reduce the rate of alloimmunization by allow-
ing preferential selection of phenotypically
similar units.96,103,104 However, particularly for
patients who are not yet alloimmunized, this
process remains controversial because pheno-
typically compatible units may be difficult to
obtain.95,104
In academic institutions in the United
States and Canada, the most common alloim-
munization protocol for nonalloimmunized
patients with SCD is pretransfusion phenotyp-
ic matching for C, E, and K antigens.105 Once
patients have developed a red cell antibody,
extension of matching to additional red cell
antigens (Fy, Jk, S) is often used to prevent
further alloimmunization.106 In a retrospective
study, children with SCD undergoing
matched-sibling-donor marrow transplanta-
tion demonstrated a decrease in RBC transfu-
sion requirements during transplantation
when they received phenotypically minor RBC
antigen-matched units.107 A recent retrospec-
tive review of patients with SCD at Children’s
Hospital of Philadelphia showed that the chil-
dren developed alloimmunization to Rh anti-
gens despite being transfused with units from
Rh-matched minority donors, and 87% of allo-
immunized patients were identified with RH
genotyping as having RH variant alleles.108 This
study’s results suggest that the role of genotyp-
ing of patients with SCD and minority donors
in alloimmunization rates needs to be stud-
ied.108 Method 2-23 can be used to perform
phenotyping on autologous red cells of recent-
ly transfused patients with SCD.
Another strategy aimed at preventing al-
loimmunization in patients with SCD is to de-
velop recruitment programs specifically for
donors of African ethnicity to decrease the
number of antigenically different RBC units
from donors of European ethnicity that are
transfused. Leukocyte reduction to prevent al-
loimmunization to red cell antigens remains
controversial.109, 110 However, HLA alloimmuni-
zation has recently been demonstrated to oc-
cur more frequently in red cell alloimmunized
patients with SCD.111
OTHER COMPLICATIONS OF RB C TRANS-
FUSIONS IN SCD. The benefits of transfu-
sion therapy in patients with SCD should be
weighed against the complications of transfu-
sion, such as iron overload and minor red cell
antigen alloimmunization, as well as the risks
of increased donor exposure during erythrocy-
tapheresis. Some practitioners have proposed
that a clinically successful course of transfu-
sions that maintains the hemoglobin S level at
<30% could, after several years, be transi-
tioned to a strategy of more limited transfu-
sions with a hemoglobin S target of 40% to 50%
to reduce the risk of iron overload.97 Patients
with SCD may also be at risk of life-threaten-
ing, delayed hemolytic transfusion reactions.
If a patient’s hemoglobin level decreases
after transfusion, the patient might have de-
veloped “hyperhemolytic” syndrome. This
poorly understood phenomenon is character-
ized by destruction of the patient’s own red
cells along with transfused cells. If hyperhe-
molytic syndrome is suspected, case reports
have shown that stopping transfusion and ad-
ministering corticosteroids in combination
with IVIG is beneficial.112,113 These patients
should also be monitored closely for the for-
mation of autoantibodies.114
Thalassemia
Thalassemia with severe anemia must be
treated with transfusion to improve tissue oxy-
genation and suppress extramedullary eryth-
ropoiesis in the liver, spleen, and marrow. This
approach also decreases many thalassemia
complications. Maintaining target hemoglo-
bin levels of 8 to 9 g/dL allows normal growth
and development in these patients. Super-
transfusion protocols aim for higher target he-
moglobin levels (11-12 g/dL). Iron overload is
a potential nonpreventable complication of
 CHAPTER 23 Neonatal and Pediatric Transfusion Practice
this RBC transfusion protocol that must be
treated with chelation therapy beginning early
in childhood.115,116
Platelet and Plasma Support
The indications for platelet and plasma trans-
fusion support are similar for older infants,
children, and adults. Tables 23-3 and 23-5 pro-
vide the indications for the transfusion of
platelets, FFP, and cryoprecipitate. A recent
study showed that plasma transfusions were
independently associated with an increased
risk of new or progressive multiple organ dys-
function, nosocomial infections, and pro-
longed length of stay in 831 children admitted
to one institution’s pediatric critical care
unit.117 Plasma transfusions should be consid-
ered with caution in this population.
In older infants and children, platelets are
most often prophylactically administered dur-
ing chemotherapy. The transfusion threshold
for these patients is usually between 10,000
and 20,000/µL, although the platelet count
should not be the sole determinant for trans-
fusion. In the Prophylactic PLAtelet DOse
(PLADO) study, Slichter et al randomly as-
signed 1351 patients with hypoproliferative
thrombocytopenia to receive one of three pro-
phylactic platelet transfusion doses (low, me-
dium, or high) with the primary endpoint of
World Health Organization Grade 2 bleed-
ing.118 The dose of prophylactic platelets ad-
ministered had no effect on the incidence of
Grade 2 bleeding.118 A subgroup analysis of
the 198 children aged 0-18 years in the PLADO
study revealed that children were at a higher
risk of bleeding over a wider range of platelet
counts than adults.119 Therefore, prophylactic
platelet transfusions with a threshold of
10,000/µL in concert with different doses of
platelets do not seem to affect the bleeding
rates of children or adults.118,119 However, when
children are transfused, the use of ABO-com-
patible platelets is associated with better clini-
cal outcomes than use of non-ABO matched
platelets.31,120-122
䡲 589
Granulocyte Support
For children older than 4 months, the indica-
tions for granulocyte transfusions—persistent
neutropenia or granulocyte dysfunction in
conjunction with a bacterial and/or fungal in-
fection(s)—are similar to those previously
mentioned for younger (<4 months) infants.
The minimum granulocyte dose for older chil-
dren and adults is 1 × 1010 cells/kg.23,78
Granulocyte components should be irra-
diated, ABO compatible, CMV seronegative (if
the recipient is CMV seronegative), cross-
match compatible with the recipient, and, ide-
ally, administered within 24 hours of collec-
tion. To obtain higher doses of granulocytes
for larger patients, donors can be mobilized
with steroids, growth factors [eg, granulocyte
colony-stimulating factor (G-CSF)], or a com-
bination. This approach can increase the
amount of granulocytes in the collection by
three or four times compared to products from
donors who receive steroid stimulation alone.
The National Heart, Lung, and Blood In-
stitute’s High Dose Granulocyte Transfusions
for the Treatment of Infection in Neutropenia:
Resolving Infection in Neutropenia with Gran-
ulocytes study is examining the effect of gran-
ulocyte transfusions plus standard microbial
therapy vs standard microbial therapy alone
for patients with neutropenia and severe in-
fections as well as the combined effect of G-
CSF and steroids on granulocyte donation
yield.
PREVE NTION OF AD VE RS E
EF F E C TS O F TR A NS F U S I O N IN
NE ONATES, OL DER INFA NTS,
AND CHILDRE N
CMV Prevention
CMV may be transmitted to neonates trans-
placentally, during the birth process or breast-
milk feeding, as a result of personal contact
with the mother or nursery staff, or from trans-
fusion. With current technologies, the risk of
acquiring CMV from transfusion is between
1% and 3%.123
590 䡲 AABB TECHNICAL MANUAL
The manifestation of CMV infection in
neonates is variable, ranging from asymptom-
atic seroconversion to death. Studies have re-
vealed that the rate of symptomatic transfu-
sion-transmitted CMV infection in infants is
low compared to the high rate of seropositivity
in adults. Moreover, symptomatic CMV infec-
tion is uncommon in neonates born to sero-
positive mothers.124
The risk of transfusion-transmitted CMV
infection is higher in multitransfused low
birthweight infants (<1200 g) born to seroneg-
ative mothers.13,123,125 For this reason, these in-
fants should receive only CMV-reduced-risk
blood. One approach is to use blood from
CMV-seronegative donors.
Leukocyte Reduction
The benefits of transfusions of leukocyte-
reduced components for neonates include re-
ducing the risk of transfusion-transmitted
CMV.13,126,127 A recent study in Canada that
evaluated clinical outcomes in premature in-
fants (weighing <1250 g) before and after na-
tionwide implementation of universal leuko-
cyte reduction revealed no change in mortality
or bacteremia rates. However, rates of retinop-
athy of prematurity and bronchopulmonary
dysplasia decreased as did length of hospital-
ization.128 Other known benefits of transfusing
leukocyte-reduced components include pre-
vention of febrile nonhemolytic transfusion
reactions and HLA alloimmunization.
TABLE 23-9. Irradiation Guidelines for Neonates and Older Children Who Require Cellular Blood
Components23,26
1. Premature infants weighing <1200 g at birth.
2. Any patient with:
a. Known or suspected cellular immune deficiency.
b. Significant immunosuppression related to chemotherapy or radiation treatment.
3. Any patient receiving:
a. Components from blood relatives.
b. HLA-matched or crossmatched platelet components.
Irradiation
Cellular blood components are irradiated to
prevent TA-GVHD in immunocompromised
recipients (see Table 23-9). Expert opinions
and practices differ on this topic. Therefore,
protocols should be based on the patient pop-
ulations served, equipment available, and best
practices. The processes of irradiation, irradia-
tor quality control, and quality assurance are
beyond the scope of this chapter but are ad-
dressed in Chapters 1 and 9.
Volume Reduction and Washing
The plasma volume of the component is usu-
ally reduced in transfusions in premature in-
fants who have renal ischemia or compro-
mised cardiac function. In 1993, the AABB
Committee on Pediatric Hemotherapy stated
that volume reduction of platelet concentrates
should be reserved for infants who have total
body fluid restrictions.63 Methods for platelet
volume reduction have been published (see
Method 6-13).129 However, optimal centrifuga-
tion rates and preparation methods remain
unclear. As with any platelet modification, the
total number of platelets typically decreases
and platelet activation may occur with these
procedures.130
Saline-washed RBCs and platelets are ad-
ministered in an effort to reduce the risk of ad-
verse reactions to certain components from
plasma, anticoagulant preservative solutions,
and high levels of potassium. Transfusion of
 CHAPTER 23 Neonatal and Pediatric Transfusion Practice 䡲 591

unwashed RBCs or platelet products procured
from the mother of an infant is strongly dis-
couraged.26 Maternal cells must be washed for
effective treatment of hemolytic disease of the
newborn and neonatal alloimmune thrombo-
cytopenia when maternal RBCs and platelets
are transfused.
Massive Transfusion in the Pediatric
Setting
Trauma is the leading cause of death in in-
fants, children, and young adults aged 1 to 21
years. Although trauma rarely leads to hemor-
rhagic shock and massive transfusion, resusci-
tation after trauma can be challenging.
The evidence to support pediatric MTP is
limited, but several pediatric institutions use
MTPs to improve the outcomes of patients
who have experienced trauma. The appropri-
ate ratios of RBCs to plasma and platelets have
not been well defined (ie, 1:1:1 or 2:1:1). How-
ever, studies indicate that an MTP in the pedi-
atric setting is feasible not only for providing
rapid and balanced blood product support but
also for decreasing the risk of thromboembolic
events.131-133 Furthermore, when Hendrickson
et al examined the impact of coagulopathy in
102 pediatric trauma patients, they found that
abnormal prothrombin time, activated partial
thromboplastin time, and platelet count at
emergency room admission were strongly as-
sociated with mortality (p = 0.005, 0.001, and
<0.0001, respectively).134 These investigators
did not examine the effect of MTP resuscita-
tion on coagulopathy and mortality, which
may provide insights into further optimiza-
tion of MTPs. Although adult studies have
demonstrated benefit from MTP, the role of
the MTP and the optimal ratio of blood com-
ponents still needs to be defined in the pediat-
ric setting.131-133

KEY PO I NT S

1. RBCs are the most frequently transfused blood product in children. Frequent blood loss, in-
cluding iatrogenic losses from repeated phlebotomy, contribute to the need for RBC trans-
fusions in the neonatal population.
2. Symptomatic anemia and/or a target hemoglobin level is a preferred strategy for neonatal
RBC transfusion rather than a milliliter-for-milliliter replacement of due to blood losses.
3. A full-term newborn has a blood volume of approximately 85 mL/kg whereas a preterm in-
fant has a total blood volume of 100 mL/kg.
4. In infants <4 months of age initial patient testing must include ABO and D typing of their
red cells and a screen for unexpected red cell antibodies, using either plasma or serum from
the infant or mother. During any one hospitalization, crossmatch compatibility testing and
repeat ABO and D typing may be omitted as long as all of the following criteria are met: the
antibody screen is negative; transfused red cells are group O, ABO-identical, or ABO-com-
patible; and transfused cells are either D negative or the same D type as the patient. Testing
the infant’s reverse type for anti-A and/or anti-B is not necessary.
5. Small-volume simple RBC (no matter what the storage solution) transfusions when admin-
istered slowly have been shown to have little effect on serum potassium concentrations in
infants less than 4 months of age despite elevated potassium levels in the plasma of stored
RBCs.
6. During component preparation if aliquots are made with a sterile docking device, it is con-
sidered a “closed system” and the original units expiration date can be used for the new ali-
quot product.
7. Dosing of RBC units with additive solutions such as AS-1, AS-3, and AS-5 should not exceed
10-15 mL/kg for neonates.
592 䡲 AABB TECHNICAL MANUAL

8. Transfusion of ABO-incompatible plasma should be avoided in pediatric patients and espe-

cially in infants because of their small blood and plasma volumes. If ABO out-of-group
platelet transfusion becomes necessary, plasma may be removed either by volume reduc-
tion or washing.
9. Chronic RBC transfusion therapy reduces the risk of stroke in patients with sickle cell dis-
ease by decreasing the percentage of red cells containing hemoglobin S in order to reduce
sickling and prevent an increase in blood viscosity. Hemoglobin levels should be main-
tained around 8 to 9 g/dL with a hemoglobin S level of <30%.
10. Patients with sickle cell disease have the highest rates of alloimmunization to red cell minor
antigens than any other patient group. The most commonly formed antibodies are to Rh,
Kell, Duffy, and Kidd system antigens. Many sickle cell treatment centers try to prevent red
cell alloimmunization by matching for phenotypically similar antigen profiles on recipients.
This strategy is not the same at all centers and is controversial because obtaining enough
phenotypically similar units may be difficult.
11. Currently it is recommended that low-birthweight infants born to CMV-seronegative moth-
ers receive CMV-reduced-risk blood for transfusion. These units could be from CMV-sero-
negative donors or CMV-positive donors whose donation has been leukocyte reduced.

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32. Winess, JA. Treatment and prevention of neo-
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69. Pisciotto P, Snyder EL, Snyder JA, et al. In vitro
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74. Goldenberg NA, Manco-Johnson MJ. Pediatric
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83. Jenson HB, Pollack BH. The role of intravenous
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87. Burch KJ, Phelps SJ, Constance TD. Effect of an
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88. Criss VR, DePalma L, Luban NLC. Analysis of a
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90. Lindemann R, Haga P. Evaluation and treat-
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91. Black VD, Rumack CM, Lubchenco LD, Koops
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93. Bartlett RH, Andrews AF, Toomasian JM, et al.
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94. Friedman DF, Montenegro LM. Extracorporeal
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95. Sharon BI. Management of congenital hemo-
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96. Cohen AR, Norris CF, Smith-Whitley K. Trans-
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97. Adams DM, Schultz WH, Ware RF, Kinney TR.
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98. Adams RJ, McKie VC, Hsu L, et al. Prevention
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transcranial Doppler ultrasonography. N Engl
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99. Aygun, B, Wruck, LM, Schultz, WH, et al.
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100. Rosse WF, Gallagher D, Kinney TR, et al. Trans-
fusion and alloimmunization in sickle cell dis-
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101. Spanos T, Karageorge M, Ladis V, et al. Red cell
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102. Rosse WF, Telen M, Ware RE. Transfusion
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103. Smith-Whitley K. Alloimmunization in pa-
tients with sickle cell disease. In: Herman JK,
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104. Hillyer KL, Hare VW, Josephson CD, et al. Part-
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tol 2006;22:108-11.
105. Afenyi-Annan A, Brecher ME. Pre-transfusion
phenotype matching for sickle cell disease pa-
tients (letter). Transfusion 2004;44:619-20.
106. Tahan HR, Holbrook CT, Braddy LR, et al. Anti-
gen-matched donor blood in the transfusion
management of patients with sickle cell dis-
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109. Blumberg N, Heal JM, Gettings KF. Leukore-
duction of red cell transfusions is associated
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111. McPherson M, Anderson A, Jessup P, et al. HLA
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112. Petz LD, Calhoun L, Shulman IA, et al. The
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drome. Transfusion 1997;37:382-8.
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lytic transfusion reaction in sickle cell disease.
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114. Garratty G. Autoantibodies induced by blood
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9.
115. Hoffbrand AV, Al-Refaie F, Davis B, et al. Long-
term trial of deferiprone in 51 transfusion-
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116. Oliveri NF, Brittenham GM. Iron-chelating
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117. Karan O, Lacroix J, Robitaille N, et al. Associa-
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118. Slichter SJ, Kaufman RM, Assmann SF, et al.
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119. Josephson CD, Granger S, Assmann SF, et al.
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121. Heal JM, Blumberg N. The second century of
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122. Blumberg N, Heal JM, Hicks GL, Risher WH.
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123. Bowden RA, Slichter SJ, Sayers M, et al. A com-
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124. Mussi-Pinhata MM, Yamamoto AY, Rego MAC,
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tion. J Pediatr 2004;145:685-8.
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125. Bradley MT, Milam JD, Anderson DC. Use of
deglycerolized red blood cells to prevent post-
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126. Gilbert GL, Hayes K, Hudson H, et al. Preven-
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remove leukocytes. Lancet 1989;i:1228-31.
127. Strauss RG. Selection of white cell-reduced
blood components for transfusions during
early infancy. Transfusion 1993;33:352-7.
128. Fergusson D, Hebert PC, Lee SK, et al. Clinical
outcomes following institution of universal
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mature infants. JAMA 2003;289:1950-6.
129. Moroff G, Friedman A, Robkin-Kline L, et al.
Reduction of the volume of stored platelet
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The functional integrity of platelets in volume-
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131. Dehmer JJ, Adamson MD. Massive transfusion
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132. Hendrickson JE, Shaz BH, Pereira G, et al. Im-
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133. Chidester SJ, Williams N, Wang W, Groner JI. A
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ma Acute Care Surg;73:1273-7.
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 C h a p t e r 2 4

Patient Blood Management

Mary Ghiglione, RN, MSN, MHA, and

Kathleen E. Puca, MD, MT(ASCP)SBB

TR A N S F U S I O N O F B L O O D compo- DEFINITION AND SCOPE OF

nents plays a critical role in clinical care.
More than 13.5 million Red Blood Cell (RBC)
transfusions were administered in US hospi-
tals in 2011 at an estimated cost of $10 bil-
lion. 1,2 Transfusion is the single most com-
monly billed procedure for patients receiving
hospital care.3
Blood transfusions can be life-saving, but
they can also be associated with complica-
tions. Transfusion-transmitted infections, al-
though rare, are of concern. In addition, a
growing body of literature demonstrates an as-
sociation between transfusions and poorer pa-
tient outcomes.4-8
The need to better understand the bene-
fits and risks associated with transfusion and
to control health-care costs has led to a grow-
ing interest by many organizations in initia-
tives to improve the quality, stewardship, and
utilization of blood components. Identifying
best practices—including provision of quality
health care without transfusion—is being ad-
dressed by professional societies, federal agen-
cies, hospital systems, and other stakeholders.
PAT IE N T BL O OD MAN AGE ME NT
Patient blood management (PBM) can be de-
fined as an evidence-based, multidisciplinary
approach to optimizing the care of patients
who might need transfusion. Evidence-based
guidelines for the use of blood components
and education for health-care providers on
best practices in blood transfusion are vital el-
ements of PBM. Employing a combination of
pharmacologic, surgical, and medical modali-
ties to conserve blood also plays an important
role in the overall care of patients.
Although the term “patient blood man-
agement” may be fairly new, the concepts have
developed over time in parallel with medical
advances. A full discussion of this history is be-
yond the scope of this chapter. However, two
examples illustrate the shared “drivers” of
blood transfusion and PBM.
One example is the influence of wartime
injuries. The discoveries of blood groups,
crossmatching, and blood preservation in the
early years of the 20th century enabled the use

Mary Ghiglione, RN, MSN, MHA, National Director, Patient Blood Management, Accumen, Inc, San Diego,
California, and Kathleen E. Puca, MD, MT(ASCP)SBB, Medical Director, BloodCenter of Wisconsin, Milwau-
kee, Wisconsin
The authors have disclosed no conflicts of interest.

599
600 䡲 AABB TECHNICAL MANUAL
of banked blood in the treatment of exsangui-
nating wounds suffered in the armed conflicts
of the same period. Yet transporting the blood
was difficult, and battlefield surgeons devel-
oped techniques to treat casualties when no
transfusion was possible.
A second example is the influence of Je-
hovah’s Witnesses, who cite Biblical passages
as the foundation for their refusal of blood
transfusion. By the middle of the 20th century,
blood transfusion had become universally ac-
cepted as a medical treatment for a wide range
of indications. Jehovah’s Witness patients had
to seek out those few physicians and hospitals
that would provide medical and surgical care
without blood transfusion. These surgeons and
hospitals became increasingly utilized by Je-
hovah’s Witnesses and other patients, and blood
conservation programs began to flourish. With
an emphasis on an individualized, patient-
centric approach, these programs led the way
toward the PBM movement seen today.
Although the focus is frequently on sur-
gery, PBM encompasses the entire scope of a
patient’s hospital experience. It includes:
1. Efforts to identify and manage anemia and
bleeding risks before any treatment begins.
2. Use of blood-sparing surgical techniques
and intraoperative blood recovery meth-
ods.
3. Adjunctive strategies during intensive care
unit (ICU) and postoperative care that
decrease the need for transfusion.
4. Blood utilization review and feedback to
ordering physicians.
5. PBM education of all health-care providers
involved in patient care.
The scope of PBM activities is discussed
in detail in the section on Basic Elements of a
Patient Blood Management Program.
TH E RAT I O NA LE F O R PAT IE N T
BL O O D MANA G E ME NT
Blood transfusion became universally accept-
ed as a mainstream medical therapy long be-
fore all the risks and complications associated
with it were recognized. The compelling rea-
sons for PBM include the risk of clerical errors,
errors in patient identification, the threat of a
new transfusion-transmitted pathogen, and
the mounting evidence that transfusions are
associated with adverse patient outcomes.
Other potential drivers of PBM include in-
creasing patient demand for improved quality
in health care, the projected shrinking of the
eligible donor base, and increasing health-care
costs.9,10
Risks of Transfusion
The infectious risks of transfusion that became
so widely known in the late 20th century,
which are now rare, as well as unknown
emerging pathogens continue to be a concern.
Improvements in donor screening, develop-
ment of effective tests, and research into
pathogen inactivation technology have all
played a role in reducing the known infectious
disease risks of transfusion. Currently, the risk
of either hepatitis C virus transmission or hu-
man immunodeficiency virus transmission is
less than 1 in 1,000,000 donations, and the risk
of hepatitis B is less than one in 300,000.11
The noninfectious risks of transfusion,
including hemolytic transfusion reactions,
transfusion-related acute lung injury, transfu-
sion-associated circulatory overload, and
allergic reactions are more common than in-
fectious adverse events and often go unrecog-
nized. Chapter 27, Noninfectious Complica-
tions of Blood Transfusion, discusses the
diagnosis, etiology, treatment, and prevention
of these risks in detail.
An emerging body of evidence indicates
that transfusion may not provide the expected
therapeutic outcomes long assumed by both
clinicians and patients. This is especially the
case in stable, nonbleeding patients.12 Ran-
domized controlled trials assessing the effica-
cy of RBC transfusion have shown that restric-
tive transfusion strategies for nonbleeding
patients have no negative effect, and may even
improve outcomes for some patients.8,13-16
Recent research is identifying a link be-
tween allogeneic transfusion and worse pa-
tient outcomes. Although many of these stud-
ies on blood transfusion are observational,
 CHAPTER 24
they have enrolled large numbers of patients
across many different specialties (eg, cardiolo-
gy, surgery, ICU, gastroenterology). Such stud-
ies have repeatedly concluded that a strong as-
sociation exists between RBC transfusion and
worse patient outcomes.5,8,9,17
Several studies have shown the risk of
nosocomial infection in ICU, surgical, and
trauma patients to be two to four times higher
in transfused patients than in nontransfused
patients.5 In addition, the risk is dose-depen-
dent (ie, the more blood the patient receives,
the greater the risk of adverse events).18,19 RBC
transfusion has been associated, in a dose-
related fashion, with higher rates of cancer re-
currence, stroke, myocardial infarctions, renal
complications, acute lung injury and respira-
tory arrest, longer ICU and hospital stays, and
mortality both in the hospital and the long
term (5 years).5,17,20-24
These deleterious effects on clinical out-
comes are not solely related to allogeneic RBC
transfusions. One single-center study of more
than 800 critically ill patients demonstrated
that 1) transfusion was independently associ-
ated with the risk of acute lung injury and 2)
the risk was higher with the transfusion of
plasma-rich blood components (platelets and
plasma) than with transfusion of RBC units.25
Plasma transfusion in critically ill patients has
shown little benefit and has been associated
with worse patient outcomes.26,27
Thus, a growing body of literature strong-
ly suggests that for the nonbleeding patient
the benefit of transfusion is outweighed by un-
expected risks and worse outcomes. The risks
can be reduced only by a shift from the more
conventional transfusion practice (where
transfusion is often a default decision) to a
PBM approach to transfusion practice (where
the decision to transfuse is an option consid-
ered after evaluation of the risks, benefits, and
alternatives specific to each patient).
The Patient’s Perspective
Despite advances in safety of the blood supply,
patients are concerned about needing a blood
transfusion during their treatment.28,29 Wheth-
er due to their underlying illness, inadequate
Patient Blood Management 䡲 601
communication, or their physician’s demean-
or, some patients trust that the provider who
orders the transfusion is acting in their best in-
terest. Too often, patients do not ask for, or are
not provided with, information on alternative
treatment options before receiving a transfu-
sion.28
Allogeneic transfusion is considered a
therapeutic intervention and, as with other
medical interventions, requires the patient to
provide informed consent.30,31 In order for the
patient to be adequately “informed,” the con-
sent discussion needs to include information
about the risks and benefits not only of the
transfusion option, but of the alternatives to
transfusion and the refusal of treatment as
well.32 The PBM approach, which encompass-
es all the options, provides an excellent avenue
for open communication, joint consideration
of treatments, and the potential for improved
patient outcomes and satisfaction.
PBM strategies are a necessity for patients
for whom blood transfusion is not an option
due to medical status, cultural influences, reli-
gious beliefs, or unavailability of compatible
blood. From the patient’s perspective, the ra-
tionale for PBM is the ability to make a more
informed choice, having access to a higher
quality of care, and potentially experiencing
less risk.
The Physician’s Perspective
After “first do no harm,” a physician’s primary
responsibility is to ensure effective treatment
with minimal risk. Clinicians intend to do
what is best for their patients. But too often
they take blood transfusion for granted, as-
suming that benefits will be achieved and per-
ceiving the risks to be minimal.
Medical school curricula devote a bare
minimum of time for education on the risks of,
indications for, and alternatives to the use of
blood.33,34 Physicians often learn transfusion
practices based on the norms and standards
developed years ago. It is difficult enough for
them to keep up to date with current research
and best practices in their own specialty, let
alone to keep abreast of developments in
transfusion medicine.
602 䡲 AABB TECHNICAL MANUAL
Studies show a wide variation in transfu-
sion practice. Whether or not a patient re-
ceives a blood transfusion is often influenced
by the hospital where the patient is admitted
or by the physician providing the clinical care,
rather than by the patient’s clinical condi-
tion.3,35-38
Transfusion based on physician behavior
can be improved with the implementation of
evidence-based clinical practice guidelines
and computer “dashboards” identifying best
practices linked to blood utilization and pa-
tient outcomes. Providing individual physi-
cians with their usage data in comparison to
that of their colleagues can be a powerful tool
for process improvement.
Audits and evaluation of transfusion
practices, along with analysis of blood utiliza-
tion data, are tools hospitals can apply to iden-
tify best practices. From these tools, guide-
lines, algorithms, and protocols can be
developed and implemented across a service
line in an effort to assist individual physicians
in meeting quality standards and improving
patient care.
Thus, a PBM approach benefits not only
patients, but the physicians who treat them.
Physicians who use the patient-centric ap-
proach of PBM will gain a reputation for better
patient outcomes, shorter lengths of stay, and
reduced costs. In some facilities, blood utiliza-
tion data are included on a physician “score-
card,” acknowledging those physicians whose
transfusion practice complies with the hospi-
tal’s guidelines.
The Hospital’s Perspective
Economic Pressures
Due to the economic pressures facing hospi-
tals today, it is estimated that by 2020 one in
three hospitals will close or reorganize into a
different type of health-care service.39 Oppor-
tunities to reduce costs are eagerly sought by
hospital administrators, and reducing blood
usage is no exception.
The acquisition cost of an RBC unit is only
a fraction of the total cost of that unit to the
hospital. Using activity cost-based modeling,
Shander et al2 reported that the total cost for
blood- and transfusion-related activities for
surgical patients ranged from $522 to $1183
per unit transfused. They also found that total
annual blood expenditures correlated not with
the volume of surgeries performed, but with
the transfusion rate. The authors concluded
that reducing the proportion of patients who
receive transfusions, the number of RBC units
transfused per patient, or both can be an im-
portant cost-saving strategy for hospitals.
In a model simulation, Zilberberg and
Shorr40 showed that universal adoption of a re-
strictive transfusion therapy (hemoglobin less
than 7 g/dL) in all ICU patients in US hospitals
could potentially result in cost savings of near-
ly $1 billion annually. Even more important,
the simulation indicated that this strategy
could improve quality of care and patient out-
comes by preventing nearly 40,000 transfu-
sion-attributable complications.
A recent blood utilization project involv-
ing 464 US hospitals identified opportunities
for significant reductions in blood utilization
and costs.3 Several elements of a PBM program
were noted as important steps for hospital
leaders to consider.
Appropriate reimbursement for health-
care supplies and services, including blood
and transfusion, is a growing concern for hos-
pitals. Medicare reimbursements have failed
to keep pace with increased costs, which have
exacerbated the revenue pressures. The Cen-
ters for Medicare and Medicaid Services (CMS)
structure for outpatient reimbursement iden-
tified that in 2013, payments for the more fre-
quently transfused blood components would
decrease.41 An understanding of all the con-
tributing factors to the cost of transfusion (in-
cluding reimbursement) is vital for hospitals to
develop. The economic pressures can be an
additional incentive for reducing unnecessary
transfusions and improving blood utilization.
Requirements for Accreditation
The Joint Commission promotes several stan-
dards related to hospital blood administration42
and requires hospitals to review the use of
blood and blood components in an effort to
monitor and improve practices. The Joint
 CHAPTER 24
Commission also has developed PBM Perfor-
mance Measures, which include seven areas
for improvement.43 Although they are not uni-
versally practiced, the measures can help hos-
pitals to monitor blood usage.
Both AABB and the College of American
Pathologists (CAP) promote standards for
transfusion service laboratories. These re-
quirements address more specific, technical
issues in quality testing and patient safety re-
lated to blood administration.44,45 Often, it is
the responsibility of the transfusion service to
ensure that requirements related to transfu-
sion are met—even when the requirements in-
volve activities outside the purview of the lab-
oratory staff.
For instance, the CAP requirement
TRM.41025-Transfusionist Training Phase II
requires all personnel who administer blood
components to be trained in proper identifica-
tion of transfusion recipients. AABB Standards
for Blood Banks and Transfusion Services re-
quires a peer-review program for monitoring
and addressing transfusion practices in all cat-
egories of blood and blood components.45(p91)
Both require collaboration of other hospital
departments for compliance. With the multi-
disciplinary, team-oriented approach of PBM,
an additional benefit to hospitals may be in-
creased ease of compliance, as well as more
readily accepted and implemented corrective
actions and process improvements.
The Community’s Perspective
Initiatives to limit the use of blood also benefit
the community by supporting a safe and ade-
quate blood supply for those who need it. The
increasing age of the population and the di-
minishing donor pool may have significant
impacts on blood availability. In the next 10 to
20 years, it is expected that the number of indi-
viduals in the United States who are more than
65 years of age will increase at a faster rate
than those aged 16 to 64, and make up 20% of
the population.46,47
A recent review of data from the Health
and Retirement Study reported that 31% of
Americans over the age of 65 received at least
Patient Blood Management 䡲 603
one transfusion in a 10-year period.48 Although
data are limited, it is estimated that patients
who are 65 or older receive 55% to 60% of RBC
transfusions.47 Similar population demograph-
ics are expected in Germany, where the pre-
dicted shortfall in the blood supply could be
47% by 2020.49
Blood availability may also be affected by
changes in donor eligibility and storage of
blood units. A better understanding of iron de-
pletion in frequent blood donors has prompt-
ed a discussion of increased donor hemoglo-
bin requirements and prolonged donation
intervals.50,51 If ongoing trials confirm a rela-
tionship between the age of stored blood units
and increased morbidity and mortality follow-
ing transfusion in acutely ill adult patients,
blood inventory management will change.52-56
Any shortening of the shelf-life of the RBC
unit could put more pressure on diminishing
blood supplies and donor recruitment efforts
may not be able to make up for the loss.57 Im-
plementing PBM strategies to reduce the need
for allogeneic blood and making sure only ap-
propriate transfusions of the correct dose are
given are two ways to support the available
blood supply for a community.
BAS IC ELE MENTS OF A PBM
PROG R A M
As noted earlier in the section on Definition
and Scope of PBM, several PBM strategies can
be implemented to decrease the use of alloge-
neic blood transfusions. They encompass all
aspects of the patient’s evaluation and clinical
management. A PBM approach is typically im-
plemented in elective surgical patients in the
preoperative, intraoperative, and postopera-
tive phases of care as outlined in Table 24-158
and in the following sections. PBM strategies
are also relevant to medical patients. Although
any one of the strategies used individually will
be successful in decreasing the use of alloge-
neic blood transfusion, they are most effective
when combined and individualized for the
specific patient.59
604 䡲 AABB TECHNICAL MANUAL

TABLE 24-1. Scope of Patient Blood Management

Nonsurgical or ICU and Blood Utilization Medical

Preoperative Intraoperative Postoperative Review Education
䡲 Anemia screening and 䡲 Replacement fluids 䡲 Replacement 䡲 Clinical prac- 䡲 In-service
management – crystalloid fluids tice guidelines – grand rounds
– iron therapy – colloid – crystalloid – indications – conferences
– cautious use of EPO 䡲 Surgical techniques – colloid – thresholds – reminders
䡲 Screen for bleeding – meticulous 䡲 Limit phlebot- – restrictive (posters, data
risks and optimize suturing omy strategies cards, etc)
coagulation – harmonic scalpel 䡲 Tolerate low 䡲 Blood Utiliza- – journal club
– discontinue antico- – suction control hemoglobin tion or Transfu- 䡲 Continuing edu-
agulants and anti- – rinsing swabs 䡲 Monitor bleed- sion Committee cation
platelet drugs 䡲 Acute normovole- ing 䡲 Audits – certification
– discontinue herbal mic hemodilution 䡲 Wound drain- – prospective – CME/CE credit
supplements, some 䡲 Intraoperative age – concurrent – online tools
vitamins blood recovery 䡲 Iron therapy – retrospective (webinars,
– address genetic 䡲 Hemostatics 䡲 Hyperbaric 䡲 Patient Blood interactive
coagulation abnor- – topical thrombin oxygen Management modules)
malities – fibrin sealant 䡲 Point-of-care Coordinator or 䡲 Medical and
䡲 Minimize crystalloids – platelet gel testing Transfusion nursing school
in acute bleeding 䡲 Point-of-care Safety Officer curricula
䡲 Limit phlebotomy testing
䡲 Preoperative autolo- 䡲 Monitor acute
gous donation bleeding and man-
age coagulation
䡲 Tolerate low hemo-
globin
䡲 Avoid hypothermia
䡲 Tolerate low blood
pressure
䡲 Positioning

Modified with permission from Becker J, Shaz B.58

ICU = intensive care unit; EPO = erythropoietin; CME = continuing medical education; CE = continuing education.

Preoperative Strategies Anemia Assessment and Management

Patient evaluation and planning are essential
first steps of a PBM program. A detailed medi-
cal history and physical examination, with
special attention to family and personal histo-
ry of spontaneous or postoperative bleeding
and anemia, are vital to identifying and sup-
porting high-risk patients. Medications that
may affect coagulation should also be as-
sessed. Structured questions can help with the
evaluation, which should be performed suffi-
ciently ahead of the planned surgery (eg, 30
days) to allow for diagnostic testing and thera-
peutic interventions, as needed.60
Recognition and management of anemia in
the medical and preoperative patient is a core
principle of PBM. The World Health Organiza-
tion (WHO) defines anemia as a hemoglobin
level less than 13 g/dL in men and less than
12 g/dL in premenopausal, nonpregnant
women.61 Clinical signs and symptoms of ane-
mia (tachycardia, chest pain, shortness of
breath, dizziness, headache, depression, cold
extremities, pale skin, fatigue, and decreased
cognitive function) occur when the oxygen-
carrying capacities of the red cells are unable
to meet the oxygen demand of the tissues.
 CHAPTER 24
Anemia is a common condition and is es-
timated to be as high as 75% depending on the
patient’s underlying conditions, reason for
surgery, and definition of anemia used.62,63
Anemia has been independently associated
with increased perioperative mortality and
morbidity in patients undergoing joint re-
placement and cardiac surgery.62,64 In a retro-
spective study of noncardiac surgery patients
65 years or older, 30-day postoperative mortal-
ity and cardiac event rates increased by 1.6%
for every percentage-point decrease from the
normal hematocrit range.65 Even mild anemia
has been shown to be an independent risk fac-
tor for adverse outcomes in patients undergo-
ing colon surgery.66
If anemia is detected, the initial test re-
sults may suggest possible etiologies.67 Addi-
tional laboratory testing such as creatinine,
reticulocyte count, iron and iron-binding ca-
pacity, ferritin, vitamin B12, and folate may
help in the diagnosis. Several published guide-
lines can assist the PBM program in develop-
ing algorithms for identifying and treating
anemia in medical and preoperative pa-
tients.67-70 Whenever possible, an elective,
high-blood-loss surgery should be delayed un-
til the anemia is explained and appropriately
treated.
Pharmacologic agents for anemia may in-
clude iron (both oral and intravenous prepara-
tions), folic acid, vitamin B12, or erythropoie-
sis-simulating agents (see Appendix 24-1). The
choice of therapeutic agent should be guided
by the underlying etiology of the anemia, the
patient’s other medical conditions, and avail-
able time to treat before any surgery.
Intravenous (IV) iron replacement using
iron sucrose, ferric gluconate, or iron dextran
has been shown to quickly correct iron defi-
ciency anemia and may be the preferred route
over oral preparations in patients scheduled
for surgery or in the postoperative period. The
efficacy of IV iron in treating anemia has been
consistently proven in reducing the need for
allogeneic blood transfusions in surgical pa-
tients.
Erythropoietic stimulating agents (ESAs)
are also effective in increasing the hemoglo-
bin level if sufficient iron stores are present.
Patient Blood Management 䡲 605
The use of ESAs to lower transfusion rates in
patients undergoing elective, orthopedic sur-
gery is well established.71 However, due to the
risk of thromboembolic events, tumor growth,
and death associated with ESAs, the Food and
Drug Administration (FDA) has restricted its
use, recommending more conservative dosing
and strict monitoring.72
Addressing Bleeding Risk
A second important aspect of PBM is minimiz-
ing the risk of bleeding. Assessing a patient’s
risk for bleeding in relation to the type and
complexity of surgery and the presence of any
pre-existing anemia and/or coagulopathy can
help formulate an individualized plan. Estab-
lishing protocols for discontinuation or dose
adjustment of antiplatelet and anticoagulant
drugs is an important function of a PBM
program and an essential preoperative plan-
ning tool.
Published guidelines such as those devel-
oped by the Society for Thoracic Surgery and
the American College of Chest Physicians can
provide a basis for development of such proto-
cols.73-75 Protocols help practitioners to opti-
mize the patient’s hemostasis before an elec-
tive surgery while minimizing risk for
thrombotic events.
Herbal supplements such as garlic, gink-
go, and ginseng, have also been known to in-
crease bleeding risk. Patients should be asked
about their use and ingestion should be dis-
continued before surgery. Readers are direct-
ed to more focused reviews on the effect of
herbal supplements on coagulation.76,77
Preoperative Autologous Blood
Donation
Historically, preoperative autologous blood
donation (PAD) had been the primary means
to reduce the use of allogeneic blood. With
PAD the patient donates his/her own blood,
ideally 4 to 6 weeks, before surgery. The goal of
the preoperative donation is for regeneration
of the patient’s red cell mass and return of the
patient’s hemoglobin to the precollection val-
ue before surgery.
606 䡲 AABB TECHNICAL MANUAL
With increasing safety of the blood sup-
ply, the number of autologous units collected
in the United States has declined.1,78 Other fac-
tors contributing to the decline include high
wastage of PAD blood (45% or more is discard-
ed),79,80 higher risk for preoperative anemia af-
ter donation,80,81 errors related to production
and handling of these units,83,84 and increasing
acquisition costs that may not be reimburs-
able.
In addition, a systematic review showed
that patients in a PAD program had a higher
likelihood of receiving transfusions (allogeneic
and/or autologous) compared to those pa-
tients who did not participate in PAD because
their hemoglobin did not completely recover
before surgery.82 Therefore, patients in PAD
programs incur a higher overall risk to their
health with a greater likelihood of being trans-
fused.
Despite these concerns, PAD may be a
reasonable option for patients with rare blood
types or multiple red cell alloantibodies or for
patients who refuse allogeneic blood. In these
situations, advanced planning and evaluation
of the patient are crucial before PAD is at-
tempted. In order to mitigate the anemia in-
duced by PAD, the collection needs to occur at
least 4 to 6 weeks before surgery (earlier if
there are freezing capabilities for the units) to
allow sufficient time for recovery of the pa-
tient’s red cell mass.85
Intraoperative Strategies
Acute Normovolemic Hemodilution
Acute normovolemic hemodilution (ANH) is
the removal of whole blood from the patient
immediately before or shortly after the begin-
ning of the surgical procedure into a standard
blood bag containing anticoagulant. Crystal-
loid or colloid solutions or both are reinfused
to maintain adequate circulatory volume.
ANH lowers the patient’s hematocrit, improves
blood fluidity, and increases cardiac output
and organ blood flow, offsetting the decline in
oxygen capacity of the diluted blood.86 Any
blood lost from the patient during the surgery
is then diluted and has a reduced red cell con-
tent.
The ANH-collected whole blood is typi-
cally stored at room temperature (for up to
8 hours) and is often reinfused near the end of
the procedure or whenever transfusion is
needed.87(p22) An added value of ANH is that
platelets and coagulation factors remain viable
when the product is stored at room tempera-
ture. Although ANH is considered a relatively
safe procedure, reports on the clinical efficacy
and benefits are mixed.88 In some studies, the
use of ANH resulted in lower allogeneic trans-
fusion rates, fewer units transfused, and de-
creased complications,89,90 while others failed
to show any benefit.91,92
ANH is more likely to be of benefit in pa-
tients undergoing high-blood-loss procedures
(1500 mL or greater) who have preoperative
hemoglobin levels of 12 g/dL or higher. Poten-
tial contraindications for ANH include active
cardiac ischemia, restrictive or obstructive
pulmonary disease, renal failure, hemoglobin-
opathy associated with hemolysis, and known
coagulation abnormalities.93 Planning and co-
ordination by the surgical team are important
for ANH to be effective.
Intraoperative Blood Recovery
Intraoperative blood recovery is another com-
mon PBM strategy. Use of recovered shed
blood is efficacious in several procedures,
such as cardiothoracic, orthopedic, neurolog-
ic, vascular, and trauma surgery. Reduction in
allogeneic RBC transfusions by 38%, with an
average savings of 0.68 unit of allogeneic RBCs
per patient has been reported.94
Intraoperative blood recovery involves
collecting shed blood from the surgical site,
centrifuging it, and washing it with normal sa-
line. During the centrifugation and washing
process, plasma, platelets, red cell stroma,
contaminants, and anticoagulant are removed.
The washed red cells are transferred to a sepa-
rate bag, and then administered to the same
patient. Ideally, washed recovered blood should
have a hematocrit of at least 45% to 55%.
Concerns regarding the safety of blood re-
covery often surround its use in cancer and
obstetric surgeries. The concern is that un-
wanted material and cells (eg, tumor cells,
 CHAPTER 24 Patient Blood Management 䡲 607

bacteria, and amniotic fluid) from the surgical lection and Administration and other AABB
field may end up in the washed product and publications.58,87,101-103
be re-infused to the patient. However, the use
of double suction setup and leukocyte reduc- Anesthesia and Surgical Techniques
tion filters has been shown to reduce these
Minimizing intraoperative bleeding is critical
risks.95,96 In addition, reviews do not support
for reducing the need for allogeneic transfu-
the theoretical concern for increased risk of ei-
sion. Obvious measures include meticulous
ther amniotic fluid emboli or cancer recur-
surgical technique and rapid and rigorous
rence, although the quality of the studies has
control of bleeding. Proper positioning of the
been questioned.97,98 Prospective, appropriate-
patient can reduce blood loss and is guided by
ly powered studies are needed to define the
two basic principles—elevating the surgical
risks and benefits of recovered blood in these
site above the level of the heart and avoiding
controversial settings.
compression of venous drainage of the surgi-
A recent novel approach for blood recov-
cal field.104 The deliberate reduction of mean
ery in surgeries involving cardiopulmonary
arterial pressure, known as “controlled or de-
bypass (CPB) is the Hemobag modified ultra-
liberate hypotension,” in selected patients can
filtration system (Global Blood Resources,
reduce blood flow to the surgical site and
LLC, Somers, CT). After completion of CPB
thereby decrease blood loss. However, the
and decannulation, residual whole blood from
benefits must be balanced against potential
the CPB circuit, which can be up to 2 liters, is
risk for organ ischemia.59,105
ultrafiltered and hemoconcentrated. Excess
Maintaining normothermia is important
water and inflammatory mediators are re-
for optimal coagulation and hemostasis. Ef-
moved, resulting in a whole blood product
forts to keep the patient warm by using warm
with hematocrit of approximately 50%. In con-
IV fluids, air warming blankets and by keeping
trast to washed, recovered blood, blood pro-
the surgical suite warmer can help to avoid hy-
cessed with the ultrafiltration system contains
pothermia and thus decrease surgical blood
platelets, plasma proteins, and coagulation
loss. Optimal fluid management, including
factors that have been preserved and concen-
choice of infusion fluids, volume, and timing
trated.99,100 The use of this device should theo-
of administration, can also affect surgical
retically reduce exposure to allogeneic blood
blood loss and transfusion requirements.
more than washed, recovered blood, especially
Modern devices for tissue dissection, such as
exposure to plasma and platelet transfusions;
harmonic scalpels, water jet dissectors, and
however, the clinically relevant impact of this
electrocautery with argon beam coagulation
newer technique remains unknown.
can minimize tissue damage and provide bet-
Quality control and quality management
ter hemostasis at the incision site. Other mea-
programs for autologous blood products pre-
sures influencing surgical blood loss include
pared by intraoperative blood recovery devices
use of tourniquets, direct control of bleeding,
are not yet as well developed and accepted
infiltration of vasoconstrictors into the surgi-
among hospitals as other aspects of transfu-
cal wound, choice of ventilation patterns, and
sion medicine. Significant opportunity exists
choice of anesthesia.59,104-106
in hospitals for collaboration between the
transfusion service, surgery, perfusion, anes-
Point-of-Care Testing and Transfusion
thesia, and nursing departments in the devel-
Algorithms
opment of blood recovery programs. Guide-
lines and regulatory requirements for Point-of-care testing (POCT) has several ad-
establishing, enhancing, and maintaining vantages over conventional laboratory testing
quality and safety for these particular transfu- in that it can 1) be utilized whenever necessary,
sion practices are described in the AABB Stan- 2) provide more rapid turnaround time, and 3)
dards for Perioperative Autologous Blood Col- use minimal sample volumes for testing. Use
608 䡲 AABB TECHNICAL MANUAL
of POCT can provide information for timely
transfusion decisions and help to avoid iatro-
genic anemia. Several POCT devices are avail-
able that provide information on coagulation,
platelet function, and clot stability; they are
used in the surgical and critical care set-
tings.107,108
Transfusion based on clinical observa-
tion of bleeding and blood loss is highly em-
pirical and variable. Transfusion algorithms
based on POCT can enhance management of
bleeding by rapidly distinguishing between
bleeding due to coagulopathy and bleeding of
surgical origin and can provide goal-directed
transfusion therapy.
In cardiovascular surgical patients, the
use of thromboelastography-based transfu-
sion algorithms has generally shown a reduc-
tion in transfusion requirements, particularly
plasma and platelets, and a decrease in blood
loss compared to clinician-directed and/or
standard laboratory-based transfusion algo-
rithms.109-111 In a single center, small prospec-
tive trial, a therapy-guided algorithm based on
viscoelastic and aggregometric POCT was su-
perior to that based on conventional laborato-
ry testing for reducing allogeneic RBC transfu-
sions in coagulopathic, complex cardiac
surgery patients.112 The authors attributed
faster turnaround time and the functional as-
sessment of coagulation by the POCT as an ad-
vantage over the conventional quantitative co-
agulation tests. It is important to note that
institutions need to establish their own algo-
rithmic decisions based on available coagula-
tion assays and therapeutic options.
Thromboelastography-guided algorithms
are also routinely used in liver transplant sur-
gery; however, the evidence to date is limited
in showing reduction in transfusion require-
ments.113 The use of thromboelastography in
trauma patients is emerging. Although recent
studies have shown its potential for early de-
tection of trauma-induced coagulopathy and
fibrinolysis for facilitating goal-directed thera-
py over conventional laboratory tests,114,115 on-
going research is needed to assess its effective-
ness in reducing transfusion requirements.
Pharmacologic Agents
Hemostatic agents, such as antifibrinolytics,
desmopressin, and topical agents, can assist
coagulation during surgery in an effort to re-
duce the need for blood transfusion. Aminoca-
proic acid and tranexamic acid inhibit fibrino-
lytic activity, preventing premature breakdown
of the blood clot. Systematic reviews have
shown both agents to be safe and effective in
reducing surgical blood loss and the need for
RBC transfusion.116 Topical agents such as he-
mostats, sealants, and adhesives are gaining
importance as useful tools during surgery and
can reduce bleeding by causing blood to clot,
sealing vessels, or gluing tissues. More focused
reviews of these topical agents and other phar-
macologic agents are available.117-119 Brief de-
scriptions of several pharmacologic therapies
that may help control hemorrhage and/or pre-
vent transfusion are found in Appendix 24-1.
PO STOPERATIVE ST RA TEGIES
Postoperative Blood Recovery
Postoperative blood recovery involves the col-
lection and reinfusion of blood from surgical
drains and/or wounds. An adequate amount
of blood needs to be collected and processed
for this technique to be effective. Thus, it is
used mainly in cardiac and orthopedic surgi-
cal cases where the volume of shed blood can
be significant (500 mL or more).
Postoperative recovered blood can be un-
washed or washed. For the unwashed product,
shed blood is collected and filtered in a device
until sufficient volume is reached; then the
blood is transferred to an infusion bag for rein-
fusion. For the washed product, once suffi-
cient shed blood is collected, it is further pro-
cessed by washing and then transferred to a
bag for reinfusion.
In the past, reinfusion of unwashed shed
blood was popular as a blood conservation
technique in joint replacement surgery. More
recently, the increasing use of other blood
management strategies, particularly antifibri-
nolytics, has led to a decline in surgical bleed-
 CHAPTER 24
ing, making postoperative blood recovery un-
necessary.116 In addition, unwashed shed
blood is less desirable because it has a hema-
tocrit ranging from 20% to 30% and contains
activated clotting and complement factors, in-
flammatory mediators, cytokines, and fat par-
ticles that can increase the risk for febrile reac-
tions.120,121
For patients with substantial postopera-
tive blood loss, improved product quality and
safety—hematocrit ranging from 60% to 80%
with removal of contaminants—can be
achieved by using devices that wash and con-
centrate the postoperative wound drainage
blood. Maintaining competency of nursing
staff and higher cost for these devices may be
disadvantages for some hospitals. However,
when compared to allogeneic blood transfu-
sions, use of postoperative washed products in
joint replacement surgery is potentially com-
parable in cost.122
Limiting Phlebotomy-Related Blood
Loss
Minimizing the impact of phlebotomy on the
development of iatrogenic anemia is a key
strategy for reducing transfusion requirement.
Iatrogenic anemia related to routine laborato-
ry testing is common. In a study of 145 West-
ern European ICUs, blood loss from phleboto-
my averaged 41.1 mL per day per patient,123
which could result in the loss of nearly a unit of
blood for an ICU stay of 1-2 weeks. A US retro-
spective database study of over 17,000 patients
with acute myocardial infarction who were not
anemic on admission showed that patients
who developed moderate to severe anemia
had experienced nearly 100 mL higher mean
blood losses from phlebotomy over the course
of their hospitalization than those who did not
develop anemia.124 In a single-center retro-
spective study of patients with prolonged ICU
stays, after day 21 the odds of being transfused
was independently associated with phleboto-
my volume.125
Reducing the quantity and frequency of
laboratory testing is a logical measure to re-
duce the amount of blood loss related to phle-
Patient Blood Management 䡲 609
botomy. Routine, standing orders are a com-
mon practice. Laboratory testing should be
ordered when clinically justified and the re-
sults are likely to change clinical management.
When ordered, collection for laboratory test-
ing should be consolidated to minimize the
number of tubes collected. Another approach
is the use of pediatric or small volume tubes
for sample collection. This strategy can reduce
blood loss from laboratory testing by up to
70%.126,127 Smaller sample volumes, often less
than 0.5 mL, can also be obtained with the use
of POCT devices.
Even the process of drawing the blood
can be a significant cause of blood loss. Pa-
tients with invasive lines (eg, central venous
catheters or arterial catheters) have a three-
fold increase in phlebotomy volume com-
pared to patients without such catheters. The
ease with which samples can be obtained and
the added requirement to discard the first few
mLs (up to 10 mL) each time the line is ac-
cessed can contribute to greater blood loss.128
Such catheters should be discontinued as
soon as they are no longer required. Orders for
specimens drawn from these catheters should
be consolidated to minimize the amount of
blood otherwise discarded. Use of a device for
blood sampling whereby the initial volume of
blood can be reinfused into the patient instead
of discarded has shown reduction in mean
phlebotomy volume by 50%, higher hemoglo-
bin levels, and fewer RBC transfusions even
when a restrictive transfusion practice is im-
plemented.127,128
Increased Tolerance of Anemia and
Transfusion Thresholds
A patient’s response to anemia is highly indi-
vidualized and dependent on his or her ability
to maintain adequate oxygen delivery to the
tissues. Tolerance is dependent on the pa-
tient’s volume status, his or her physiologic re-
serve (including cardiac, pulmonary, and renal
function), and the dynamics of the anemia.
The response becomes one of the most impor-
tant factors in determining the need for trans-
fusion.129,130
610 䡲 AABB TECHNICAL MANUAL
Patients with chronic anemia due to
chronic renal failure or slow gastrointestinal
bleeding often have physiologically adapted to
a lower hemoglobin level by increasing their
cardiac output, heart rate, or stroke volume.
Rapid blood loss from surgical bleeding or
trauma often results in patients displaying he-
modynamic instability, shock, and other
symptoms that require more rapid volume re-
placement.
Increasing a patient’s tolerance for lower
hemoglobin includes increasing oxygen deliv-
ery or decreasing oxygen consumption, which
can limit the need for RBC transfusion. Strate-
gies to optimize the patient’s hemodynamic
status and oxygenation may include maintain-
ing normovolemia with intravenous fluids, use
of appropriate vasopressor agents, use of sup-
plemental oxygen or mechanical ventilation,
adequate pain control and/or sedation, main-
taining normothermia, and avoidance and
prompt treatment of any infections.131
Incorporating evidence-based transfu-
sion protocols may further aid in reducing un-
necessary transfusions. Historically, hemoglo-
bin levels of 10 g/dL or less have been used as
the threshold for transfusion. Recent random-
ized control trials involving nonbleeding criti-
cally ill patients, post-cardiac-surgery patients,
elderly hip fracture patients with cardiac dis-
ease, and patients with upper gastrointestinal
bleeding have established evidence-based
transfusion thresholds. In all these trials, the
use of a more restrictive transfusion threshold
(eg, hemoglobin of 7 to 8 g/dL) has been
shown to decrease RBC transfusions without
increasing morbidity or mortality.8,132 In
addition, an arbitrary hemoglobin level or
“threshold” should not be the sole driver for
transfusion. Transfusion decisions should be
individualized and based not only on the he-
moglobin level but also on the patient’s clini-
cal signs and symptoms of anemia and their
ability to tolerate and compensate for the ane-
mia.133
Traditionally, physicians have ordered 2
units for RBC transfusion, a practice that
evolved without clear rationale. A unit of blood
can have varying effects on hemoglobin and
hematocrit, depending on the patient’s total
blood volume and shifts in fluid between fluid
compartments. In the nonbleeding patient,
single-unit RBC transfusion followed with
clinical reassessment is advocated.134,135 Often
a single RBC unit provides an adequate in-
crease in hemoglobin and relieves the patient’s
symptoms. The potential impact of imple-
menting a single-unit transfusion policy can
be significant. Based on a transfusion thresh-
old of 8 g/dL of hemoglobin and adoption of a
single-unit strategy, one center predicted that
half an RBC unit or more could be saved per
patient.136 When combined with strict adher-
ence to a restrictive transfusion threshold, a
single-unit transfusion policy can have an
even greater impact.
BL O O D UT I L IZ A T I O N REV IE W
AND CHA NGING PHYSICIAN
BEH AV IO R
Changing the behavior of physicians whose
transfusion practices are embedded in tradi-
tion and habit can be challenging. The ele-
ments required to create a culture change in
physician transfusion practice include: 1) a de-
sire for change, 2) the provision of a new be-
havior practice, 3) a perception of the new
practice as safe and straightforward, and 4) a
presentation of change that is nonthreatening
to autonomy.119
More and more hospital administrators
are recognizing that the use of blood products
may worsen patient outcomes and that the
cost of allogeneic blood is significant. Thus, a
desire for change is already under way. Strate-
gies focused on education of appropriate
transfusion practice, providing sound alterna-
tives to transfusion, and communication and
feedback by respected colleagues or “champi-
ons” are fundamental elements of change in
transfusion practices. Building a team of dedi-
cated stakeholders and champions as the net-
work for change management is crucial for
success of any change and a key element for a
successful PBM program.
Studies have demonstrated the ability of
several interventions for changing transfusion
 CHAPTER 24
practice. Although a systematic review did not
find one intervention more effective than an-
other, the authors concluded that even simple
interventions appear to be effective.137 Inter-
ventions can be broadly grouped into: 1) edu-
cation, 2) adoption of guidelines, 3) reminders,
and 4) audits with feedback.
Education can be in the form of sched-
uled conferences or one-on-one teaching.
One-on-one education with physicians, al-
though more time consuming, is likely to have
a more sustained effect. The educational con-
tent needs to be evidence-based, provided by
respected colleagues or opinion leaders, and
be ongoing. Providing repeated sessions and
easy access to educational information, such
as on the hospital website, can help support
physicians as they consider changing their
practices.
Evidence-based transfusion guidelines
are the basis for improving appropriateness in
transfusion. Introduction of the guidelines
needs to be combined with education and re-
minders such as posters and pocket guide-
lines. Issuing a memo of the transfusion guide-
lines to physicians without follow-up typically
fails to reduce blood usage.138 Transfusion
guidelines can be reinforced when they are
used with computerized provider order entry
(CPOE) systems. Incorporating decision sup-
port tools into CPOE transfusion order sets
and requiring physicians to document the in-
dication when outside of the guidelines can be
effective in improving practice.139,140
Interventions to change transfusion prac-
tice go hand in hand with auditing or monitor-
ing of transfusion practice. Audits that occur at
the time of the transfusion order or during the
24 hours after the transfusion and are accom-
panied with education provide a clear oppor-
tunity for changing behavior.141 Prospective
audits (approval before issuing the product)
performed manually require resources and
can be time consuming. The implementation
of CPOE can provide a more practical ap-
proach to monitoring each transfusion order.
Chapter 28 provides a detailed discussion of
blood utilization auditing.
Blood utilization reports that incorporate
benchmarking with the goal of continuous
Patient Blood Management 䡲 611
quality improvement are powerful tools to
change transfusion practice. Providing physi-
cians with outcome data in relationship to
their transfusion rates for a surgical procedure
or specific treatment as well as comparison to
their peers and best practices are likely to mo-
tivate changes. Providing physicians with reg-
ular reports helps to maintain awareness and
encourages physicians’ interest in looking for
additional strategies to reduce avoidable
transfusions.142
A recent initiative to improve transfusion
practice has been the employment of transfu-
sion safety officers or patient blood manage-
ment coordinators. As change agents they can
provide regular education to all staff involved
in transfusion practice, increase awareness of
and access to transfusion alternatives, develop
a network of caregivers and “champions” for
the promotion of optimal transfusions, and
provide utilization reports for continuous im-
provement. Working with the local “champi-
on,” these coordinators can be instrumental in
changing the culture and reducing allogeneic
transfusions.143
Medical Education
Education is an integral part of any PBM initia-
tive. Because behavioral change is sometimes
difficult to achieve, repetition and reinforce-
ment over time are typically required for suc-
cess. Because PBM is multidisciplinary, differ-
ent audiences may respond to different
approaches.
Educational delivery options may in-
clude journal club, grand rounds, webinars,
online courses, guest lecturers, conference at-
tendance, one-to-one instruction, self-study,
and blood usage review feedback. Provision of
continuing education credits for educational
activities can be an effective motivator for par-
ticipation.
Although ordering physicians and nurs-
ing staff need the greatest understanding of
PBM, other groups of hospital personnel also
benefit from educational opportunities. For
instance, information technology and finan-
cial personnel may not need to know the most
612 䡲 AABB TECHNICAL MANUAL
technical details, but they do need to under-
stand the intent, benefits, and outcomes of
PBM efforts so they can effectively support
those efforts.
PRO GR AM DEVE LO P MEN T
Successful implementation of a PBM program
requires planning, education, and teamwork.
It is important to first analyze the current
transfusion practices within the hospital. A
needs assessment can help to evaluate the
current behaviors and understand the hospital
culture and readiness for change.
Demonstrating the clinical benefits as
well as potential cost savings with a PBM pro-
gram is key to ensuring support and buy-in at
all levels. Care must be taken to ensure that
clinical staff understand that the primary ob-
jective of a PBM program is to improve patient
outcomes, even though hospital administra-
tors may also focus on the cost savings.
The specific activities that are carried out
as part of PBM program implementation can
vary by institution, and are defined by the ex-
ecutive management team of the facility.
Guidance is available from a variety of profes-
sional organizations. The Society for the Ad-
vancement of Blood Management offers Ad-
ministrative and Clinical Standards for Patient
Blood Management Programs, which outlines
12 standards related to the activities of a for-
mal, comprehensive, organization-wide PBM
program.144 A PBM program can be designated
as an activity level 1, 2, or 3 program as out-
lined in AABB Standards for a Patient Blood
Management Program.145(pp2-3) The responsibil-
ities for oversight and monitoring at each ac-
tivity level are described in Appendix 24-2.
An effective PBM program involves a mul-
tidisciplinary effort with input and participa-
tion from administration, physician and
nursing leadership, laboratory, transfusion
medicine, ethics, registration, surgery, finance,
and technology personnel. Identifying cham-
pions who are willing to be spokespersons and
drivers of the program is essential when start-
ing a program. A general strategy for imple-
TABLE 24-2. General Strategy for Implementing
a PBM Program
1. Education
a. A knowledgeable advocate
b. Executives
c. Core group (pharmacy, nursing, blood
bank)
d. Any department involved in PBM activi-
ties (ordering clinicians, finance, informa-
tion technology)
2. Buy-in from executives
a. “C” suite (CEO, COO, CFO, CMO)
b. Medical executive committee
c. Blood utilization/transfusion committee
d. Surgical services committee
3. Business proposal
a. Background/current environment
b. Program description
c. Financial aspects
d. Risk/benefit analysis
e. Summary/conclusion
4. Teamwork
a. Broad-based stakeholder group of those
affected by PBM program (multidisci-
plinary emphasis, identification of con-
cerns, details to be addressed, enhanced
buy-in)
b. Smaller, more focused steering commit-
tee (baseline usage data, relationship to
strategic plan, implementation steps,
timeline, policy review, monitor progress
checks)
c. Effective meetings
d. Milestone celebrations
5. Evaluation
a. Possible improvements/lessons learned
b. Study of metrics/outcome data
c. Analysis/report of program success
d. Possible program expansion
menting a PBM program is summarized in Ta-
ble 24-2. More detailed descriptions of PBM
program implementation are available.146,147
 CHAPTER 24 Patient Blood Management 䡲 613

KEY PO I NT S

1. Patient blood management (PBM) is an evidence-based, multidisciplinary approach to op-

timizing the care of patients who might need transfusion.
2. Several factors have been drivers of PBM, including transfusion-associated risks, demand
for improved quality of care, promotion of evidence-based practice, economic pressures,
overutilization of blood components, and a projected shrinking of the blood supply.
3. Elements of PBM include: 1) managing anemia and bleeding risks before treatment begins,
2) intraoperative blood recovery and blood-sparing surgical techniques, 3) ICU and postop-
erative strategies to reduce the need for transfusion, 4) blood utilization review, and 5) edu-
cation of health-care providers.
4. PBM can provide benefits to medical as well as surgical patients.
5. Preoperative anemia is common. Identifying and treating preoperative anemia is one of the
fundamental elements of PBM.
6. PBM involves more than just transfusion avoidance. It involves use of pharmaceutical
agents, blood recovery techniques, surgical tools to limit blood loss, limiting phlebotomy
for laboratory testing, adherence to transfusion guidelines, and medical education.
7. A multidisciplinary team with “champions” is critical for success of the PBM program.
8. Ongoing blood utilization reviews with benchmarking and emphasis on patient outcomes
are key elements of a successful program.

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 CHAPTER 24
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93. Shander A, Rijhwani TS. Acute normovolemic
hemodilution. Transfusion 2004;44:26S-34S.
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vage for minimising perioperative allogeneic
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96. Waters JH. The future of blood management.
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98. Waters JH, Yazer M, Chen YF, Kloke J. Blood
salvage and cancer surgery: A meta analysis of
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99. Colli A, Balduzzi S, Ruyra X. The Hemobag:
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100. Beckmann S, Lynn P, Miller S, et al. Evaluation
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101. Waters JH, Shander A, eds. Perioperative blood
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102. Waters JH, Dyga RM, Yazer MH. Guidelines for
blood recovery and reinfusion in surgery and
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103. Berte L. Quality manual preparation workbook
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104. Anesthesia – more than sleeping. In: Seeber P,
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107. Enriquez LJ, Shore-Lesserson L. Point-of-care
coagulation testing and transfusion algo-
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phy-based transfusion algorithm reduces
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113. Gurusamy KS, Pissanou T, Pikhart H, et al.
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114. Holcomb JB, Minei KM, Scerbo ML, et al. Ad-
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115. Kashuk JL, Moore EE, Wohlauer M, et al. Initial
experiences with point-of-care rapid throm-
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116. Henry DA, Carless PA, Moxey AJ, et al. Anti-fi-
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121. Sinardi D, Marino A, Chillemi S, et al. Compo-
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125. Chant C, Wilson G, Friedrich JO. Anemia,
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131. Physiology of anemia and oxygen transport.
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132. Carson JL, Carless PA, Hebert PC. Transfusion
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134. Administrative and clinical standards for pa-
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136. Ma M, Eckert K, Ralley F, Chin-Yee I. A retro-
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137. Tinmouth A. Reducing the amount of blood
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138. Tinmouth A, MacDougall L, Fergusson D, et al.
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Arch Intern Med 2005;165:845-52.
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fectiveness of a prospective physician self-au-
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140. Fernandez Perez ER, Winters JL, Gajic O. The
addition of decision support into computer-
ized physician order entry reduces red blood
cell transfusion resource utilization in the in-
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141. Damiani G, Pinnarelli L, Sommella L, et al. Ap-
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sion 2010;50:139-44.
 CHAPTER 24
142. Toy P. Effectiveness of transfusion audits and
practice guidelines. Arch Pathol Lab Med 1994;
118:435-7.
143. Brevig J, McDonald J, Zelinka ES, et al. Blood
transfusion reduction in cardiac surgery: Mul-
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144. Freedman J, Luke K, Escobar M, et al. Experi-
ence of a network of transfusion coordinators
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Coordinators (OnTraC)]. Transfusion 2008;
48:237-50.
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145. Holcomb J, ed. Standards for a patient blood
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146. Thomas J. Building a business case. In: Puca K,
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Press, 2008:33-82.
䡲 APPENDIX 24-1
Pharmacologic Therapies for Supporting Patient Blood Management*
Pharmacologic Agent Specific Drug
Intravenous (IV) Iron Iron high-molecular-
Therapy weight dextran; iron low-
molecular-weight dex-
tran; iron gluconate; iron
sucrose; iron carboxy-
methyl dextran.
Erythropoietic Stimulat- Epoetin alfa: darbepoetin 䡲 Approved for treatment
ing Agents (ESAs) alfa.
Primary Use Mechanism of Action
Used to treat iron defi- Iron is an essential ele-
ciency and iron-defi- ment of hemoglobin and
ciency anemia. the actual site of oxygen
binding. It helps trans-
port oxygen to the tis-
sues.
There is substantial evi-
dence that IV iron is
effective in treating iron-
deficiency anemia in
many chronic condi-
tions.
䡲 ESAs stimulate differ-
of anemia resulting from entiation of stem cells
chronic kidney failure, into immature red cells;
chemotherapy, certain increase rate of mitosis
treatments for human and release of reticulo-
immunodeficiency virus, cytes into the circula-
and also to reduce the tion; and induce
number of blood transfu- hemoglobin forma-
sions during and after tion.
certain elective noncar- 䡲 A glycoprotein hor-
diac, nonvascular sur- mone produced in the
geries (hemoglobin kidney, a growth factor
>10 g/dL but 13 g/dL). for red cell precursors
in marrow.
620
Considerations
䡲
䡲 Blood loss is a major cause of iron defi-
ciency.
䡲 Even under the best of circumstances,
oral iron is not well tolerated, and
patients are often noncompliant due to
gastrointestinal (GI) symptoms.1
䡲 IV iron given concurrently with ESAs
provides better response than oral iron
and allows lowering of ESA dose in cer-
tain patient populations.1
䡲 High-molecular-weight dextran is asso-
ciated with serious side effects.1
䡲 Response seen by day 5 to 7 in iron-
replete patients.
AABB TECHNICAL MANUAL
䡲 One unit equivalent hematocrit increase
by day 7; 3-5 units by day 28.
䡲 Darbepoetin is an alternative used in
renal and oncology settings.2,3
 䡲 Synthetic erythropoie-
tin produced by recom-
binant DNA technology.
Antifibrinolytics Epsilon-aminocaproic 䡲 Enhances hemostasis 䡲 Blocks fibrinolysis by
䡲 Society for Thoracic Surgery 2011
guidelines report “It is reasonable to
use preoperative erythropoietin plus
iron, given several days before cardiac
surgery, for preoperative anemia, in
candidates who refuse transfusion (eg,
Jehovah’s Witnesses), or in patients
who are at high risk for postoperative
anemia.”4
䡲 Black box warning due to increased risk
for death, myocardial infarction, stroke,
venous thromboembolism, thrombosis
of vascular access, and tumor progres-
sion or recurrence.5
䡲 Reduces blood loss and transfusion
CHAPTER 24

acid (EACA) associated with surgical
complications following
heart surgery; orthope-
Tranexamic acid (TA)6 dic surgery; some hema-
(Similar to EACA, but 10 tologic disorders
times as potent) associated with throm- 䡲 TA provides equivalent
bocytopenia; and mas-
sive traumatic bleeding
in the presence of fibri-
nolysis.
inhibiting activation of requirements in cardiac and joint
plasminogen to plas- replacement surgeries.8
min, which is responsi- 䡲 Rapid intravenous administration
ble for limiting and should be avoided; may induce hypo-
dissolving clots. tension, bradycardia, and/or arrhyth-
mia.
antifibrinolytic effect as 䡲 Use with caution in hematuria of upper
EACA but at only 1/10 urinary tract origin, unless the possible
the concentration and benefits outweigh risk.
has a slower renal 䡲 Contraindicated in patients with dis-
Patient Blood Management

clearance (6-8 hours vs seminated intravascular coagulation.

<3 hours with EACA).7 䡲 Skeletal muscle weakness with necrosis
of muscle fibers has been reported fol-
䡲

lowing prolonged use.9

䡲 Must use renal dosing guidelines for
patients with impaired renal function.
(Continued)
621
䡲 APPENDIX 24-1
Pharmacologic Therapies for Supporting Patient Blood Management* (Continued)
Pharmacologic Agent Specific Drug Primary Use
Antifibrinolytics 䡲 Dysfunctional uterine
(Continued) bleeding, menorrhagia
associated with intrauter-
ine device, conization of
cervix, post-/antepartum
hemorrhage, uterine/
vaginal surgery.
䡲 Oral bleeding, post-
dental extraction bleed-
ing in patients with
hemophilia, von Wille-
brand disease (vWD), or
thrombocytopenia.
Reversal Agents Vitamin K Reverses the anticoagu-
lant effect of warfarin.
Protamine 䡲 Neutralizes unfraction-
ated heparin (UFH) after
cardiac surgery.
䡲 Antidote when bleeding
complications are asso-
ciated with excessive
heparin anticoagulation.
Mechanism of Action
Vitamin K is necessary
for the hepatic synthesis
of coagulation factors
(Factors II, VII, IX, and X)
and anticoagulant pro-
teins (Protein C and S).
Binds to heparin and
displaces antithrombin
from heparin-antithrom-
bin complex.12
622
Considerations
䡲 Adverse effects: nausea, vomiting, diar-
䡲
rhea, dizziness, disturbance of color
vision, theoretic risk of thrombosis.9
See clinical practice guidelines outlining
the evidence-based management of
vitamin K antagonist initiation, monitor-
AABB TECHNICAL MANUAL
ing, and treatment of complications.10,11
䡲 Adverse events associated with admin-
istration may include hypotension, pul-
monary edema and anaphylaxis.
䡲 Excess protamine can lead to anticoag-
ulation effect.
䡲 In cardiac surgery, dosing based on
point-of-care testing allows more
appropriate dose and reduces postop-
erative bleeding.12
 䡲 May be used for reversal of low-molec-
ular-weight heparin, but less effective.13
Coagulation Factor Prothrombin complex 䡲 3-factor PCCs have only 䡲 PCCs are derived from 䡲 3-factor PCCs do not effectively lower
Concentrates concentrate (PCC) one approved indication pooled human plasma. the international normalized ratio (INR);
for the prevention and 䡲 3-factor PCCs contain addition of small amount of Fresh Fro-
control of bleeding three Vitamin-K-depen- zen Plasma (mean 2 units) increases
related to hemophilia B. dent coagulation fac- likelihood of satisfactory INR lowering.15
䡲 4-factor PCCs are tors (Factors II, IX, and 䡲 4-factor PCCs contain heparin and
approved for urgent X) and a small amount should not be used in patients with
reversal of Vitamin K of Factor VII. heparin allergies or heparin-induced
antagonist (warfarin) 䡲 4-factor PCCs contain thrombocytopenia.14
in life-threatening therapeutic levels of 䡲 Efficacy and safety of 3-factor PCCs or
bleeding.14 Factor VII (in addition 4-factor PCCs in the setting of patients
to Factors II, IX, and X). with severe or life-threatening bleeding
associated with newer oral anticoagu-
CHAPTER 24

lants (eg, dabigatran, rivaroxaban,

apixaban) is unclear.16
Recombinant Factor VIIa Approved by the Food Enhances thrombin gen- 䡲 Black box warning issued in 2005
(rFVIIa) and Drug Administration eration at the site of vas- regarding the risk of thromboembolic
(FDA) for treatment of cular injury. complications. Efficacy of rFVIIa as a
hemophilia A and B with general hemostatic drug remains
inhibitors. Acceptable to unproven. Study results indicate
use in patients with con- increased risk of arterial thromboem-
genital Factor VII defi- bolic events. Use of rFVIIa outside its
ciency and patients with current licensed indications should be
Patient Blood Management

Glanzmann thrombasthe- restricted to clinical trials.17

nia with antibodies to
glycoprotein IIb/IIIa.
䡲

(Continued)
623
䡲 APPENDIX 24-1
624

Pharmacologic Therapies for Supporting Patient Blood Management* (Continued)

Pharmacologic Agent Specific Drug Primary Use Mechanism of Action Considerations
Coagulation Factor Fibrinogen concentrate Approved by FDA for 䡲 Derived from pooled 䡲 Adverse reactions have included aller-
䡲

Concentrates (Continued) treatment of bleeding human plasma. gic and hypersensitivity reactions.18
only in patients with con- 䡲 Precursor to fibrin; 䡲 Thromboembolic events have been
genital fibrinogen defi- thrombin converts reported.18
ciency.18 fibrinogen to fibrin to 䡲 Use of fibrinogen concentrate in obstet-
form soluble fibrin clot, ric hemorrhage and cardiac surgery has
which is then stabilized been reported but is considered off-
by activated Factor XIII. label and future studies are needed to
prove efficacy.12
Other pharmaceuticals Desmopressin (DDAVP) 䡲 Useful in patients with 䡲 Raises circulating Fac- 䡲 Responsiveness to DDAVP in mild
that can help with hemo- synthetic analog of mild hemophilia A or tor VIII and vWF; hemophilia A and type 1 VWD is usu-
stasis vasopressin19 Type I vWD (DDAVP unidentified effect on ally confirmed by elective challenge
is contraindicated in platelets and endothe- (trial).
Type 2B and platelet- lium.12 䡲 Should be used with caution in patients
type VWD). 䡲 Synthetic analog of the with fluid or electrolyte imbalance, as
natural pituitary hor- with cystic fibrosis; patients may
AABB TECHNICAL MANUAL

䡲 May be useful in patients

with uremia or cirrhosis;
these patients have pro-
longed bleeding times
due to complex disor-
ders of hemostasis.
mone 8-arginine vaso-
pressin, an antidiuretic 䡲 Patients who may benefit from DDAVP
hormone affecting renal
water conservation.
develop hyponatremia.
include:
– Patients undergoing prolonged sur-
gery with cardiopulmonary
bypass.20
– Patients experiencing excessive
postoperative bleeding or those who
are at high risk for bleeding.21-23
– Patients taking platelet-inhibiting
drugs.24
– Patients with chronic renal failure or
liver dysfunction.
 Conjugated estrogen
Topical Hemostatic Mechanical hemostats
Agents (porcine gelatin, bovine
collagen, oxidized regen-
erated cellulose); biologi-
cally active hemostats
(bovine thrombin, human
thrombin, recombinant
human thrombin); fibrin
sealants, polyethylene
glycol polymer sealants;
synthetic adhesives
Pharmaceuticals used Oxytocin
primarily in obstetrics to
control bleeding
Methergine
䡲 Dysfunctional uterine
bleeding.
䡲 Uremia.
Hemostats, sealants, and
adhesives are primarily
used during surgery for a
variety of applications to
help with hemostasis
when ligation, sutures,
compression, or cautery
is not effective. The types
of bleeding where topical
agents may be used
include: diffuse raw sur-
face bleeding, oozing
venous-type bleeding,
bone bleeding, and nee-
dle-hole bleeding.
䡲 Obstetric hemorrhage.
䡲 Used to treat uterine
atony.
䡲 Obstetric hemorrhage.
䡲 Used to treat uterine
atony.
䡲 Precise mechanism of 䡲 Possible alternative or adjunct for cryo-
action not known. precipitate or desmopressin for the
䡲 May involve effect on treatment of bleeding associated with
mucopolysaccharide renal failure.
content of vessel wall; 䡲 Effectiveness of use in uremia patients
increase synthesis of is mixed.
vWF by endothelial
cells.12
Generally act by com- 䡲 Topical hemostatic agents can be highly
pressing the bleeding effective, but they must be used care-
vessel, activating/aggre- fully to avoid systemic reactions.1
gating platelets, and/or 䡲 Full descriptions and uses of the vari-
providing a scaffold for ous topical hemostatic agents are avail-
clot formation. Some able.25
applications include
CHAPTER 24
thrombin, which speeds
clot formation.
Stimulates uterine con- Oxytocin is the standard treatment for
Patient Blood Management
tractions. postpartum hemorrhage.26-28
Increases the strength, Hypertension and toxemia are contrain-
䡲
duration, and frequency dications.26,28
of uterine contractions.
(Continued)
625
䡲 APPENDIX 24-1
626

Pharmacologic Therapies for Supporting Patient Blood Management* (Continued)

Pharmacologic Agent Specific Drug Primary Use Mechanism of Action Considerations
Pharmaceuticals used Carboprost 䡲 Obstetric hemorrhage. Stimulates uterine con- 䡲 Contraindicated in patients with pulmo-
䡲

primarily in obstetrics to 䡲 FDA-approved to treat
control bleeding uterine atony that has
(Continued) not responded to con-
ventional treatment.
Misoprostol 䡲 Obstetric hemorrhage.
䡲 Used to treat uterine
atony.
Other drugs that help Proton pump inhibitors 䡲 Peptic ulcer.
with hemostasis 䡲 Upper GI hemorrhage.
tractions. nary, cardiac, renal, and hepatic dis-
ease.
䡲 Asthma is a relative contraindica-
tion.26,28
Synthetic prostaglandin. Works most effectively when used with
other medications listed in this table
(synergistic action).26,27
Reduces incidence of Proton pump inhibitors are more effec-
upper GI rebleeding tive than H2-antagonists in preventing
after sclerotherapy by persistent or recurrent bleeding from
increasing pH to above peptic ulcer, although this advantage
4, which is necessary seems to be more evident in patients
for clot formation and not having adjunct sclerosis therapy.29
stabilization.
AABB TECHNICAL MANUAL

Octreotide 䡲 Variceal hemorrhage. 䡲 An octapeptide that Octreotide and somatostatin are at least
䡲 Acute non-variceal upper mimics natural soma- as effective as the conventional vasoac-
GI bleeding. tostatin pharmacologi- tive drugs and balloon tamponade in the
cally. treatment of variceal hemorrhage with
䡲 Long-acting analog of the advantage of fewer side effects.30
somatostatin (has a
much longer half-life —
approximately 90 min-
utes, compared to 2 to
3 minutes for soma-
tostatin).
 䡲 Reduces splanchnic
blood flow via multifac-
torial mechanism.
䡲 Most effective when
combined with sclero-
therapy.
䡲 Eliminates vasospasm
complications seen
with vasopressin.
*Drugs approved for use in the United States as of April 2014. The table is intended to provide general information. Professionals seeking additional information and individuals seeking
personal medical advice should consult a qualified physician.

1. Goodnough LT, Shander A. Current status of pharmacologic therapies in patient blood management. Anesth Analg 2013;116:15-34.
2. Goodnough LT, Monk TG, Andriole GL. Erythropoietin therapy. N Engl J Med 1997;336:933-8.
3. Ross SD, Allen IE, Henry DH, et al. Clinical benefits and risk associated with epoetin and darbepoetin in patient with chemotherapy-induced anemia: A systematic review of the literature. Clin Ther
CHAPTER 24

2006;28:801-31.
4. Ferraris VA, Brown JR, Despotis GJ, et al. Society of Thoracic Surgeons Blood Conservation Guideline Task Force; Society of Cardiovascular Anesthesiologists Special Task Force on Blood Transfusion; Inter-
national Consortium for Evidence Based Perfusion. 2011 update to the Society of Thoracic Surgeons and the Society of Cardiovascular Anesthesiologists blood conservation clinical practice guidelines. Ann
Thorac Surg 2011;91:944-82.
5. Epogen (epoetin alfa) injection for intravenous or subcutaneous use package insert. Thousand Oaks, CA: Amgen, 2012. [Available at: http://pi.amgen.com/united_states/epogen/epogen_pi_hcp_english.pdf
(accessed on March 10, 2013).]
6. Cyklokapron tranexamic acid tablets and tranexamic acid injection package insert. New York, NY: Pfizer, 2014.
7. Use of desmopressin, antifibrinolytics, and conjugated estrogens in hemostasis. In: Goodnight SH, Hathaway WE. Disorders of hemostasis and thrombosis a clinical guide. 2nd ed. New York: McGraw-Hill,
2001:528-42.
8. Henry DA, Carless PA, Moxey AJ, et al. Anti-fibrinolytic use for minimising perioperative allogeneic blood transfusion. Cochrane Database Syst Rev 2011;(3):CD001886.
9. Ipema HJ, Tanzi M. Use of topical tranexamic acid or aminocaproic acid to prevent bleeding after major surgical procedures. Ann Pharmacother 2012;46:97-107.
10. Holbrook A, Schulman S, Witt DM, et al. Evidence-based management of anticoagulant therapy. Antithrombotic therapy and prevention of thrombosis. 9th ed. American College of Chest Physicians evidence-
Patient Blood Management

based clinical practice guidelines. Chest 2012;141(Suppl):e152S-e184S.

11. Patriquin C, Crowther M. Treatment of warfarin-associated coagulopathy with Vitamin K. Expert Rev Hematol 2011;4(6):657-67.
12. Bolan CD, Klein HG. Blood component and pharmacologic therapy for hemostatic disorders. In: Kitchens CS, Kessler CM, Konkle BA, eds. Consultative hemostasis and thrombosis. 3rd ed. Philadelphia: Else-
vier Saunders, 2013:496-525.
䡲

13. Garcia DA, Baglin TP, Weitz JI, et al. Parenteral anticoagulants. Antithrombotic therapy and prevention of thrombosis. 9th ed. American College of Chest Physicians evidence-based
clinical practice guidelines. Chest 2012;141(Suppl):e24S-e43S.
14. Kcentra (Prothrombin Complex Concentrate, Human). Silver Spring, MD: Food and Drug Administration, 2013. [Available at: http://www.fda.gov/BiologicsBloodVaccines/BloodBloodProducts/ApprovedProd
ucts/LicensedProductsBLAs/FractionatedPlasmaProducts/ucm350130.htm (accessed April 1, 2014).]

(Continued)
627
 628

䡲 APPENDIX 24-1
Pharmacologic Therapies for Supporting Patient Blood Management* (Continued)
䡲

15. Holland L, Warkentin TE, Refaai M, et al. Suboptimal effect of a three-factor prothrombin complex concentrate (Profilnine-SD) in correcting supratherapeutic international normalized ratio due to warfarin
overdose. Transfusion 2009;49:1171-7.
16. Siegal DM, Garcia DA, Crowther MA. How I treat target-specific oral anticoagulant-associated bleeding. Blood 2014;123:1153-8.
17. Simpson E, Lin Y, Stanworth S, et al. Recombinant factor VIIa for the prevention and treatment of bleeding in patients without haemophilia. Cochrane Database Syst Rev 2012;(3):CD005011.
18. RiaSTAP, Fibrinogen Concentrate (Human) for Intravenous Use, Lyophilized Powder for Reconstitution. Package insert. Kankakee, IL: CSL Behring, 2011. [Available at http://labeling.cslbehring.com/PI/US/Ri-
aSTAP/EN/RiaSTAP-Prescribing-Information.pdf (accessed on April 6, 2014).]
19. Desmopressin acetate injection package insert. Irvine, CA: Sicor Pharmaceuticals, 2014.
20. Salzman EW, Weinstein MJ, Weintraub RM, et al. Treatment with desmopressin acetate to reduce blood loss after cardiac surgery: A double-blind randomized trial. N Engl J Med 1986;314:1402-6.
21. Cattaneo M, Harris AS, Stromberg U, et al. The effect of desmopressin on reducing blood loss in cardiac surgery: A meta-analysis of double-blind, placebo-controlled trials. Thromb Haemost 1995;74:1064-
70.
22. Crescenzi G, Landoni G, Biondi-Zoccai G, et al. Desmopressin reduces transfusion needs after surgery: A meta-analysis of randomized clinical trials. Anesthesiology 2008;109:1063-76.
23. Mongan PD, Hosking MP. The role of desmopressin acetate in patients undergoing coronary artery bypass surgery: A controlled clinical trial with thromboelastographic risk stratification. Anesthesiology
1992;77:38-46.
24. Laupacis A, Fergusson D. Drugs to minimize perioperative blood loss in cardiac surgery: Meta-analyses using perioperative blood transfusion as the outcome. The International Study of Peri-operative Trans-
fusion (ISPOT) investigators. Anesth Analg 1997;85:1258-67.
25. Spotnitz WD. Hemostats, sealant, and adhesives: A practical guide for the surgeon. The American Surgeon 2012;78:1305-21.
26. Esler MD, Douglas J M. Planning for hemorrhage: Steps an anesthesiologist can take to limit and treat hemorrhage in the obstetric patient. Anesthesiol Clin North Am 2003;21:127- 44.
AABB TECHNICAL MANUAL

27. Soltani H, Hutchon DR, Poulose TA. Timing of prophylactic uterotonics for the third stage of labour after vaginal birth. Cochrane Database Syst Rev 2010;(8):CD006173.
28. Shields L. Uterotonic agents fact sheet. Obstetric hemorrhage toolkit. Stanford, CA: California Maternal Quality Care Collaborative, 2010. [Available at http://www.cmqcc.org/resources/934/download (ac-
cessed January 1, 2013).]
29. Gisbert JP, González L, Calvert X, et al. Proton pump inhibitors versus H2-antagonists: A meta-analysis of their efficacy in treating bleeding peptic ulcers. Aliment Pharmacol Ther 2001;15:917-26.
30. Abid S, Jafri W, Hamid S, et al. Terlipressin vs. octreotide in bleeding oesophageal varices as an adjuvant therapy with endoscopic band ligation: A randomized double-blind placebo-controlled trial. Am J Gas-
troenterol 2009;104:617-23.
 CHAPTER 24 Patient Blood Management 䡲 629

䡲 APPENDIX 24-2
Responsibilities for Activity Levels 1, 2, and 3 PBM Programs*
Activity Activity Activity
Item Responsibility Level 1 Level 2 Level 3

1 Evidence of institutional support for the patient blood management pro- X X X

gram at the executive level.
2 Patient outcomes related to transfusion. X X X
3 Budgeting to the level of care required by the implementation of these X X X
PBM Standards.
4 Pretransfusion patient testing and evaluation. X X X
5 Assessment of potential need for blood usage. X X X
6 Ordering of blood, including completion of typing and antibody testing X X X
before procedure start time with a plan for antibody-positive patients.
7 Identification and management of presurgical anemia before elective proce- X X X
dures for which type and screen or type and crossmatch is recommended.
8 Preprocedure optimization of patient coagulation function including dis- X X X
continuation of medications and herbal supplements that impair coagu-
lation function.
9 Percentage of blood components wasted by component type (such as gen- X X X
eral red cells, rare unit red cells, general platelets, matched platelets, plasma,
AB plasma, cryoprecipitate, and granulocytes) and cause (misordering, mis-
handling, not released in a timely manner, outdating in stock, etc).
10 Minimize blood loss due to laboratory testing. X X X
11 Process for identifying patients lacking identification. Standard 6.2.3 X X X
applies.
12 Processes to identify, prior to or upon admission, patients who may X X X
refuse transfusion under any circumstances.
13 Adverse events and incidents related to transfusion. X X X
14 Processes and/or equipment to facilitate rapid decision making with X X N/A
regard to anemia and coagulation management.
15 A plan by each service line to reduce perioperative blood loss. X X N/A
16 Strategies to reduce blood loss and manage anemia and coagulopathy in X X N/A
nonsurgical patients.
17 Treatment of massive blood loss (massive transfusion) including timely X X N/A
delivery of proper ratios of blood components.
18 Use of perioperative techniques consistent with current AABB Standards X N/A N/A
for Perioperative Autologous Blood Collection and Administration.
19 An active program with evidence-based metrics and clinician feedback to X N/A N/A
ensure compliance with transfusion guidelines.
20 A formal program to care for patients who decline the use of blood or X N/A N/A
blood-derived products.
*Holcomb J, ed. Standards for a patient blood management program. Bethesda, MD: AABB, 2014:2-3.
 C h a p t e r 2 5

Transfusion Support for

Hematopoietic Stem Cell
Transplant Recipients

Christopher A. Tormey, MD, and Jeanne E. Hendrickson, MD

ALLOGENEIC HEMATOPOIETIC STEM transfusion support for HSCT recipients are no

cell transplantation (HSCT) recipients
present a distinct set of challenges for blood
banks and transfusion services. When consid-
ering transfusion for an HSCT recipient, one
has to take into account not only the complex-
ities associated with the patient’s underlying
condition, but also potential problems associ-
ated with recipient alloantibodies, donor pas-
senger lymphocytes, and different blood
group systems. Over the past two decades,
HSCT has become significantly more com-
mon. Moreover, many patients now receive
posttransplant care in community hospitals.
Thus, issues pertaining to the complexity of
longer relegated solely to academic medical
centers.
Given the increasing numbers of patients
undergoing HSCT from related and unrelated
donors and the wide variety of clinical condi-
tions that are currently treated with this ap-
proach, transfusion medicine specialists must
be prepared to address the challenges associ-
ated with transfusion support for these pa-
tients.1 This chapter provides an up-to-date
summary of the most common and important
issues faced by blood bank physicians and
other medical practitioners at transfusion ser-
vices in their treatment of HSCT recipients.

Christopher A. Tormey, MD, Assistant Professor of Laboratory Medicine, Yale University School of Medicine,
New Haven, and Medical Director, Transfusion Service, VA Connecticut Healthcare System, West Haven, Con-
necticut and Jeanne E. Hendrickson, MD, Assistant Professor of Laboratory Medicine, Yale University School
of Medicine, and Associate Medical Director, Transfusion Medicine/Apheresis Service, Yale-New Haven Hos-
pital, New Haven, Connecticut
C. Tormey has disclosed no conflicts of interest. J. Hendrickson has disclosed a financial relationship with
Terumo BCT.
The views expressed in this article are those of the authors and do not necessarily reflect the position or policy
of the Department of Veterans Affairs or the United States government.

631
632 䡲 AABB TECHNICAL MANUAL

IM PLICATIONS OF ABO- AND tion to blood component selection for transfu-

N ON - A B O- A N T I G E N -
INCO MPATIBLE RED BLOOD
CELL TRANSPL ANTAT ION FOR
TR ANSF USI O N
Incompatibilities in ABO blood group antigens
are not necessarily a barrier to successful
HSCT. In solid organ transplantation, ABO
compatibility may be essential. However, plu-
ripotent and early committed hematopoietic
progenitor cells (HPCs) lack ABO antigens;
thus, engraftment of HPCs is uninhibited even
in the presence of circulating ABO antibodies.
Nonetheless, discrepancies in ABO and non-
ABO antigens between a donor and a recipient
do play an important role in transplantation
and can become a major complicating factor
in the transfusion support of HSCT recipients.
In allogeneic transplantation, the rela-
tionships between the ABO types of the donor
and recipient fall into four categories: compat-
ible, incompatible in the major crossmatch,
incompatible in the minor crossmatch, and bi-
directionally incompatible.2-4 Table 25-1 lists
potential ABO combinations between donors
and recipients and indicates the associated
compatibility or incompatibility. ABO incom-
patibilities are present in 25% to 50% of donor/
recipient pairs, and, therefore, careful atten-
sion is necessary.2,4-6 Although the terms
“major,” “minor,” and “bidirectional incom-
patibility” are most frequently used in the con-
text of the ABO system, they are also used to
describe the presence of other red cell alloan-
tibodies, such as anti-K or anti-D, in the plas-
ma of the donor and/or recipient.
Major ABO Incompatibility
Major ABO incompatibility creates two chal-
lenges: 1) the potential for acute intravascular
hemolysis when ABO-incompatible donor red
cells within the graft are infused to a recipient
who has high ABO antibody titers and 2) the
ongoing production of ABO antibodies by host
immune cells directed against erythroid pro-
genitors and mature red cells produced by the
engrafting HPCs.
The first challenge is typically addressed
during the collection and/or processing of
HPCs. Techniques, such as red cell reduction
of a marrow graft product, minimize the risk of
hemolysis during infusion. Some HPC prod-
ucts, including all umbilical cord blood cells,
are cryopreserved before administration, and
incompatible donor red cells may be lysed
during the freeze/thaw process. There is no
general consensus on the threshold level of

TABLE 25-1. Compatibility for HSCT by ABO Blood Group of the Donor and the Recipient

Donor ABO Group

Recipient
ABO Group O A B AB
O Identical Major* Major* Major*
A Minor Identical Bidirectional Major*
(major/minor)‡
B Minor† Bidirectional Identical Major*
(Major/minor)‡
AB Minor† Minor† Minor† Identical
*Due to a naturally occurring antibody (or antibodies) in the recipient (eg, the donor is group A and the recipient is group O
and has naturally occurring anti-A).
†
Due to a naturally occurring antibody (or antibodies) in the donor graft product (eg, the donor is group O and has naturally
occurring anti-A, and the recipient is group A).
‡
Due to naturally occurring antibodies in both the donor and recipient (eg, the donor is group A and the recipient is group B).
 CHAPTER 25 HSCT Recipient Transfusion Support
incompatible red cells that may be safely in-
fused, with currently acceptable volumes
ranging from 10 to 20 mL. As suggested by
some, the recipient’s antibody titer may also
be used in guiding facility policy.2 In the ab-
sence of red cell depletion or cryopreserva-
tion, plasmapheresis of the recipient immedi-
ately before graft infusion may be warranted to
reduce the circulating titer of ABO antibodies.
The second challenge, the continuous
production of antibodies against the A and/or
B antigens of engrafted donor red cells and
erythroid precursors, can continue for up to 3
to 4 months after HPC infusion. As a result,
erythropoiesis is often delayed, with red cell
engraftment potentially occurring >40 days af-
ter transplantation. Time to red cell engraft-
ment may be even further prolonged with the
use of reduced-intensity or nonmyeloablative
conditioning regimens.5,7 In severe cases, pure
red cell aplasia (PRCA) can develop.5 There-
fore, HSCT involving major ABO incompatibil-
ity may render some recipients transfusion
dependent for several months following trans-
plantation. Fortunately, major incompatibility
tends not to affect engraftment or the produc-
tion of other myeloid-lineage cells.
Minor ABO Incompatibility
Analogous to red cell depletion in major in-
compatibility, plasma depletion of the graft
product can significantly decrease donor allo-
antibodies in minor ABO incompatibility.
However, even if isoagglutinins are not com-
pletely removed, the ensuing hemolysis is typ-
ically mild and self-limited.4 A more significant
problem with minor-incompatible HSCT re-
sults from the rapid generation of anti-A and/
or -B by donor lymphocytes against residual
recipient red cells. This so-called “passenger
lymphocyte syndrome” occurs approximately
5 to 16 days after infusion of HPCs. The patient
experiences acute, immune-mediated hemo-
lysis that can result in morbidity and even
mortality. Fortunately, in most cases, the he-
molysis is not severe and eventually subsides
with the clearance of recipient red cells.4 If the
hemolysis is severe and potentially life threat-
ening, therapeutic erythrocytapheresis can be
䡲 633
used to exchange incompatible recipient red
cells with donor-compatible red cells. In pa-
tients believed to be at particularly high risk of
the passenger lymphocyte syndrome (usually
based on the type of posttransplant immuno-
suppression), prophylactic erythrocytaphere-
sis may be warranted before transplantation.8
Bidirectional ABO Incompatibility
In bidirectional ABO incompatibility, compli-
cations arising from both major and minor
ABO-incompatible HSCT can occur in the re-
cipient. To prevent these complications, the
processing of HPC grafts might include the re-
duction of both red cell and plasma content.
The posttransplant period can be complicated
by the onset of hemolysis within days or weeks
(as occurs in minor incompatibility), delayed
engraftment of red cells, and/or, in some cas-
es, PRCA (as occurs in major incompatibility).
Incompatibility Related to Non-ABO
Antigens
Red cell antigens of other blood group systems
can present challenges similar to the ones de-
scribed for ABO-incompatible transplants.
Overall, these incompatibilities are generally
less frequently encountered, but the presence
of red cell antibodies in the patient (more
common) and/or donor (less common) re-
quires attention and approaches that are simi-
lar to those outlined above for ABO incompati-
bility. Special consideration of graft donor
selection may also be needed when reduced-
intensity or nonmyeloablative conditioning
regimens are used in alloimmunized recipi-
ents, and cognate graft donor antigen/recipi-
ent alloantibody pairs should be avoided if at
all possible.
B LO OD CO M P O N E NT
CO N SI D E R AT I ON S
Selection of Blood Components
The selection of appropriate blood compo-
nents for recipients of allogeneic HSCT is
particularly problematic. When determining
634 䡲 AABB TECHNICAL MANUAL
transfusion requirements for an allogeneic
HSCT recipient, it is vital that the blood bank
or transfusion service keep detailed records of
the patient’s pretransplant ABO group and
ABO antibody titers and the donor’s ABO
group. It is also imperative to determine the
stage of the transplant (ie, preparative period,
immediate posttransplant period, or posten-
graftment period with absence of recipient red
cells and/or antibodies).9 Recommendations
for optimal component selection at each point
in the transplant process are shown in Table
25-2.
With regard to Rh(D), the recipient can
continue to receive the type of components
transfused before the transplantation as long
as the HSCT donor and recipient match. How-
ever, if the recipient is Rh negative and the do-
nor is Rh positive or vice versa, only Rh-nega-
tive Red Blood Cell (RBC) units should be
transfused to prevent alloimmunization to the
highly immunogenic D-antigen. Given the
small risk of D alloimmunization from red
cells contained in Rh-positive platelet units,
Rh-negative platelets may be preferred if the
blood supply allows this. However, it has re-
cently been suggested that this practice be re-
evaluated.10
RBC Support
The majority of HSCT recipients require trans-
fusion support in the peritransplant period,
regardless of incompatibilities or blood group
antigen mismatches. Symptomatic anemia is
one of the most common indications for RBC
transfusion in HSCT recipients. In the absence
of symptomatic anemia, a patient’s status and
underlying comorbid conditions can be used
to determine whether RBC transfusion may be
warranted. Because specific RBC transfusion
thresholds in HSCT populations are lacking,
clinicians can rely on general RBC transfusion
guidelines. For instance, a threshold hemoglo-
bin level of 7 g/dL is likely appropriate for
most stable, nonpostoperative adult HSCT re-
cipients. However, a slightly higher threshold
of 8 g/dL is likely appropriate for adults with
preexisting heart disease, those at risk of end-
stage organ damage, or HSCT recipients in the
postoperative stage.11,12
The mechanisms underlying anemia and
RBC transfusion dependence in HSCT are suf-
ficiently exceptional to warrant brief further
discussion. After intensive chemotherapy, se-
rum erythropoietin (EPO) levels increase rap-
idly and peak in the first week after treat-
ment.13-15 Although recipients of autologous
HSCT maintain adequate EPO levels through-
out the posttransplantation period, allogeneic
recipients do not.16-20 Allogeneic HSCT recipi-
ents experience a prolonged period of inap-
propriately low endogenous EPO levels, which
often necessitate prolonged RBC transfusion
support. Interestingly, mixed results have been
obtained by the various researchers who have
examined recombinant EPO dosing after allo-
geneic HSCT.13-20 Larger studies are necessary
to determine whether EPO is capable of reduc-
ing the RBC transfusion burden and prevent-
ing the complications of HSCT, such as PRCA.
It is noteworthy that the US Food and Drug
Administration has recently strengthened its
warnings about EPO use after some studies
showed that EPO treatment had adverse ef-
fects (decreased survival and/or tumor pro-
gression) in some patients with cancer.16 Al-
though no studies have specifically linked EPO
use to poor outcomes in HSCT patients, clini-
cians should be mindful of the potential risks
of EPO usage.
Platelet Support
Platelet recovery after allogeneic HSCT has
been studied extensively. Several factors are
strongly associated with the rate at which pa-
tients become platelet transfusion indepen-
dent, including: 1) the relationship between
the donor and the recipient (ie, whether they
are related vs unrelated), 2) conditioning regi-
men used, 3) presence of graft-vs-host disease
(GVHD) or cytomegalovirus (CMV) infection,
and 4) HPC CD34+ cellular source/dose.17-23 To
summarize broadly, the findings of several
studies suggest that slower platelet engraft-
ment tends to be more common in patients
TABLE 25-2. Transfusion Support for Patients Undergoing HSCT by Type of ABO Incompatibility and Stage of Transplant

ABO Blood Group Selection

Type of Incompatibility Transplant Stage RBCs Platelets* Plasma

Major incompatibility Preparative regimen Recipient Donor Donor

Transplantation Recipient Donor Donor
Recipient antibodies detected Recipient Donor Donor
Recipient antibodies no longer detected Donor Donor Donor
CHAPTER 25

Minor incompatibility Preparative regimen Donor Recipient Recipient

Transplantation Donor Recipient Recipient
Recipient cells circulating Donor Recipient Recipient
Recipient cells no longer circulating Donor Donor Donor

Bidirectional incompatibility Preparative regimen Group O Group AB Group AB

Transplantation Group O Group AB Group AB
Recipient antibodies detected/recipient cells Group O Group AB Group AB
circulating
HSCT Recipient Transfusion Support

Recipient antibodies no longer detected/ Donor Donor Donor

recipient cells no longer circulating
䡲

*Due to the short shelf life and limited availability of group AB platelets, it may not always be possible to provide fully matched ABO platelet products as recommended in the table. Therefore,
blood banks and transfusion services may consider providing ABO-mismatched platelets that have been volume reduced to diminish their plasma content.
RBCs = Red Blood Cells.
635
636 䡲 AABB TECHNICAL MANUAL
receiving grafts from unrelated donors, who
have higher-grade GVHD, or who have pre-
transfusion viral infections.17-20 There is also
increasing evidence to suggest that the source
of an allogeneic transplant—such as cord
blood HPCs [HPCs(C)] vs HPCs from apheresis
[HPC(A)]—is predictive of time to platelet en-
graftment. Several studies have shown that the
median time to platelet engraftment for
HPC(C) transplants is, on average, significant-
ly longer than for recipients of HPC(A) or HPCs
from marrow.20-23 Thus, longer-term depen-
dence on platelet transfusion is more likely for
individuals in any of the above categories.
One major consideration for platelet
transfusion in HSCT recipients is compatibili-
ty. Plasma (including the significant volume of
plasma in platelets) contains variable amounts
of isoagglutinins. Although transfusion of lim-
ited quantities of ABO-incompatible plasma
and platelets is common in routine transfu-
sion, this practice cannot be readily applied to
the recipients of allogeneic HSCT without
careful consideration.24 In ABO-incompatible
HSCT, the plasma-containing components
should be compatible with both the donor and
the recipient whenever possible. Recommen-
dations for component selection are more ful-
ly described in Table 25-2.
In addition to the risk of hemolysis, some
researchers have found other morbidities as-
sociated with ABO-incompatible platelet
transfusions. For instance, one pediatric study
showed that such transfusions, when com-
bined with the use of melphalan, correlated
with the development of hepatic venoocclu-
sive disease, possibly resulting from the bind-
ing of A- and/or B-antigens expressed on the
surface of hepatic endothelial cells.25 There-
fore, some centers have an institutional policy
requiring the transfusion of ABO-identical
RBCs and platelets only to HSCT recipients; at
least one group has noted that this policy may
be associated with improved patient survival.26
There is an obvious need for additional pro-
spective studies to address this issue.
In the context of platelet transfusion
support in HSCT recipients, the following ad-
ditional areas are discussed more extensively
below: 1) prophylactic vs therapeutic transfu-
sions, 2) the transfusion “threshold,” 3) the ap-
propriate transfusion dose, and 4) HLA or
platelet antibodies.
Prophylactic vs Therapeutic Platelet
Transfusions
Platelet transfusions are frequently adminis-
tered for prophylaxis of bleeding, rather than
to treat acute hemorrhage, in HSCT recipients.
Although this has been a traditional manage-
ment approach for many years, several recent
studies have questioned the utility and clinical
benefit of this practice. Two studies, both pub-
lished by Wandt and colleagues,27,28 examined
the assumption that prophylactic platelet
transfusion could be replaced by therapeutic
transfusion in recipients of autologous trans-
plants. The researchers found that although
patients assigned to a therapeutic (rather than
prophylactic) transfusion regimen had some
bleeding, there was no evidence of life-threat-
ening hemorrhage. Moreover, therapeutic in-
terventions resulted in dramatic reductions in
platelet usage. However, Stanworth and
colleagues29 concluded, based on a recently
published trial, that prophylactic platelet
transfusions were more effective in preventing
hemorrhage in patients with hematologic ma-
lignancies than in a nontransfused control
population. Therefore, questions still remain
regarding the nature of appropriate, evidence-
based prophylactic transfusion strategies for
patients with thrombocytopenia. The results
of the studies conducted to date and future tri-
als will help shape prophylactic platelet trans-
fusion practice for HSCT recipients.30
Platelet Transfusion Threshold
The acceptable threshold for prophylactic
transfusion of platelets has been studied ex-
tensively for >20 years. One of the earliest es-
tablished thresholds to prevent spontaneous
hemorrhage was a platelet count of <20,000/µL
for patients undergoing chemotherapy/HSCT.31
Over time and as clinical trial data have ac-
crued, studies have consistently revealed that
a platelet count <10,000/µL in uncomplicated
thrombocytopenia (ie, in patients without co-
 CHAPTER 25 HSCT Recipient Transfusion Support
existing conditions such as fever, bleeding, or
bacteremia/sepsis) is a reasonable threshold
to prevent increased bleeding or hemorrhage-
related morbidity.30,32-35 However, a study by
Nevo and colleagues36 raises questions about
this threshold. In this study, HSCT recipients
who received transfusions at a platelet count
<10,000/µL had significantly increased non-
hemorrhagic mortality rates and reduced sur-
vival compared to those infused at counts
<20,000/µL. Although changes in the 10,000/
µL threshold should not be based on this study
alone, it is imperative that data continue to be
collected to examine the appropriateness of
this target platelet count and, as noted previ-
ously, whether prophylactic platelet transfu-
sions are necessary at any platelet count.30
Platelet Dose
Another question that frequently arises with
platelet transfusion is what the optimal plate-
let dose is per transfusion episode. To date,
three large-scale clinical trials have examined
the question of platelet transfusion dosing.37-39
A meta-analysis of aggregate data from these
three studies showed no increased risk of sig-
nificant bleeding between low and standard
platelet doses.30 It is important to note that, as
best illustrated in the PLADO trial, selecting
platelet doses for patients based on body sur-
face area may be a critical factor in the provi-
sion of lower platelet doses. Drawbacks to a
lower dosing regimen may include a lower
platelet increment after infusion and the po-
tential for a greater number of platelet transfu-
sions over time.30,39 Clearly, as more data are
collected, lower platelet transfusion doses may
become accepted as routine clinical practice
for HSCT recipients.
HSCT Recipients with HLA or HPA
Antibodies
Some patients scheduled to undergo HSCT
possess antibodies against HLA and/or human
platelet antigen (HPA); both of these types of
antibodies may reduce the efficacy of platelet
transfusion, resulting in lower corrected count
increments.40 For more thorough discussions
䡲 637
of the evaluation and treatment of platelet re-
fractoriness driven by HLA and HPA antibod-
ies, see Chapters 18 and 19.
In addition to leading to potential prob-
lems with platelet transfusion, recipient allo-
antibodies against HPA or HLA could delay
platelet or white cell engraftment in some
transplant settings. This is analogous to what
occurs in the red cell lineage with major ABO-
mismatched transplants. The issue of HPA and
HLA matching in HSCT donor-recipient pairs
has also been raised by several groups because
of concerns that mismatches between donors/
recipients for HPA (which may serve as minor
histocompatibility antigens) may increase the
risk of GVHD, although two studies have found
otherwise.41,42
Plasma, Cryoprecipitated AHF, and
Factor Concentrate Support
There are no specific recommendations re-
garding the transfusion of plasma, cryoprecip-
itated antihemophilic factor, or factor concen-
trates in HSCT patients; therefore, adherence
to general guidelines and/or expert recom-
mendations for the transfusion of these com-
ponents is advised.43 As discussed above and
outlined in Table 25-2, the most important is-
sue for plasma transfusion is the ABO group of
the recipient and engrafting cells.
For HSCT complicated by GVHD, there
has been interest in determining whether re-
combinant factor concentrates could be used
to treat bleeding complications. Dysregula-
tion of hemostasis is a well-known complica-
tion of GVHD. The utility of recombinant acti-
vated Factor VII (NovoSeven, Novo Nordisk,
Zurich, Switzerland), a potent procoagulant,
has been investigated. A multicenter, random-
ized controlled trial found no differences in
bleeding score for any of three doses (40, 80, or
160 µg/kg) of Factor VIIa compared to control
treatment for hemorrhage associated with
GVHD in the setting of HSCT.44 The results of
this trial suggest that recombinant Factor VIIa
is likely to be ineffective as a first-line therapy
for GVHD-associated bleeding.44 However,
Factor VIIa may still be useful as a last-resort
638 䡲 AABB TECHNICAL MANUAL
treatment for patients with intractable bleed-
ing.
Another possible hemostasis therapy aris-
es from the fact that GVHD often results in the
depletion of Factor XIII, increasing the risk and
severity of gastrointestinal hemorrhage.45 The
high concentration of Factor XIII in cryopre-
cipitate makes cryoprecipitate a potentially
useful therapy for GVHD-related bleeding.
Formal studies are required to determine the
efficacy of this approach.
Transfusion Support for Autologous
Transplant Recipients
Because recipients of autologous transplants
are not exposed to foreign graft products from
donors, virtually all of the concerns regarding
major/minor incompatibility (and their im-
pact on transfusion support) are not applica-
ble to this patient group. Before, during, and
after HSCT, autologous recipients should be
supported by transfusions of blood compo-
nents as needed and in compliance with stan-
dard protocols that are applicable to any trans-
fusion recipient. However, because of their
underlying immunosuppression, autologous
recipients may require specialized products or
components involving additional processing
steps. Such issues and concerns (applicable to
both autologous and allogeneic transplant re-
cipients) are discussed in greater detail below.
PAT I E N TS W I T H N E U T RO PEN I A
A N D I N F E C T IO N T H AT I S
U N R ES P O N S IV E TO
ANTI MIC RO BIA L T H E R A P Y
Traditionally, infusions of fresh granulocytes
have been used to treat severe, antibiotic-
refractory bacterial or fungal infections in pa-
tients with absolute neutrophil counts less
than 500/µL.46,47 (For a more in-depth discus-
sion of granulocyte therapy, see Chapter 18.)
To date, one study has demonstrated that neu-
trophil transfusions can be efficacious in treat-
ing infection in HSCT recipients with neutro-
penia; however, this Phase I/II study included
only 11 participants.48 To more fully establish
the clinical efficacy of transfused granulocytes
in the setting of HSCT, a multicenter random-
ized controlled trial is currently under way.49
The results of this study will be helpful in un-
derstanding the potential benefit of granulo-
cyte infusion for severely ill HSCT recipients.
S PE C I A L P ROC ES SI N G O F
B LO OD CO M P O N E NTS F O R
H S C T R E C I PI E N TS
The irradiation of cellular blood products (eg,
RBCs, platelets, and granulocytes) is intended
to inhibit the proliferation of donor lympho-
cytes, thereby preventing transfusion-associ-
ated GVHD, a reaction that is almost uniformly
fatal.9,50 Although it is generally accepted that
HSCT recipients require irradiated compo-
nents during, and for at least 1 year after,
transplantation, it is unclear whether these pa-
tients require irradiated components after this
time. Despite the absence of evidence indicat-
ing that irradiation is essential after this time
has elapsed, many institutions provide irradi-
ated products indefinitely to HSCT recipients.
This is likely a prudent policy given the poten-
tial for lifelong immunosuppression associat-
ed with HSCT and the possibility of relapse of
the malignancy for which the patient initially
underwent the HSCT.
Blood banks and transfusion services
must rigorously ensure irradiation of compo-
nents transfused to HSCT recipients. Part of
this vigilance is the development of systems or
policies to identify patients who require irradi-
ated products. One approach to help reduce
the possibility of inadvertently releasing a
nonirradiated unit for transfusion to an HSCT
recipient is to require universal irradiation of
selected blood components. In some institu-
tions, all platelet products are irradiated upon
receipt from the regional blood center. Al-
though this strategy may not be practical for
other components, such as RBCs, additional
approaches may be developed, such as irradi-
ation of all components issued to in- and out-
patient hematology/oncology wards.
 CHAPTER 25 HSCT Recipient Transfusion Support
The situation is equally complex with re-
gard to CMV, a pathogen linked to high mor-
bidity and mortality rates in HSCT recipients.
It has been proposed that transfusions of com-
ponents that were leukocyte reduced before
storage are as efficacious in the prevention of
CMV spread as components collected from
donors lacking antibodies to CMV (CMV-sero-
negative donors).9 However, a meta-analysis of
829 recipients of CMV-seronegative compo-
nents and 878 recipients of leukocyte-reduced
components revealed that the risks of CMV in-
fection were 1.63% and 3.01%, respectively,
following transfusion.51 Because CMV is also
acquired in the community, conducting stud-
ies addressing this issue definitively remains
logistically difficult.
The major shortcomings of CMV-sero-
negative products include their limited avail-
ability and the fact that seronegative donors
may have recently acquired CMV with viremia
that is not detected by serologic testing. In the
absence of a large supply of CMV-seronega-
tive products or antigen testing, leukocyte-
reduced units can reasonably be considered
to be “CMV reduced risk.” To better triage re-
quests for CMV-seronegative components, the
CMV status of recipients and donors should
also be considered. Some hospitals provide
CMV-seronegative units to HSCT recipients
who are CMV seronegative and received a
transplant from a CMV-seronegative donor, al-
though leukocyte-reduced components may
be equally efficacious.52
Special processing of transfusion units
may also be needed for HSCT recipients who
repeatedly experience common transfusion
reactions.50 For instance, HSCT recipients may
develop allergic symptoms, such as pruritis,
urticaria, or wheezing; the severity of such
symptoms may increase over time. One ap-
proach to prevent repeated allergic reactions is
to wash components to remove supernatant
plasma using automated methods for RBCs or
by centrifugation and saline resuspension for
platelets. These modifications, in conjunction
with timely premedication regimens, can help
reduce the risk or severity of recurrent allergic
or febrile transfusion reactions.
䡲 639
S PE C I A L CO N SI D E R AT I ON S
F O R T R A NS F U S I O NS I N
PEDIAT RIC HSCT RECIPI ENTS
In general, the considerations regarding trans-
fusion support of pediatric HSCT recipients
are similar to those for adults.53 However, indi-
cations for transplant, stem cell source, and
donor selection may be subtly different in
these patient populations. For example, chil-
dren with inherited diseases (such as sickle cell
disease or thalassemia major) may be more
likely to be treated with HSCT than adults. Fur-
thermore, high-dose chemotherapy with au-
tologous stem cell rescue is more often utilized
to treat some childhood malignancies (includ-
ing advanced stage neuroblastoma) than ma-
lignancies in adults. In addition, cord blood
may be utilized for transfusions to treat some
diseases in pediatric recipients. For example,
HPC(C) transfusion often provides a sufficient
dose for a child, but not for an adult, because
of a child’s small size.
Special considerations are necessary for
transplantation in children (or adults) with
sickle cell disease during both the pre- and
peritransplant periods. Red cell phenotyping
of the stem cell donor is recommended to pre-
vent alloimmunizaton in the recipient against
the donated cells by guiding both donor selec-
tion and product processing. If possible, stem
cell donors who are negative for cognate red
cell antigens against which recipients have red
cell alloantibodies should be considered.54
This is particularly important for reduced-
intensity or nonmyeloablative conditioning
regimens, which can induce long-term mixed
chimerism.54 If antigen-positive donors are
utilized, then red cell reduction of the stem cell
product is recommended (even in the absence
of ABO incompatibility) to avoid a transfusion
reaction during stem cell infusion.
Because of the association between RBC
components and HLA/HPA alloimmunization,
consideration should be given to pretrans-
plant HLA/HPA antibody screening.55-57 The
results of this screening should be taken into
consideration in planning posttransplant
platelet transfusions to comply with the gener-
al recommendations to keep platelet counts
640 䡲 AABB TECHNICAL MANUAL
above 50,000/µL (to prevent cerebrovascular
bleeding).58,59 Such results may also be impor-
tant in HPC donor selection for non-HLA-
matched, nonmyeloablative transplants. An
additional consideration is to perform red cell
exchange transfusion before transplantation
with a goal of <30% to 45% hemoglobin S to
prevent vasoocclusive events resulting from
granulocyte colony-stimulating factor admin-
istration.60
Beta-thalassemia major and severe aplas-
tic anemia may be cured by matched sibling
HSCT in childhood, yet prior transfusion ex-
posure may adversely influence engraftment
and outcomes in subsequent transfusions.61-63
Potential reasons for this association include
humoral or cellular immunization to trans-
fused antigens (whether they are defined or
other minor histocompatibility antigens) or
iron overload. Early transplantation for eligible
patients may decrease the risk of graft rejec-
tion, and careful attention to iron status at the
time of transplantation is also recommended.
The intensity of conditioning regimens is also
likely a key factor in outcomes of HSCT in pa-
tients with beta-thalassemia major or severe
aplastic anemia, and their long-term risk/ben-
efit ratios deserve careful consideration.
IN F OR MATI O N PO RTA B IL I T Y
FOR HSCT RECIPI ENTS
Many patients undergo HSCT far from their
usual medical institution. Before transplanta-
tion procedures, all of the patient’s transfusion
history (including red cell, platelet, or leuko-
cyte alloantibody testing history) should be
communicated to the transplanting facility.
Eventually, these patients may return for fol-
low-up care to their primary care physicians,
KEY POINTS
1. Allogeneic HSCT recipients face distinct transfusion challenges because of their immuno-
suppression, preexisting diseases, and potential for changes in their expressed blood group
systems.
2. ABO compatibility is not critical in the selection of potential HSCT donors because pluripo-
tent and early-committed HPCs lack ABO antigens. ABO incompatibility does, however, in-
fluence transfusion decisions during the peritransplant period.
and supporting such patients can be a signifi-
cant challenge for the transfusion service.
The flow of information from the trans-
plant center to local providers is rarely perfect;
with increased complexity of care, critical data
can be overlooked. Some institutions have cre-
ated a process to deal with such situations. For
example, the nationally networked Depart-
ment of Veterans Affairs medical center data-
base can be consulted to detect any ABO or Rh
discrepancies and determine whether a partic-
ular patient has ever had an alloantibody de-
tected at another network hospital. If a trans-
fusion service or blood bank does have an
interconnected hospital laboratory informa-
tion system, such databases can be useful for
gathering information on transplant recipi-
ents.
Regardless of the availability of an inter-
connected information system, the transfu-
sion service should consider developing a pol-
icy with its hematology-oncology colleagues
that clearly outlines the approach to patients
who receive their HSCT at an outside facility.
This policy should call for communicating the
following important information to a patient’s
usual health-care facility after that patient re-
turns from an off-site transplant facility: 1)
whether the transplant was autologous or allo-
geneic; and, 2) if the transplant was allogeneic,
the ABO type of the donor, and any auto- or al-
loantibodies developed by the patient during
care at that facility. In some cases, if such in-
formation is not completely communicated,
the transfusion service can contact the trans-
plant center and obtain the required informa-
tion at the time of the patient’s first posttrans-
plant visit. A portable worksheet with these
data should also be made available to patients.
 CHAPTER 25 HSCT Recipient Transfusion Support 䡲 641

3. ABO incompatibilities, which are present in 20% to 40% of donor/recipient pairs, fall into
three categories: 1) incompatible in the major crossmatch, 2) incompatible in the minor
crossmatch, and 3) bidirectionally incompatible. Such incompatibilities have the potential
to produce acute and, in the case of major incompatibility, ongoing intravascular hemolysis
and pure red cell aplasia. They can be partially mitigated by red cell and/or plasma deple-
tion of the graft before infusion.
4. Management approaches to transfusion are similar for adult and pediatric HSCT recipients;
however, indications for transplantation, stem cell source, and donor selection may be sub-
tly different.
5. When transfusion requirements for allogeneic HSCT recipients are determined, it is vital
that the blood bank or transfusion service keep detailed records of the recipient’s pretrans-
plant ABO group and the donor’s ABO group. It is also imperative to determine the stage of
the transplant.
6. Platelet concentrates are most frequently transfused to HSCT recipients to prevent an acute
hemorrhage. A platelet count threshold of 10,000/µL in uncomplicated thrombocytopenia
is widely accepted as safe for allogeneic recipients.
7. Cellular blood components are irradiated to inhibit the proliferation of donor lymphocytes,
thereby preventing transfusion-associated GVHD. Many services provide irradiated compo-
nents indefinitely to HSCT recipients.
8. RBC and platelet components that have been leukocyte reduced before storage are general-
ly considered to be equivalent to CMV-seronegative units in terms of CMV transmission
risk. Some studies, however, suggest that seronegative units may be marginally safer.

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myeloablative conditioning. Bone Marrow
Transplant 2003;32:821-4.
42. Garcia-Malo MD, Corral J, Gonzalez M, et al.
Human platelet antigen systems in allogeneic
peripheral blood progenitor cell transplanta-
tion: Effect of human platelet antigen mis-
match on platelet engraftment and graft-ver-
sus-host disease. Transfusion 2004;44:771-6.
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43. Roback JD, Caldwell S, Carson J, et al. Evi-
dence-based practice guidelines for plasma
transfusion. Transfusion 2010;50:1227-39.
44. Pihusch M, Bacigalupo A, Szer J, et al, F7BMT-
1360 trial investigators. Recombinant activat-
ed Factor VII in treatment of bleeding compli-
cations following hematopoietic stem cell
transplantation. J Thromb Haemost 2005;3:
1935-44.
45. Pihusch M. Bleeding complications after he-
matopoietic stem cell transplantation. Semin
Hematol 2004;41(Suppl 1):93-100.
46. Hubel K, Carter RA, Liles WC, et al. Granulo-
cyte transfusion therapy for infections in can-
didates and recipients of HPC transplantation:
A comparative analysis of feasibility and out-
come for community donors versus related
donors. Transfusion 2002;42:1414-21.
47. Grigull L, Pulver N, Goudeva L, et al. G-CSF
mobilised granulocyte transfusions in 32 pae-
diatric patients with neutropenic sepsis. Sup-
port Care Cancer 2006;14:910-16.
48. Price TH, Bowden RA, Boeckh M, et al. Phase
I/II trial of neutrophil transfusions from do-
nors stimulated with G-CSF and dexametha-
sone for treatment of patients with infections
in hematopoietic stem cell transplantation.
Blood 2000;95:3302-9.
49. Safety and effectiveness of granulocyte trans-
fusions in resolving infection in people with
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627393 (accessed December 2, 2013).]
50. Torres R, Kenney B, Tormey CA. Diagnosis,
treatment, and reporting of adverse effects of
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51. Vamvakas EC. Is white blood cell reduction
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ing transmission of cytomegalovirus by trans-
fusion? A review of the literature and meta-
analysis. Transfus Med Rev 2005;19:181-99.
52. Nash T, Hoffmann S, Butch S, et al. Safety of
leukoreduced, cytomegalovirus (CMV )-un-
tested components in CMV-negative allogene-
ic human progenitor cell transplant recipients.
Transfusion 2012;52:2270-2.
53. Luban NL, McBride E, Ford JC, Gupta S. Trans-
fusion medicine problems and solutions for
the pediatric hematologist/oncologist. Pedi-
atr Blood Cancer 2012;58:1106-11.
54. Petz LD. Immune hemolysis associated with
transplantation. Semin Hematol 2005;42:145-
55.
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55. McPherson ME, Anderson AR, Haight AE, et al.
HLA alloimmunization is associated with RBC
antibodies in multiply transfused patients
with sickle cell disease. Pediatr Blood Cancer
2010;54:552-8.
56. Buetens O, Shirey RS, Goble-Lee M, et al. Prev-
alence of HLA antibodies in transfused pa-
tients with and without red cell antibodies.
Transfusion 2006;46:754-6.
57. Lo SC, Chang JS, Lin SW, Lin DT. Platelet allo-
immunization after long-term red cell transfu-
sion in transfusion-dependent thalassemia
patients. Transfusion 2005;45:761-5.
58. Walters MC, Sullivan KM, Bernaudin F, et al.
Neurologic complications after allogeneic
marrow transplantation for sickle cell anemia.
Blood 1995;85:879-84.
59. McPherson ME, Anderson AR, Haight AE, et al.
Transfusion management of sickle cell pa-
tients during bone marrow transplantation
with matched sibling donor. Transfusion 2009;
49:1977-86.
60. Tormey CA, Snyder EL, Cooper DL. Mobiliza-
tion, collection, and transplantation of periph-
eral blood hematopoietic progenitor cells in a
patient with multiple myeloma and hemoglo-
bin SC disease. Transfusion 2008;48:1930-3.
61. Lucarelli G, Gaziev J. Advances in the allogene-
ic transplantation for thalassemia. Blood Rev
2008;22:53-63.
62. Lucarelli G, Clift RA, Galimberti M, et al. Mar-
row transplantation for patients with thalas-
semia: Results in class 3 patients. Blood 1996;
87:2082-8.
63. McCann SR, Bacigalupo A, Gluckman E, et al.
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13:233-7.
 C h a p t e r
Therapeutic Apheresis
Robertson D. Davenport, MD
THERAPEUTIC APHERESIS IS the
treatment of diseases through removal
or extracorporeal manipulation of blood com-
ponents or specific blood substances. It is dis-
tinct from blood component collection by
apheresis, which is covered in Chapter 7. Stan-
dards and guidelines for therapeutic aphere-
sis, clinical privileging, and documentation are
provided by AABB and The American Society
for Apheresis (ASFA).1-4
P R I N C IP L E S A N D M O D A L I T IE S
The goal of therapeutic apheresis is to remove
a pathologic element from blood or to modu-
late cellular function by manipulation such as
through extracorporeal photopheresis (Table
26-1), with or without replacement of the re-
moved element. Apheresis can be performed
manually, but automated techniques are faster
and more efficient. In therapeutic plasma ex-
change (TPE), 1.0 or 1.5 plasma volumes are
typically processed. Larger volume procedures
can increase the risk of coagulopathy, citrate
toxicity, or electrolyte imbalance, depending
on the replacement fluid. The effectiveness of
apheresis in removing pathologic substances
depends on the concentration of those sub-
Robertson D. Davenport, MD, Medical Director, Blood Bank and Transfusion Service, and Associate Professor,
University of Michigan Health System, Ann Arbor, Michigan
The author has disclosed no conflicts of interest.
2 6
stances in the blood, the volume of blood pro-
cessed, and the equilibrium between blood
and the substance’s extravascular volume of
distribution. The procedure is most efficient at
the beginning because the amount of the com-
ponent removed decreases exponentially with
time (Fig 26-1). Continued production or mo-
bilization from the extravascular space will re-
sult in a less-than-expected reduction. A 1.0
plasma volume exchange typically removes
approximately two-thirds of a substance if it
does not move significantly from the extravas-
cular to the intravascular space.
Continuous flow centrifuge apheresis de-
vices have a rotating channel designed to in-
troduce whole blood at one site, and the blood
elements subsequently separate by density as
blood flows through the channel. The resulting
layers of plasma, platelets, leukocytes, or red
cells can be removed selectively. The compo-
nent to be removed is diverted into a collec-
tion bag, while the remaining blood compo-
nents are mixed with the replacement fluid
and returned to the patient. The device con-
trols the flow rates of blood withdrawal, anti-
coagulant solution and replacement fluid in-
fusion, as well as centrifuge speed to achieve
optimal separation.
645
646 䡲 AABB TECHNICAL MANUAL

TABLE 26-1. Apheresis Modalities

Blood Component Replacement

Procedure Removed Typical Indication Fluid

Therapeutic plasma Plasma Reduction of an abnor- Albumin or plasma

exchange mal plasma protein, eg,
autoantibody
Red cell exchange Red cells Sickle cell disease-related Red cells
complications
Leukapheresis Buffy coat Leukemia with As needed
leukostasis
Thrombocytapheresis Platelet-rich plasma Thrombocytosis As needed
Erythrocytapheresis Red cells Erythrocytosis None
Extracorporeal Buffy coat (reinfused) Chronic graft-vs-host None
photopheresis disease
Selective adsorption Specific plasma protein Hypercholesterolemia None
Rheopheresis High-molecular-weight Age-related macular None
plasma proteins degeneration

FIGURE 26-1. Theoretical removal of a substance by apheresis. For a substance that is strictly intravascular,
36.8% of the initial concentration remains after a single blood volume exchange. However, if the substance is
also present in the extravascular space with a total volume of distribution equal to twice the blood volume,
60.6% will remain after a single blood volume has been processed.
 CHAPTER 26
Intermittent centrifugation devices use
an alternative method in which a specified vol-
ume of whole blood is first withdrawn into a
centrifuge bowl. Blood withdrawal is then
stopped, and the extracorporeal blood prod-
uct is processed. The rest of the procedure is
similar to the continuous flow process in that
the blood is centrifuged, the selected compo-
nent is diverted into a waste bag, and the re-
mainder of the blood is returned to the patient
with appropriate replacement fluid. The pro-
cess can be repeated for several cycles. Inter-
mittent centrifugation is most commonly used
for extracorporeal photopheresis (ECP), al-
though a continuous flow ECP device has been
introduced.5
Filtration devices also operate by contin-
uous flow. Anticoagulated whole blood is
passed through a microporous filter that al-
lows plasma to pass through but retains the
blood cells. The separated plasma can then be
diverted into a waste bag, or, as in the case of
selective adsorption, further processed and re-
turned to the patient. This type of device is not
suitable for cytapheresis. A variant of this tech-
nique is rheopheresis, or double-filtration TPE,
in which high-molecular-weight molecules—
primarily fibrinogen, low-density lipoprotein
(LDL), fibronectin, and von Willebrand
factor (vWF)—are removed, reducing plasma
viscosity.
In selective adsorption, blood or plasma
is passed over a column that has a high affinity
for a specific component, such as immune
globulin G (IgG) or LDL, and the effluent is re-
turned to the patient. This has the advantage
of highly specific removal of the element of in-
terest. However, it is restricted in the United
States to only a few conditions for which affini-
ty adsorbers are available. Such techniques are
more widely employed outside of the United
States.
IN D ICA TI O N S
Although there are many case reports of suc-
cessful treatment of a variety of diseases and
conditions by apheresis, there have been few
high-quality clinical trials. ASFA has published
Therapeutic Apheresis 䡲 647
clinical guidelines categorizing the indications
for apheresis in 78 disease states.2
1. Category I: Disorders for which apheresis is
accepted as first-line therapy, either as a
primary stand-alone treatment or in con-
junction with other modes of treatment.
2. Category II: Disorders for which apheresis
is accepted as second-line therapy, either as
a stand-alone treatment or in conjunction
with other modes of treatment.
3. Category III: Disorders in which the opti-
mal role of apheresis therapy is not estab-
lished. Decision making for patients should
be individualized.
4. Category IV: Disorders in which published
evidence demonstrates or suggests that
apheresis is ineffective or harmful. Institu-
tional review board approval is desirable if
apheresis treatment is undertaken in these
circumstances.
TPE
The goal of TPE is to remove a pathogenic mol-
ecule, protein, or high-molecular-weight com-
plex from plasma. In addition, TPE may be
used to provide a deficient normal substance,
such as an enzyme or coagulation factor. Indi-
cations for TPE with the recommended treat-
ment course are listed in Table 26-2. The fre-
quency and duration of treatment are guided
by clinical judgment, although laboratory test-
ing may be helpful for some indications.
Replacement fluids for TPE are compared
in Table 26-3. For most indications, albumin is
the preferred replacement fluid, because it is
isosmotic with blood and has a smaller risk of
adverse reactions and infectious disease trans-
mission than plasma. Plasma is indicated for
thrombotic thrombocytopenic purpura (TTP)
or if coagulopathy is a concern. Red Blood
Cells (RBCs) may also be used to prime the
apheresis circuit when treating small patients
(ie, <10-20 kg) in whom the extracorporeal vol-
ume may exceed 10% to 15% of the patient’s
blood volume.
Diseases in which a pathogenic autoanti-
body is targeted include acute and chronic
inflammatory demyelinating polyradiculo-
648 䡲 AABB TECHNICAL MANUAL

TABLE 26-2. Indications for Therapeutic Plasma Exchange

Typical Course
(Number of
Indication Modifying Conditions Category treatments)

Acute disseminated encephalomyelitis II QOD (3-6)

Acute inflammatory demyelinating I QOD (5-6)
polyneuropathy (Guillain-Barré
After IVIG III
syndrome)
Acute liver failure III Daily (variable)
Amyloidosis, systemic IV
Amyotrophic lateral sclerosis IV
ANCA-associated rapidly progressive Dialysis dependence I Daily or QOD
glomerulonephritis (Granulomatosis (6-9)
DAH I
with polyangiitis; Wegener granuloma-
tosis) Dialysis independence III

Antiglomerular basement membrane Dialysis dependent and no DAH III Daily or QOD
disease (Goodpasture syndrome) (7-10)
DAH I
Dialysis independence I
Aplastic anemia; pure red cell aplasia Aplastic anemia III Daily or QOD
(variable)
Pure red cell aplasia III
Autoimmunic hemolytic anemia: Severe WAIHA III Daily or QOD
WAIHA; cold agglutinin disease (variable)
Severe cold agglutinin disease II
Burn shock resuscitation III Once
Cardiac transplantation Desensitization, positive cross- III Daily or QOD
match due to donor specific HLA (variable)
antibody
Antibody-mediated rejection III
Catastrophic antiphospholipid II Daily (3-5)
syndrome
Chronic focal encephalitis (Rasmussen III QOD (3-6)
encephalitis)
Chronic inflammatory demyelinating I 2-3/week
polyradiculoneuropathy (variable)
Coagulation factor inhibitors Alloantibody IV
Autoantibody III Daily (variable)
Cryoglobulinemia Symptomatic/severe I QOD (3-8)
Dermatomyositis or polymyositis IV
Dilated cardiomyopathy, idiopathic NYHA II-IV III QOD (5)
 CHAPTER 26 Therapeutic Apheresis 䡲 649

TABLE 26-2. Indications for Therapeutic Plasma Exchange (Continued)

Typical Course
Number of
Indication Modifying Conditions Category treatments)

Familial hypercholesterolemia Homozygotes with small blood II Weekly

volume (indefinite)
Focal segmental glomerulosclerosis Recurrent in transplanted kidney I Daily or QOD
(variable)
HSCT, ABO incompatible Major HPC, Marrow II Daily (1-3)
Major HPC, Apheresis II
Hemolytic uremic syndrome, atypical Complement gene mutations II Daily (variable)
Factor H antibodies I
MCP mutations IV
Hemolytic uremic syndrome, Shiga toxin associated IV Daily (variable)
infection-associated
S. pneumonae associated III
Henoch-Schonlein purpura Crescentric III QOD (4-11)
Severe extrarenal disease III
Heparin-induced thrombocytopenia Precardiopulmonary bypass III Daily or QOD
(variable)
Thrombosis III
Hypertriglyceridemic pancreatitis III Daily (1-3)
Hyperviscosity in monoclonal Symptomatic I Daily (1-3)
gammopathies
Prophylaxis for rituximab I
Immune complex rapidly progressive III QOD (3-6)
glomerulonephritis
Immune thrombocytopenia Refractory IV
Immunoglobin A nephropathy Crescentic III QOD (6-9)
Chronic progressive III
Inclusion body myositis IV
Lambert-Eaton myasthenic syndrome II Daily or QOD
(variable)
Liver transplantation, ABO Desensitization, live donor I Daily or QOD
incompatible (variable)
Desensitization, deceased donor III
Antibody-mediated rejection III
Lung allograft rejection Antibody-mediated rejection III Daily or QOD
(variable)
(Continued)
650 䡲 AABB TECHNICAL MANUAL

TABLE 26-2. Indications for Therapeutic Plasma Exchange (Continued)

Typical Course
(Number of
Indication Modifying Conditions Category treatments)

Multiple sclerosis Acute CNS inflammatory demye- II QOD (5-7)

linating disease
Chronic progressive III Weekly (variable)
Myasthenia gravis Moderate-severe I QOD (3-7)
Before thymectomy I
Myeloma cast nephropathy II Daily or QOD (5-7)
Nephrogenic systemic fibrosis III Daily or QOD (10-14)
Neuromyelitis optica (Devic syndrome) Acute II Daily or QOD
(variable)
Maintenance III Weekly (variable)
Overdose, envenomation, and Mushroom poisoning II Daily (variable)
poisoning
Envenomation III
Natalizumab and PML III
Paraneoplastic neurologic syndromes III QOD (5-6)
Paraproteinemic demyelinating IgG/IgA I QOD (5-6)
polyneuropathies
IgM I
Multiple myeloma III
PANDAS; Sydenham chorea PANDAS exacerbation I QOD (5-6)
Sydenham chorea I
Pemphigus vulgaris Severe III Daily or QOD
(variable)
Phytanic acid storage disease (Refsum II Daily or QOD
disease) (variable)
POEMS syndrome IV
Posttransfusion purpura III Daily (variable)
Psoriasis IV
Red cell alloimmunization in pregnancy Before IUT availability III QOD (variable)
Renal transplantation, ABO compatible Antibody-mediated rejection I Daily or QOD
(5-6)
Desensitization, living donor, I
positive crossmatch due to
donor-specific HLA antibody
Desensitization, high PRA III
deceased donor
 CHAPTER 26 Therapeutic Apheresis 䡲 651

TABLE 26-2. Indications for Therapeutic Plasma Exchange (Continued)

Typical Course
(Number of
Indication Modifying Conditions Category treatments)

Renal transplantation, ABO Desensitization, live donor I Daily of QOD

incompatible (variable)
Antibody-mediated rejection II
Group A2/A2B into B, deceased IV
donor
Schizophrenia IV
Scleroderma (Progressive systemic III 2/week (6)
sclerosis)
Sepsis with multiorgan failure III Daily (variable)
Stiff-person syndrome III QOD (4-5)
Sudden sensorineural hearing loss III QOD (3)
Systemic lupus erythematosus Severe II Daily or QOD
(3-6)
Nephritis IV
Thrombotic microangiopathy, drug Ticlopidine I Daily (variable)
associated
Clopidogrel III
Cyclosporine/tacrolimus III
Gemcitabine IV
Quinine IV
Thrombotic microangiopathy, HSCT Refractory III Daily (variable)
associated
Thrombotic thrombocytopenic purpura I Daily (variable)
Thyroid storm III Daily (2-3)
Toxic epidermal necrolysis Refractory III Daily or QOD
(variable)
Voltage-gated potassium channel II QOD (5-7)
antibodies
Wilson disease Fulminant I Daily or QOD (3-5)
QOD = every other day; IVIG = intravenous immunoglobulin; DAH = diffuse alveolar hemorrhage; WAIHA = warm autoim-
mune hemolytic anemia; NYHA = New York Heart Association class; HSCT = hematopoietic stem cell transplantation;
MCP = membrane cofactor protein; CNS = central nervous system; PML = progressive multifocal leukoencephalopathy;
PANDAS = pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections; IUT = intrauterine
transfusion.

neuropathy (AIDP and CIDP), antiglomerular clude renal transplantation with presensitiza-
basement membrane antibody disease, and tion, and antibody-mediated organ transplant
myasthenia gravis. Examples of conditions in rejection.
which the goal is to remove an alloantibody in-
652 䡲 AABB TECHNICAL MANUAL

TABLE 26-3. Comparison of Replacement Fluids

Replacement Solution Advantages Disadvantages

Crystalloids Low cost 2-3 volumes required

Nonallergenic Hypo-oncotic
No viral risk Lacks coagulation factors and
immunoglobulins
Albumin Iso-oncotic Higher cost
Low risk of reactions Lacks coagulation factors and
immunoglobulins
Plasma Iso-oncotic Viral transmission risk
Normal levels of coagulation Citrate load
factors, immunoglobulins and ABO compatibility required
other plasma proteins Risk of allergic reactions
Cryoprecipitate-reduced plasma Iso-oncotic Same as plasma
Reduced high-molecular-weight
von Willebrand factor and
fibrinogen
Normal levels of most other
plasma proteins

In myeloma with hyperviscosity, the goal
is to remove an excessive paraprotein (M pro-
tein). Measurement of plasma viscosity may
not be useful in guiding therapy in some pa-
tients because plasma viscosity may not corre-
late with symptoms. Normal plasma viscosity
ranges from 1.4 to 1.8 centipoise (cP). Because
most patients are not symptomatic until the
plasma viscosity is more than 4.0 or 5.0 cP, pa-
tients with mild elevations may not require
treatment. In general, hyperviscosity becomes
a concern when M protein concentrations
reach 3 g/dL for IgM, 4 g/dL for IgG, and 6 g/dL
for IgA.6 Patients receiving rituximab (anti-
CD20) therapy for IgM myeloma may experi-
ence a transient increase in M protein levels.
Patients with pretreatment IgM greater than
5 g/dL are at particular risk of developing
symptomatic hyperviscosity.7
There are conflicting data regarding the
efficacy of TPE for treatment of acute renal
failure in myeloma. A randomized controlled
trial of TPE vs. conventional care showed no
difference in mortality or renal function at 6
months.8 However, among dialysis-dependent
patients, 43% in the TPE group and none in the
control group recovered renal function. An-
other randomized clinical trial showed no
benefit of TPE in a composite outcome mea-
sure of death, dialysis dependence, and glo-
merular filtration rate.9 However, biopsy con-
firmation of the renal diagnosis was not
required in this trial. Similarly, a retrospective
cohort study showed no benefit of TPE in ei-
ther reducing mortality or preserving renal
function.10 If TPE is to be undertaken, biopsy
confirmation of cast nephropathy may be ad-
visable.
Diseases in which immune complexes
may be pathogenic and can be removed by
apheresis include rapidly progressive glomer-
ulonephritis, cryoglobulinemia, and vasculi-
tis. Other indications include conditions treat-
ed by removal of protein-bound drugs or
toxins or high-concentration lipoproteins.
In TTP, a deficiency of the vWF-cleaving
metalloprotease ADAMTS-13 results in accu-
mulation of high-molecular-weight vWF mul-
timers with subsequent intravascular platelet
activation and platelet-rich thrombi in the
 CHAPTER 26
microvasculature.11 In many cases, an inhibi-
tor of ADAMTS-13 can be demonstrated. Plas-
ma exchange is first-line treatment for TTP,
with the goal of removing both the inhibitor
and large vWF multimers while simultaneous-
ly replacing the deficient enzyme. Secondary
forms of microangiopathic hemolytic anemia
associated with systemic lupus erythemato-
sus, hematopoietic progenitor cell transplan-
tation, chemotherapy, or immunosuppressive
medications may be clinically indistinguish-
able from idiopathic TTP. However, in many
cases, ADAMTS-13 activity has been shown to
be normal or only moderately reduced, and
the response to plasma exchange is typically
poor in these secondary forms of microangio-
pathic hemolytic anemias. Transplant-associ-
ated microangiopathic hemolysis rarely re-
sponds to apheresis and probably represents a
different disease process.12
TPE to treat TTP is typically performed
daily until the platelet count and lactate dehy-
drogenase level are in the normal range, but
the intensity and duration of treatment should
be guided by the individual patient’s course.
After response has been achieved, intermittent
apheresis or plasma infusion taper may be in-
stituted, but the efficacy of this approach in
preventing relapse has not been established.13
TPE therapy has greatly improved the survival
rate in TTP; however, treatment failures do oc-
cur and cause major morbidity or death.14
Hemolytic uremic syndrome (HUS) is a
similar condition that occurs more commonly
in children than adults. HUS may follow diar-
rheal infections with verotoxin-secreting
strains of Escherichia coli (strain 0157:H7) or
Shigella. Compared to patients who have clas-
sic TTP, those with HUS have more renal dys-
function and less prominent neurologic and
hematologic findings. Most patients with HUS
do not have antibody to ADAMTS-13 and have
normal activity of this protease. Although diar-
rhea-associated HUS rarely responds to TPE,
atypical HUS (aHUS) caused by complement
factor deficiencies or autoantibodies to Factor
H may respond; however, patients with aHUS
caused by membrane cofactor protein muta-
tions may not respond to TPE. A terminal
complement inhibitor, eculizumab, may be ef-
Therapeutic Apheresis 䡲 653
fective in the treatment of individuals with
aHUS.15
TPE is increasingly being used in the
treatment of central nervous system acute dis-
seminated encephalomyelitis. Experience in
treating chronic progressive multiple sclerosis
with TPE has been discouraging. However, a
randomized clinical trial in acute CNS inflam-
matory demyelinating diseases unresponsive
to steroids showed that TPE was beneficial.16
Early initiation of TPE is predictive of re-
sponse, and some clinical responses may not
manifest until later in follow-up.17 TPE may be
effective in neuromyelitis optica, even in the
absence of NMO antibodies.18
In focal segmental glomerulosclerosis
(FSGS), a circulating factor that increases glo-
merular permeability with resultant protein-
uria has been demonstrated.19 A recent study
has suggested that circulating urokinase re-
ceptor may be involved in the pathogenesis of
this disease.20 FSGS frequently recurs after re-
nal transplantation and can result in allograft
failure. TPE may effectively remove the perme-
ability factor and induce remission in recur-
rent FSGS following renal transplantation. Re-
sponse to TPE in the primary form of the
disease has not been well studied.
TPE may be an adjunct to immunosup-
pression in the treatment or prevention of an-
tibody-mediated rejection (AMR) of solid or-
gan transplants. AMR presenting in the early
posttransplantation period may respond bet-
ter to TPE than later AMR.21 TPE before renal
transplantation of an ABO-incompatible kid-
ney may be used to prevent hyperacute rejec-
tion, and posttransplantation TPE is often
used to treat AMR that occurs in this set-
ting.22,23 TPE in conjunction with immuno-
modulatory therapies, such as intravenous im-
mune globulin (IVIG), before transplantation
can reduce the risk of rejection in HLA-alloim-
munized patients.24
Cytapheresis
The goal of cytapheresis is to remove excessive
or pathogenic leukocytes, platelets, or red
cells. In addition, in red cell exchange, donor
red cells are used to restore oxygen-carrying
654 䡲 AABB TECHNICAL MANUAL
capacity. Indications for cytapheresis are listed
in Table 26-4.
In acute leukemia, high blast counts (typ-
ically >100,000/L) can result in microvascu-
lar stasis with headache, mental status chang-
es, visual disturbances, or dyspnea. The
leukocyte count at which a patient may be-
come symptomatic is variable. Typically, pa-
tients with acute or chronic lymphocytic leu-
kemia tolerate higher cell counts than patients
with myelogenous leukemia, and cytapheresis
may not be required. Cytapheresis commonly
results in a less-than-predicted reduction in
leukocyte count, despite excellent collection,
because of mobilization and re-equilibration
of cells from extravascular sites. Myelogenous
leukemia cells commonly have a higher densi-
ty than lymphocytic cells and can be difficult
to separate from red cells. Use of hydroxyethyl
starch enhances red cell sedimentation by
rouleaux formation and can improve the effi-
ciency of cytapheresis in acute myelogenous
leukemia. Massive thrombocytosis, typically
>1,000,000/L, can occur in essential throm-
bocythemia, polycythemia vera, or as a reac-
tive phenomenon. Such patients may be at risk
of thrombosis or hemorrhage. Reduction in
platelet count is commonly less than predicted
because of mobilization of platelets to the pe-
ripheral blood, primarily from the spleen.
Red cell exchange is most commonly per-
formed in the setting of sickle cell disease
(SCD). The goal is both to reduce the burden of
hemoglobin S and to provide donor red cells
containing hemoglobin A. Acute chest syn-
drome is a serious complication of SCD, pre-
senting as dyspnea, chest pain, and cough, of-
ten accompanied by fever, leukocytosis,
decreasing hematocrit, and pulmonary infil-
trates. Respiratory failure can develop, and
death occurs in about 3% of cases.25 Red cell
exchange is indicated for progressive infil-
trates and hypoxemia refractory to conven-
tional therapy and simple transfusion.26,27 A
common goal is to reduce hemoglobin S to
less than 30% with a final hematocrit not to ex-
ceed approximately 30%. Red cell exchange
may also be indicated for prevention of stroke
in sickle cell anemia. For patients with elevat-
ed cerebral blood flow velocity determined by
transcranial Doppler imaging, transfusion re-
duces the risk of stroke.28,29 Chronic red cell ex-
change, typically every 4 to 6 weeks, can be ef-
fective in normalizing cerebral blood flow
while minimizing iron overload associated
with simple transfusions. Red cell exchange
may have a role in other sickle cell syndromes,
including priapism, multiorgan failure, hepat-
ic/splenic sequestration, intrahepatic cho-
lestasis. In addition, the technique may be
used for prevention of iron overload, and pre-
vention or management of vaso-occlusive
pain crisis.
RBCs for replacement must be ABO com-
patible and negative for known clinically sig-
nificant alloantibodies. For sickle cell disease,
the RBCs should be matched for C, E, and K, if
possible.30 It is desirable to use relatively fresh
units to maximize posttransfusion red cell
survival. Units containing either citrate-phos-
phate-dextrose-adenine (CPDA)-1 or additive
solutions (AS) may be used. It is desirable that
all units used in a given procedure contain the
same anticoagulant solution so that they have
similar hematocrits. Chronic red cell ex-
change may carry a lower risk of alloimmuni-
zation than simple transfusion in sickle cell
disease.31
ECP
ECP is a specialized procedure in which the
buffy-coat layer is collected from peripheral
blood, treated with 8-methoxypsoralen and ul-
traviolet A light, and re-infused into the pa-
tient. The treatment causes cross-linking of
leukocyte DNA, which prevents replication
and induces apoptosis. The procedure was de-
veloped for the treatment of cutaneous T-cell
lymphoma, although it is increasingly used for
other indications (Table 26-5). ECP has com-
plex immunomodulatory effects, including in-
duction of monocyte differentiation to den-
dritic cells, alteration of T-cell subsets, and
changes in cytokine production profiles.32 ECP
may be effective in acute and chronic skin
graft-vs-host disease (GVHD), although the
role in non-skin GVHD is less well defined.33
ECP for solid organ transplant rejection
has been best studied in cardiac and lung
 CHAPTER 26 Therapeutic Apheresis 䡲 655

TABLE 26-4. Indications for Cytapheresis

Indication Modifying Conditions Procedure Category

Babesiosis Severe RCE I

High-risk population RCE II

Dermatomyositis or Leukocytapheresis IV
polymyositis

HSCT, ABO incompatible Minor HPC, Apheresis RCE III

Hereditary hemochroma- Erythrocytapheresis I

tosis

Hyperleukocytosis Leukostasis Leukocytapheresis I

Prophylaxis Leukocytapheresis III

Inclusion body myositis Leukocytapheresis IV

Inflammatory bowel Ulcerative colitis Adsorptive cytapheresis III/II

disease
Crohn’s disease Adsorptive cytapheresis III

Malaria Severe RCE II

Overdose Tacrolimus RCE III

Polycythemia vera and Polycythemia vera Erythrocytapheresis I

erythrocytosis
Secondary erythrocytosis Erythrocytapheresis III

Psoriasis Disseminated pustular Adsorptive cytapheresis III

Lymphocytapheresis III

Sickle cell disease, acute Acute stroke RCE I

Acute chest syndrome, severe RCE II

Priapism RCE III

Multi-organ failure RCE III

Splenic sequestration; hepatic seques- RCE III

tration; intrahepatic cholestasis

Sickle cell disease, Stroke prophylaxis/ iron overload RCE II

Non-acute prevention

Vaso-occlusive pain crisis RCE III

Preoperative management RCE III

Thrombocytosis Symptomatic Thrombocytapheresis II

Prophylactic or secondary Thrombocytapheresis III

RCE = red cell exchange; HSCT = hematopoietic stem cell transplantation; HPC = hematopoietic progenitor cells.
656 䡲 AABB TECHNICAL MANUAL

TABLE 26-5. Indications for Photopheresis

Indication Modifying Conditions Category

Cardiac transplantation Rejection prophylaxis II

Cellular or recurrent rejection II
Cutaneous T-cell lymphoma; mycosis Erythrodermic I
fungoides; Sézary syndrome
Nonerythrodermic III
Graft-vs-host disease Skin (chronic) II
Skin (acute) II
Non-skin (acute/chronic) III
Inflammatory bowel disease Crohn's disease III
Lung allograft rejection Bronchiolitis obliterans syndrome II
Nephrogenic sytemic fibrosis III
Pemphigus vulgaris Severe III
Psoriasis III
Scleroderma (Progressive systemic III
sclerosis)

transplantation. In a randomized clinical trial,
prophylactic photopheresis in conjunction
with previous generation immunosuppres-
sion (not including calcinurin inhibitors or
mycophenolate) resulted in fewer rejection
episodes, decreased HLA antibodies, and re-
duced coronary artery intimal thickness, but
no difference in time to first episode, inci-
dence of hemodynamic compromise, or sur-
vival at 6 or 12 months.34,35 In recurrent cardiac
rejection, photopheresis may decrease severi-
ty of rejection and allow for reduced dosage of
immunosuppressives.36 ECP may have a role in
the setting of cardiac rejection with hemody-
namic compromise and for bronchiolitis oblit-
erans syndrome status after lung transplanta-
tion.37,38
Selective Adsorption
There are currently few established indica-
tions for selective adsorption of plasma pro-
teins (Table 26-6) and few devices have been
approved by the Food and Drug Administra-
tion (FDA).
In the two FDA-approved LDL apheresis
devices, selective removal of LDL can be ac-
complished by passing heparinized plasma
over a dextran sulfate column or beads coated
with anionic polyacrylate ligands, or by pre-
cipitation of heparin-LDL complexes in acidi-
fied plasma. Because LDL production contin-
ues, LDL apheresis treatments must be
repeated, typically at 2-week intervals, indefi-
nitely. There is evidence that LDL apheresis re-
duces the incidence of major coronary events
and stroke.39 In addition, atherosclerotic
plaque regression can occur in some pa-
tients.40 Secondary effects of LDL apheresis
that may be beneficial include reduction in
levels of C-reactive protein, fibrinogen, tissue
factor, and soluble adhesion molecules.41,42
LDL apheresis may also be used in the treat-
ment of primary or recurrent FSGS, although
the mechanism of action has not been well de-
fined.43
 CHAPTER 26 Therapeutic Apheresis 䡲 657

TABLE 26-6. Indications for Selective Adsorption

Treatment
Indication Modifying Conditions Modality Category

Age-related macular degeneration, dry Rheopheresis I

Chronic focal encephalitis (Rasmussen IA III
encephalitis)
Coagulation factor inhibitors Alloantibody IA III
Autoantibody IA III
Cryoglobulinemia Symptomatic/severe IA II
Dilated cardiomyopathy, idiopathic NYHA II-IV IA II
Familial hypercholesterolemia Homozygotes LDL apheresis I
Heterozygotes LDL apheresis II
Focal segmental glomerulosclerosis Primary, treatment resistant LDL apheresis *
Recurrent after renal trans- LDL apheresis
plantation
Immune thrombocytopenia Refractory IA III
Liprotein (a) hyperlipoproteinemia LDL apheresis II
Multiple sclerosis Acute CNS inflammatory IA III
demyelinating disease
Paraneoplastic neurologic syndromes IA III
Paraproteinemic demyelinating IgG/IgA/IgM IA III
polyneuropathies
Pemphigus vulgaris Severe IA III
Peripheral vascular diseases LDL apheresis III
Phytanic acid storage disease LDL apheresis II
(Refsum disease)
Sudden sensorineural hearing loss LDL apheresis III
Rheopheresis III
*FDA approved but ASFA category not yet assigned.
IA = immunoadsorption; LDL = low-density lipoprotein; NYHA = New York Heart Association class; CNS = central nervous
system.

IgG can be selectively removed by passing
plasma over a column of staphylococcal pro-
tein A bound to silica. The putative mecha-
nism of action is the removal of pathogenic au-
toantibodies or immune complexes, although
an alternative mechanism has been proposed
in ITP.44 Staphylococcal protein A adsorption
treatment can be performed manually or in
conjunction with automated TPE. Affinity col-
umns containing anti-IgG or ABO blood group
658 䡲 AABB TECHNICAL MANUAL

substances have been tested in clinical trials, TABLE 26-7. Reported Frequency of Adverse
but are not currently approved for use in the Reactions to Apheresis*
United States.
Reaction Frequency (%)

ANTICOAGUL ATION Paresthesia 1.30

Acid-citrate-dextrose solution A (ACD-A), is Hypotension 0.91

the most commonly used anticoagulant, al-
though heparin in combination with ACD-A is
also used, particularly in the setting of large-
volume leukapheresis for hematopoietic pro-
genitor cell collection. Current automated
apheresis devices control the administration
rate of citrate to achieve anticoagulation while
minimizing the risk for hypocalcemia. Hepa-
rin anticoagulation is necessary for LDL
apheresis and may be desirable for selected
patients undergoing TPE who are particularly
susceptible to hypocalcemia, such as small
children, or in the setting of severe metabolic
alkalosis or renal failure. With citrate anticoag-
ulation, coagulation monitoring is not gener-
ally necessary, although monitoring of ionized
calcium may be helpful for selected patients.
The infused citrate in the returned blood is
rapidly metabolized and rarely causes system-
ic anticoagulation.
A DV ER SE E F F E C TS
Although therapeutic apheresis is very safe,
complications do occur. An adverse event oc-
curs in about 4% of procedures (Table 26-7),
but the majority are mild.45,46 Symptomatic hy-
pocalcemia from infusion of citrate with the
returned blood is the most common adverse
effect of apheresis. Perioral and digital pares-
thesias are the most common symptoms. Nau-
sea may also occur. Tetany is rare. Cardiac ar-
rhythmia is very rare, but patients with
preexisting hypocalcemia or significant pro-
longation of the QT interval should be moni-
tored carefully. Calcium supplementation may
alleviate the symptoms of citrate toxicity. A
typical supplementary dose is 10 mL of 10%
calcium gluconate per liter of albumin in-
fused. Citrate also chelates ionized magne-
sium, so it is possible that hypomagnesemia
contributes to the symptoms of citrate toxicity.
Urticaria 0.63
Nausea 0.39
Shivering 0.29
Flushing 0.16
Dyspnea 0.15
Vertigo 0.17
Arrhythmia 0.11
Abdominal pain 0.12
Anaphylaxis 0.02
Total 4.25
*Adapted from Matsuzaki.40
However, one randomized clinical trial
showed no benefit to adding magnesium dur-
ing leukapheresis with continuous IV calcium
supplementation.47 Metabolism of citrate
leads to a mild metabolic alkalosis, which can
exacerbate hypocalcemia, and may cause hy-
pokalemia.48
Allergic reactions are most common with
plasma replacement, although they may oc-
cur with albumin as well. Most reactions are
mild, characterized by urticaria or cutaneous
flushing. More severe reactions can involve the
airways with dyspnea, wheezing, and (rarely)
stridor. Most allergic reactions respond quick-
ly to intravenous diphenhydramine. Anaphy-
laxis is very rare but can occur. Patients with
TTP who receive large volumes of plasma are
most at risk of allergic reactions. Premedica-
tion with an antihistamine, or possibly ste-
roids, is not necessary for routine apheresis
but may be indicated for patients with repeat-
ed or previous severe reactions.
Respiratory difficulty during or immedi-
ately following apheresis can have many
 CHAPTER 26
causes, such as pulmonary edema, pulmo-
nary embolism, air embolism, obstruction of
the pulmonary microvasculature, anaphylac-
tic reactions, and transfusion-related acute
lung injury (TRALI).49 Hemothorax or hemo-
pericardium resulting from vascular erosion
due to a central venous catheter is rare, but
when it occurs it is typically unsuspected, and
may be fatal.50 Pulmonary edema that results
from volume overload or cardiac failure is usu-
ally associated with dyspnea, an increase in di-
astolic blood pressure, and characteristic chest
radiograph findings. Acute pulmonary edema
may also arise from damage to alveolar capil-
lary membranes secondary to an immune re-
action or to vasoactive substances in plasma
or colloid solutions prepared from human
plasma. Predominantly ocular reactions (peri-
orbital edema, conjunctival swelling, and tear-
ing) have occurred in donors sensitized to the
ethylene oxide gas used to sterilize disposable
plastic apheresis kits. 51,52
Hypotension during apheresis can be a
sign of citrate toxicity; hypovolemia; or a vaso-
vagal, allergic, drug, or transfusion reaction.
Hypovolemia can occur early in a treatment of
a small patient when the return fluid consists
of the saline used to prime the apheresis cir-
cuit. Vasovagal reactions are characterized by
bradycardia and hypotension. Such reactions
usually respond well to a fluid bolus and plac-
ing the patient in the Trendelenburg position.
When hypotension occurs during plasma
or red cell exchange, a transfusion reaction
such as TRALI, acute hemolysis, bacterial con-
tamination, or anti-IgA-related anaphylaxis
should be considered. Hypotension is more
frequent in children, the elderly, neurology pa-
tients, anemic patients, and those treated with
intermittent-flow devices that have large ex-
tracorporeal volumes. Continuous-flow devic-
es typically do not have large extracorporeal
volumes but can produce hypovolemia if re-
turn flow is inadvertently diverted to a waste
collection bag, either through operator over-
sight or device malfunction. Hypovolemia may
also be secondary to inadequate volume or
protein replacement. During all procedures, it
is essential to maintain careful and continuous
records of the volumes removed and returned.
Therapeutic Apheresis 䡲 659
When plasma is exposed to foreign sur-
faces of plastic tubing or filtration devices, the
kinin system can be activated, resulting in pro-
duction of bradykinin. Infusion of plasma con-
taining bradykinin can cause abrupt hypoten-
sion. Patients taking angiotensin-converting
enzyme (ACE) inhibitors are more susceptible
to the hypotensive reactions because the
drugs block enzymatic degradation of bradyki-
nin.53 Hypotensive reactions are more likely
during selective adsorption procedures be-
cause the devices expose plasma to a very
large surface area. Because some ACE inhibi-
tors have a long duration of action, stopping
the drug only the day before the procedure
may not be sufficient to prevent a reaction.
Intensive TPE without plasma replace-
ment causes depletion of coagulation factors.
A 1.0 plasma volume exchange will typically
reduce coagulation factor levels by 25% to
50%, though Factor VIII levels are less affect-
ed.54 Levels of fibrinogen, a large molecule
without extravascular distribution, are re-
duced by about 66%. If the patient has normal
hepatic synthetic function, coagulation factor
levels typically return to near normal within 2
days. Thus, many patients can tolerate TPE ev-
ery other day for 1 or 2 weeks without develop-
ing significant coagulopathies that require
plasma replacement.
Bleeding as a consequence of coagula-
tion factor depletion is rare but has been re-
ported. For patients at risk, plasma may be
used for replacement at the end of the proce-
dure. Apheresis can also cause thrombocyto-
penia, particularly with HPC collection. Inten-
sive TPE can cause hypogammaglobulinemia.
Serum levels of IgG and IgM recover to about
40% to 50% of the preapheresis level at 48
hours.54 The absolute immunoglobulin level at
which a patient becomes at risk of infections
has not been established.
Albumin-bound drugs are removed by
TPE. This can result in subtherapeutic levels of
medications unless dosage adjustments are
made. High-molecular-weight biologicals
such as IVIG, antithymocyte globulin, and
monoclonal antibodies have a long intravas-
cular half-life and are readily removed by
apheresis. TPE shortly after administration of
660 䡲 AABB TECHNICAL MANUAL
such drugs should be avoided because it may
significantly impair their effectiveness. In ad-
dition, intensive apheresis reduces the con-
centration of potentially diagnostic plasma
constituents, so blood for testing should be
drawn before initiating a course of treatment.
Collapsed or kinked tubing, malfunction-
ing pinch valves, or improper threading of tub-
ing may damage red cells in the extracorporeal
circuit. Instrument-related hemolysis has
been reported in 0.06% of therapeutic aphere-
sis procedures.45 Hemolysis can also occur
with the use of incompatible replacement flu-
ids such as 5% dextrose solution (eg, D5W
used to dilute 25% albumin) or ABO-incom-
patible plasma. The operator should carefully
observe plasma collection lines for pink dis-
coloration suggestive of hemolysis. Other
types of equipment failure such as problems
with the rotating seal, leaks in the plastic, and
roller pump failure are rare.
Fatalities during apheresis are rare. Death
has been reported in 0.006% to 0.09% of thera-
peutic procedures.45,55 Most fatalities are at-
tributable to underlying medical conditions.
VAS C U L A R AC C E S S
Therapeutic apheresis requires good vascular
access to achieve adequate flow rates. Periph-
eral access generally requires at least a 17-
gauge needle for blood withdrawal and at least
an 18-gauge catheter for return. Patients with-
out adequate peripheral veins or who require
multiple frequent procedures may require
central venous catheters. Central venous cath-
eters for apheresis must have rigid walls to ac-
commodate the negative pressure generated
in the withdrawal line. Peripherally inserted
central catheters (PICC lines) typically do not
support the high flow rates used in apheresis
and should not be used. At least two lumens
are required for continuous-flow procedures,
although single-lumen catheters can be used
for intermittent-flow procedures. An implant-
ed subcutaneous port may be an option for
some patients requiring long-term treatment,
such as chronic red cell exchange.
The choice of placement site for a central
catheter is influenced by the expected dura-
tion of treatment. Subclavian or internal jugu-
lar access is generally preferable for treat-
ments lasting up to several weeks. Femoral
access should be used only temporarily be-
cause of the higher risk of infection. Patients
requiring long-term treatment usually have a
tunneled catheter. With proper care, tunneled
catheters can be used for prolonged periods.
Good catheter care is very important to
maintain patency and prevent complications.
Catheters need to be flushed regularly. Hepa-
rin or 4% trisodium citrate is usually placed in
each lumen after each use to prevent occlu-
sion by clots. If a port becomes clotted, instil-
lation of a fibrinolytic agent such as urokinase
or recombinant tissue plasminogen activator
may restore patency. Routine dressing care is
essential to prevent insertion site infections.
An arteriovenous (AV) fistula may also be used
for apheresis, but apheresis personnel should
be suitably trained before attempting to access
an AV fistula.
Venous access devices may cause further
vascular damage, sometimes resulting in
thrombosis. Infrequently, they may result in
severe complications such as pneumothorax
or perforation of the heart or great vessels.
Other complications include arterial puncture,
deep hematomas, and arteriovenous fistula
formation. Bacterial colonization often com-
plicates long-term placement and may lead to
catheter-associated sepsis, especially in pa-
tients who are receiving immunosuppressants.
Inadvertent disconnection of catheters may
produce hemorrhage or air embolism.
PAT IE N T EVA L U A T IO N
All patients should be evaluated by a physician
familiar with apheresis before treatment be-
gins. The indication, type of procedure, fre-
quency and number of treatments, and the
goal or endpoint should be documented in the
patient’s medical record. The nature of the
procedure, the expected benefits, the possible
risks, and the available alternatives must be
explained to the patient, and the patient’s con-
 CHAPTER 26 Therapeutic Apheresis 䡲 661

sent must be documented. The procedure cluding hypocalcemia, hypokalemia, and

should be performed only in a setting where
there is ready access to care for untoward reac-
tions, including equipment, medications, and
personnel trained in managing serious ad-
verse events such as anaphylaxis.
The assessment should include evalua-
tion of the indication, medical conditions that
may affect the patient’s ability to tolerate
apheresis, vascular access, and medications.
Some points to consider include the following:
䡲 Transfusion history: History of transfusion
reactions and special blood component re-
quirements.
䡲 Neurologic status: Mental status and the
ability to consent and cooperate.
䡲 Cardiorespiratory status: Adequate ventila-
tion and oxygenation, hyper- or hypovole-
mia, and cardiac arrhythmia.
䡲 Renal and metabolic status: Fluid balance,
alkalosis, and electrolyte abnormalities, in-
hypomagnesemia.
䡲 Hematologic status: Significant anemia or
thrombocytopenia, coagulopathy, bleed-
ing, or thrombosis.
䡲 Medications: Drugs with high albumin
binding, immunoglobulins, and biologics.
Appropriate laboratory monitoring is dic-
tated by the indication, type and frequency of
procedures, and concomitant medical condi-
tions. In general, it is wise to obtain a complete
blood cell count, type and screen, and electro-
lytes assessment before starting treatment. If
at all possible, diagnostic studies such as tests
for infectious disease, pregnancy, ADAMTS-13
activity, glomerular basement membrane anti-
body, or acetylcholine receptor antibody
should be completed before the first treat-
ment, particularly if plasma is used for re-
placement. Coagulation monitoring may be
appropriate when albumin is used for replace-
ment during frequently repeated procedures.

KEY PO I NT S

1. Therapeutic apheresis treats disease by removal or extracorporeal manipulation of patho-

logic plasma substances, white cells, platelets, or red cells, and may be accomplished by
continuous centrifugation, filtration, selective adsorption, or photopheresis.
2. The American Society for Apheresis (ASFA) has published guidelines and recommendations
for the use of therapeutic apheresis in clinical practice.
3. Anticoagulation is usually accomplished with citrate, although heparin may be used, partic-
ularly for selective adsorption, hematopoietic progenitor cell collection, and photopheresis.
4. Albumin is the most commonly used replacement fluid for therapeutic plasma exchange,
but plasma may be indicated for patients with TTP or coagulopathy.
5. Adverse effects of apheresis are usually mild but may include symptomatic hypocalcemia,
hypotension, urticaria, and nausea. Complications of apheresis can include coagulopathy,
hypogammaglobulinemia, and removal of certain drugs and biologicals.
6. Vascular access for apheresis may be accomplished through peripheral veins, but a central
venous catheter or an AV fistula are required for some patients.
7. Medical evaluation of the apheresis patient should focus on the indication, type of proce-
dure, frequency and number of treatments, therapeutic goal, ability of the patient to toler-
ate apheresis, vascular access, and medications. Appropriate laboratory monitoring is dic-
tated by the indication, type and frequency of procedures, and concomitant medical
conditions.
662 䡲 AABB TECHNICAL MANUAL
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pheresis: A focus on apoptosis and cytokines.
J Dermatol Sci 2006;43:85-94.
33. Del Fante C, Scudeller L, Viarengo G, et al. Re-
sponse and survival of patients with chronic
graft-versus-host disease treated by extracor-
poreal photochemotherapy: A retrospective
study according to classical and National Insti-
tutes of Health classifications. Transfusion
2012;52:2007-15.
34. Barr ML, Baker CJ, Schenkel FA, et al. Prophy-
lactic photopheresis and chronic rejection: Ef-
fects on graft intimal hyperplasia in cardiac
transplantation. Clin Transplant 2000;14:162-
6.
35. Barr ML, Meiser BM, Eisen HJ, et al. Photo-
pheresis for the prevention of rejection in car-
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diac transplantation. Photopheresis Trans-
plantation Study Group. N Engl J Med 1998;
339:1744-51.
36. Dall’Amico R, Montini G, Murer L, et al. Extra-
corporeal photochemotherapy after cardiac
transplantation: A new therapeutic approach
to allograft rejection. Int J Artif Organs 2000;
23:49-51.
37. Kirklin JK, Brown RN, Huang ST, et al. Rejec-
tion with hemodynamic compromise: Objec-
tive evidence for efficacy of photopheresis.
J Heart Lung Transplant 2006;25:283-8.
38. Jaksch P, Scheed A, Keplinger M, et al. A pro-
spective interventional study on the use of ex-
tracorporeal photopheresis in patients with
bronchiolitis obliterans syndrome after lung
transplantation. J Heart Lung Transplant 2012;
31:950-7.
39. Masaki N, Tatami R, Kumamoto T, et al. Ten-
year follow-up of familial hypercholesterol-
emia patients after intensive cholesterol-low-
ering therapy. Int Heart J 2005;46:833-43.
40. Matsuzaki M, Hiramori K, Imaizumi T, et al.
Intravascular ultrasound evaluation of coro-
nary plaque regression by low density lipopro-
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Coronary Morphology and Reserve Trial (LAC-
MART). J Am Coll Cardiol 2002;40:220-7.
41. Wang Y, Blessing F, Walli AK, et al. Effects of
heparin-mediated extracorporeal low-density
lipoprotein precipitation beyond lowering
proatherogenic lipoproteins—reduction of
circulating proinflammatory and procoagula-
tory markers. Atherosclerosis 2004;175:145-50.
42. Kobayashi S, Oka M, Moriya H, et al. LDL-
apheresis reduces P-Selectin, CRP and fibrino-
gen—possible important implications for im-
proving atherosclerosis. Ther Apher Dial 2006;
10:219-23.
43. Muso E, Mune M, Yorioka N, et al. Beneficial
effect of low-density lipoprotein apheresis
(LDL-A) on refractory nephrotic syndrome
(NS) due to focal glomerulosclerosis (FGS).
Clin Nephrol 2007;67:341-4.
44. Silverman GJ, Goodyear CS, Siegel DL. On the
mechanism of staphylococcal protein A im-
munomodulation. Transfusion 2005;45:274-
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45. McLeod BC, Sniecinski I, Ciavarella D, et al.
Frequency of immediate adverse effects asso-
ciated with therapeutic apheresis. Transfusion
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664 䡲 AABB TECHNICAL MANUAL
46. Norda R, Berseus O, Stegmayr B. Adverse
events and problems in therapeutic hema-
pheresis. A report from the Swedish registry.
Transfus Apher Sci 2001;25:33-41.
47. Haddad S, Leitman SF, Wesley RA, et al. Place-
bo-controlled study of intravenous magne-
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leukapheresis in healthy allogeneic donors.
Transfusion 2005;45:934-44.
48. Marques MB, Huang ST. Patients with throm-
botic thrombocytopenic purpura commonly
develop metabolic alkalosis during therapeu-
tic plasma exchange. J Clin Apher 2001;16:120-
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49. Askari S, Nollet K, Debol SM, et al. Transfu-
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50. Duntley P, Siever J, Korwes ML, et al. Vascular
erosion by central venous catheters. Clinical
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51. Leitman SF, Boltansky H, Alter HJ, et al. Aller-
gic reactions in healthy plateletpheresis do-
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52. Purello D’Ambrosio F, Savica V, Gangemi S, et
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Nephrol Dial Transplant 1997;12:1461-3.
53. Owen HG, Brecher ME. Atypical reactions as-
sociated with use of angiotensin-converting
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54. Orlin JB, Berkman EM. Partial plasma ex-
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55. Kiprov DD, Golden P, Rohe R, et al. Adverse re-
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 C h a p t e r 2 7

Noninfectious Complications of
Blood Transfusion

Catherine A. Mazzei, MD; Mark A. Popovsky, MD; and

Patricia M. Kopko, MD

S T A T I S T I C A L L Y, T H E G R E A T E S T sion practices. One of the main purposes of

risk of morbidity and mortality from
transfusion is from a transfusion reaction or
the noninfectious complications of blood
transfusion. In fact, transfusion-related acute
lung injury (TRALI), hemolytic transfusion re-
actions (HTRs), and transfusion-associated
circulatory overload (TACO) are the three most
commonly reported causes of transfusion-
related mortality.1 It is these noninfectious
complications that are addressed in this chap-
ter.
HE MOVI GI L AN CE
Hemovigilance may be defined as the collec-
tion of information on the complications of
transfusion, analysis of these data, and subse-
quent data-driven improvements in transfu-
developing a hemovigilance program is to im-
prove reporting of transfusion-related adverse
events. It is widely believed that the major
noninfectious complications of transfusion
are both underrecognized and underreported.
The US Biovigilance Network is a nation-
al collaboration between government and
nongovernment organizations. The network
develops and enhances surveillance systems
designed to track adverse reactions and inci-
dents associated with blood collection; blood
transfusion; and the transplantation of cells,
tissues, and organs. Transfusion safety was the
first issue that the network addressed.
Definitions and classification schemes
are outlined in detail in the appendices of the
National Healthcare Safety Network Biovigi-
lance Component protocol with the goal of

Catherine A. Mazzei, MD, Medical Director, American Red Cross, Northern California Blood Services Region,
Oakland, California; Mark A. Popovsky, MD, Associate Clinical Professor, Harvard Medical School, Boston,
Massachusetts, and Vice President and Chief Medical Officer, Haemonetics Corporation, Braintree, Massa-
chusetts; and Patricia M. Kopko, MD, Clinical Professor of Pathology, University of California, San Diego, Cali-
fornia
C. Mazzei and P. Kopko have disclosed no conflicts of interest. M. Popovsky has disclosed a financial relation-
ship with Haemonetics Corporation.

665
666 䡲 AABB TECHNICAL MANUAL
improving the quality of national surveillance
data.2 The Centers for Disease Control and
Prevention intends to publish the results of
system participation to allow benchmarks and
best practices to be established.
REC O G N IT I O N A N D
EVALUATION OF A SUSPECTED
TRANSF USION RE ACTION
Identification of a Transfusion Reaction
As with many necessary medical therapies, ad-
verse effects cannot always be accurately pre-
dicted or avoided. The transfusing physician
should be aware of such risks when discussing
the need for transfusion with a patient. In-
formed consent for transfusion may include a
discussion of the risks of infectious disease
and serious noninfectious complications, such
as TRALI, and HTRs.3 Furthermore, medical
staff administering blood components should
be well aware of the signs and symptoms of
possible reactions. These staff should be pre-
pared to mitigate the current episode and pre-
vent future similar reactions when possible.
Many common clinical signs and symp-
toms are associated with more than one type
of adverse reaction (see Table 27-1). Early rec-
ognition, prompt cessation of the transfusion,
and further evaluation are key to a successful
outcome. The signs and symptoms that may
be indicators of a transfusion reaction include
the following:
䡲 Fever, generally defined as a 1 C rise in
temperature above 37 C [the most common
sign of an acute HTR (AHTR)].4(p907)
䡲 Chills with or without rigors.
䡲 Respiratory distress, including wheezing,
coughing, and dyspnea.
䡲 Hyper- or hypotension.
䡲 Abdominal, chest, flank, or back pain.
䡲 Pain at the infusion site.
䡲 Skin manifestations, including urticaria,
rash, flushing, pruritus, and localized edema.
䡲 Jaundice or hemoglobinuria.
䡲 Nausea/vomiting.
䡲 Abnormal bleeding.
䡲 Oliguria/anuria.
Clinical Evaluation and Management
of a Transfusion Reaction
The evaluation of a suspected transfusion re-
action involves a two-pronged investigation
combining clinical evaluation of the patient
with laboratory verification and testing. The
nurse or transfusionist must treat the patient
by administering supportive care at the direc-
tion of the physician, discontinue the transfu-
sion of the implicated component, and con-
tact the blood bank for directions on the
investigation. When an AHTR is suspected,
several steps must be taken immediately.
Patient-focused steps are as follows:
1. Stop the transfusion immediately but keep
the line open with saline.
2. Document the clerical recheck between the
patient and the component. The labels on
the component, patient records, and
patient identification should be examined
to detect any identification errors. Trans-
fusing facilities may require repeat ABO
and Rh typing of the patient using a new
sample.5(p88) (See “Standard Laboratory
Investigation of a Transfusion Reaction”
section below.)
3. Contact the treating physician immediately
for instructions on patient care.
Component-focused steps are as follows:
1. Contact the transfusion service for direc-
tions on investigating the cause of the
AHTR. Most transfusion services use a stan-
dardized form to document all of the infor-
mation available on both the patient and
the component.
2. Obtain instructions concerning the return
of any remaining component, associated
intravenous fluid bags, and tubing.
3. Identify appropriate samples (blood and
urine) to send to the laboratory.
The transfusion service determines
whether the blood donor center should be no-
tified of the AHTR.
TABLE 27-1. Categories of Adverse Transfusion Reactions and Their Management*

Therapeutic/Prophylactic
Type Incidence Etiology Presentation Diagnostic Testing Approach

Acute (<24 hours) Transfusion Reactions—Immunologic

Hemolytic ABO Rh mismatch: Red cell Chills, fever, hemoglobinuria, Clerical check Keep urine output >1 mL/kg/hr
1 in 40,000 incompatibility hypotension, renal failure with DAT with fluids and IV diuretic
AHTR: 1 in 76,000 oliguria, DIC (oozing from IV Visual inspection (free Hb) (furosemide)
CHAPTER 27

Fatal HTR: sites), back pain, pain along Repeat patient ABO, pre- and Analgesics (may need mor-
1 in 1.8 million infusion vein, anxiety posttransfusion sample phine)
Further tests as indicated to Pressors for hypotension
define possible incompatibil- (low-dose dopamine)
ity Hemostatic components
Further tests as indicated to (platelets, cryoprecipitate, or
detect hemolysis (LDH, FFP) for bleeding
bilirubin, etc)
Febrile, nonhemo- 0.1 to 1% with uni- Accumulated cyto- Fever, chills/rigors, head- Rule out hemolysis (DAT, Leukocyte-reduced blood
lytic versal leukoreduction kines in platelet unit ache, vomiting inspect for hemoglobinemia, Antipyretic premedication
Antibody to donor repeat patient ABO) (acetaminophen, no aspirin)
WBCs Rule out bacterial
contamination
WBC antibody screen†
Urticarial 1:100-1:33 Antibody to donor Urticaria, pruritis, flushing Rule out hemolysis (DAT, Antihistamine, treatment or
(1%-3%) plasma proteins inspect for hemoglobinemia, premedication (PO or IV)
Noninfectious Complications of Blood Transfusion

repeat patient ABO) May restart unit slowly after

antihistamine if symptoms
resolve
䡲

(Continued)
667
TABLE 27-1. Categories of Adverse Transfusion Reactions and Their Management* (Continued)
668

Therapeutic/Prophylactic
Type Incidence Etiology Presentation Diagnostic Testing Approach
䡲

Anaphylactic 1:20,000-1:50,000 Antibody to donor Hypotension, urticaria, bron- Rule out hemolysis (DAT, Trendelenburg (feet-up) posi-
plasma proteins chospasm (respiratory dis- inspect for hemoglobinemia, tion
(includes IgA, tress, wheezing), local edema, repeat patient ABO) Fluids
haptoglobin, C4) anxiety Anti-IgA Epinephrine (adult dose: 0.2-
Cytokines IgA, quantitative 0.5 mL of 1:1000 solution SC or
IM; in severe cases, 1:10,000
IV, initial rate 1mcg/minute)
Antihistamines, corticosteroids,
beta-2 agonists
IgA-deficient blood components
TRALI 1:1,200-1:190,000 WBC antibodies in Hypoxemia, respiratory fail- Rule out hemolysis (DAT, Supportive care until recovery
donor (occasionally ure, hypotension, fever, inspect for hemoglobinemia, Deferral of implicated donors
in recipient), other bilateral pulmonary edema repeat patient ABO)
WBC-activating Rule out cardiogenic
AABB TECHNICAL MANUAL

agents in compo- pulmonary edema

nents WBC antibody screen in
donor and recipient. If
positive, antigen typing may
be indicated
WBC crossmatch
Chest X-ray
Acute (<24 hours) Transfusion Reactions—Nonimmunologic
Transfusion- Varies by component Bacterial contamina- Fever, chills, hypotension Gram’s stain Broad spectrum antibiotics
associated (see Infectious Dis- tion Culture of component (until sensitivities completed)
sepsis ease Screening, Patient culture Treat complications (eg, shock)
Chapter 8) Rule out hemolysis (DAT,
inspect for hemoglobinemia,
repeat patient ABO)
CHAPTER 27

Hypotension asso- Dependent on clinical Inhibited metabolism Flushing, hypotension Rule out hemolysis (DAT, Withdraw ACE inhibition
ciated with ACE setting of bradykinin with inspect for hemoglobinemia, Avoid albumin volume replace-
inhibition infusion of brady- repeat patient ABO) ment for plasmapheresis
kinin (negatively Avoid bedside leukocyte filtra-
charged filters) or tion
activators of
prekallikrein
Circulatory over- <1% Volume overload Dyspnea, orthopnea, cough, Chest X-ray Upright posture
load tachycardia, hypertension, Rule out TRALI Oxygen
headache IV diuretic (furosemide)
Phlebotomy (250-mL incre-
ments)
Nonimmune hemo- Rare Physical or chemical Hemoglobinuria, hemoglobi- Rule out patient hemolysis Identify and eliminate cause
lysis destruction of blood nemia (DAT, inspect for hemoglobi-
(heating, freezing, nemia, repeat patient ABO)
hemolytic drug or Test unit for hemolysis
Noninfectious Complications of Blood Transfusion

solution added to
blood)
Air embolus Rare Air infusion via line Sudden shortness of breath, X-ray for intravascular air Place patient on left side with
䡲

acute cyanosis, pain, cough, legs elevated above chest and

hypotension, cardiac arrhyth- head
mia
(Continued)
669
TABLE 27-1. Categories of Adverse Transfusion Reactions and Their Management* (Continued)
670

Therapeutic/Prophylactic
Type Incidence Etiology Presentation Diagnostic Testing Approach
䡲

Hypocalcemia (ion- Dependent on clinical Rapid citrate infusion Paresthesia, tetany, Ionized calcium PO calcium supplement for mild
ized calcium; citrate setting (massive transfusion arrhythmia Prolonged Q-T interval on symptoms during therapeutic
toxicity) of citrated blood, electrocardiogram apheresis procedures
delayed metabolism Slow calcium infusion while
of citrate, apheresis monitoring ionized calcium lev-
procedures) els in severe cases
Hypothermia Dependent on clinical Rapid infusion of cold Cardiac arrhythmia Central body temperature Employ blood warmer
setting blood
Delayed (>24 hours) Transfusion Reactions—Immunologic
Alloimmuni- 1:100 (1%) Immune response to Positive blood group Antibody screen Avoid unnecessary transfusions
zation, red cell foreign antigens on antibody screening test DAT Leukocyte-reduced blood
antigens RBCs
Alloimmuni- 1:10 (10%) WBCs and Platelet refractoriness, Platelet antibody screen Avoid unnecessary transfusions
AABB TECHNICAL MANUAL

zation, HLA anti- platelets (HLA) delayed hemolytic reaction, HLA antibody screen Leukocyte-reduced blood
gens hemolytic disease of the
newborn
Hemolytic 1:2500-11,000 Anamnestic immune Fever, decreasing Antibody screen Identify antibody
response to red cell hemoglobin, new DAT Transfuse compatible RBCs as
antigens positive antibody screening Tests for hemolysis (visual needed
test, mild jaundice inspection for hemoglobine-
mia, LDH, bilirubin, urinary
hemosiderin as clinically
indicated)
Graft-vs-host Rare Donor lymphocytes Erythroderma, maculopapu- Skin biopsy Corticosteroids, cytotoxic
disease engraft in recipient lar rash, anorexia, nausea, HLA typing agents
and mount attack on vomiting, diarrhea, hepatitis, Molecular analysis for Irradiation of blood components
host tissues pancytopenia, fever chimerism for patients at risk (including
components from related
donors and HLA-selected com-
ponents)
CHAPTER 27

Posttransfusion Rare Recipient platelet Thrombocytopenic Platelet antibody screen and IVIG
purpura antibodies (apparent purpura, bleeding 8-10 days identification HPA-1a-negative platelets
alloantibody, usually after transfusion Plasmapheresis
anti-HPA-1a) destroy
autologous platelets
Delayed (>24 hours) Transfusion Reactions—Nonimmunologic
Iron overload Typically after >100 Multiple transfusions Diabetes, cirrhosis, cardiomy- Serum ferritin Iron chelators
RBC units with obligate iron opathy Liver enzymes
load in transfusion- Endocrine function tests
dependent patient
*For platelet refractoriness, see chapter on platelet and granulocyte antigens and antibodies; for septic transfusion reactions, see chapter on transfusion-transmitted diseases. For a recent
summary of transfusion reactions, see Popovsky.4
†
Blood group antibody screening test.
AHTR = acute hemolytic transfusion reaction; HTR = hemolytic transfusion reaction; DIC = disseminated intravascular coagulation; DAT = direct antiglobulin test; IV = intravenous; Hb =
hemoglobin; LDH = lactate dehydrogenase; CRYO = cryoprecipitated antihemophilic factor; FFP = fresh frozen plasma; WBC = white blood cell; PO = by mouth; SC = subcutaneous; IM =
intramuscular; IgA = immunoglobulin A; ACE = angiotensin-converting enzyme; TRALI = transfusion-related acute lung injury; RBC = Red Blood Cell; IVIG = intravenous immunoglobulin;
HPA = human platelet antigen.
Noninfectious Complications of Blood Transfusion
䡲
671
672 䡲 AABB TECHNICAL MANUAL
Standard Laboratory Investigation of a
Transfusion Reaction
When the laboratory receives notice of a possi-
ble transfusion reaction, several steps are per-
formed by technologists:
1. Clerical check of the component bag, label,
paper work, and patient sample.
2. Repeat ABO testing on the posttransfusion
sample.
3. Visual check of pre- and posttransfusion
samples to look for evidence of hemolysis
(which may not be visible if <50 mg/dL of
hemoglobin is present).
4. Direct antiglobulin test (DAT) on a post-
transfusion sample.
5. Report of findings to the blood bank super-
visor or medical director, who may request
additional studies, tests, or quarantine of
cocomponents generated from the same
donor collection.
The transfusion service must retain any
patient records that are related to transfusion
reactions, clinically significant antibodies, or
special transfusion requirements.5(pp74-77) Addi-
tional information regarding special products,
such as irradiated or washed components re-
quired for a particular patient, may be re-
tained by the transfusion service as well.
In an increasingly mobile society, patients
often present for treatment at a facility that
has no ready access to the patient’s medical re-
cords. When the patient is able to give a thor-
ough history, transfusion services are able to
share medical information in a timely manner.
When this is not possible, medical warning
bracelets or wallet identification cards may
benefit patients with multiple alloantibodies
whose strength may decrease over time.
Specialized Laboratory Investigations
for Selected Reactions
Additional laboratory evaluation may be re-
quired for investigations of some nonhemoly-
tic transfusion reactions, such as anaphylaxis,
sepsis, or TRALI, as described in their respec-
tive sections.
Acute or Immediate Transfusion
Reactions
Acute or immediate transfusion reactions oc-
cur within 24 hours of the administration of a
component and often during the transfusion.
Acute transfusion reactions include hemolysis,
both immune and nonimmune mediated;
transfusion-related sepsis; TRALI; severe aller-
gic reactions, including anaphylaxis; TACO;
complications of massive transfusion; air em-
bolism; febrile nonhemolytic transfusion reac-
tions (FNHTRs); and mild allergic reactions,
such as urticaria or rash. The clinical signifi-
cance of an acute transfusion reaction often
cannot be determined by the patient’s clinical
history or signs and symptoms alone but re-
quires laboratory evaluation.
AHTRs
Presentation
Rapid hemolysis of as little as 10 mL of incom-
patible blood can produce symptoms of
AHTR. The most common presenting symp-
tom is fever with or without accompanying
chills or rigors. A patient with a mild reaction
may have abdominal, chest, flank, or back
pain. If a patient has a severe AHTR, hypoten-
sion, dyspnea, and flank pain may be present
and, in some cases, progress to shock with or
without accompanying disseminated intravas-
cular coagulation (DIC). Red or dark urine may
be the first sign of intravascular hemolysis,
particularly in the anesthetized or uncon-
scious patient, who may also present with oli-
guria or, in rare cases, DIC. The severity of the
symptoms of this reaction is related to the
amount of incompatible blood transfused.
Prompt recognition of the reaction and imme-
diate cessation of the transfusion can prevent
grave consequences.
Differential Diagnosis
Many of the signs and symptoms of an im-
mune-mediated AHTR also occur in other
acute transfusion reactions. Fever with or
without chills and accompanied by hypoten-
sion may also develop in transfusion-related
 CHAPTER 27 Noninfectious Complications of Blood Transfusion
sepsis and TRALI. Hemolysis may not be de-
tected immediately and does not occur in sep-
sis and TRALI. Respiratory difficulty is not typ-
ically a symptom of an AHTR. Immediate
treatment requirements for acute hemolysis
are identical to those for sepsis—stop the
transfusion and maintain hemodynamic sta-
bility—and can be instituted while the diagno-
sis is being determined.
The patient’s underlying disease process
can make the diagnosis of any AHTR extremely
difficult. Patients with glucose-6-phosphate
dehydrogenase (G6PD) deficiency, autoim-
mune hemolytic anemia, or sickle cell disease
present particularly complicated situations
when symptoms such as fever and hypoten-
sion occur. In these patients, autoantibodies
and multiple alloantibodies delay the serolog-
ic diagnosis of an AHTR and make identifica-
tion of the responsible antibody a challenge.
Acute hemolysis may also result from nonim-
mune mechanisms, as described in the “Non-
immune-Mediated Hemolysis” section below.
Pathophysiology
The interaction of preformed antibodies with
red cell antigens is the immunologic basis for
AHTRs. The most severe reactions are associ-
ated with transfusions of red cells that are ABO
incompatible with the recipient, resulting in
acute intravascular destruction of the trans-
fused cells. Transfusion of ABO-incompatible
plasma, as can occur with apheresis platelets,
may also cause hemolysis of the patient’s red
cells. Although this form of acute hemolysis is
not usually clinically significant or character-
ized by typical hemolytic symptoms, it can be
severe if donors have high titers of ABO anti-
bodies. Although rare, the most common cir-
cumstance is when group O platelets from do-
nors with high titers of anti-A are transfused to
group A recipients.6,7
When preformed immunoglobulin M
(IgM) or IgG antibodies recognize correspond-
ing red cell antigens, complement activation
may occur, resulting in intravascular hemoly-
sis, hemoglobinemia, and, eventually, hemo-
globinuria. IgM antibodies are strong activa-
䡲 673
tors of complement, and IgG antibodies, when
present at sufficient concentrations and of the
relevant subclass, may activate complement as
well.
Complement activation involves C3
cleavage with the ensuing production of C3a,
an anaphylatoxin, which is released into the
plasma, and C3b, which coats the red cells. If
complement activation proceeds to comple-
tion, which is characteristic of ABO antibodies,
a membrane attack complex is assembled on
the red cell surface, and intravascular lysis oc-
curs. C5a, an anaphylatoxin that is 100 times
more potent than C3a, is produced as part of
this hemolysis. C3a and C5a promote the re-
lease of histamine and serotonin from mast
cells, leading to vasodilation and smooth-
muscle contraction, particularly of bronchial
and intestinal muscles. C3a and C5a are recog-
nized by many other cell types as well, includ-
ing monocytes, macrophages, endothelial
cells, platelets, and smooth muscle, and are in-
volved in the production and release of cyto-
kines, leukotrienes, free radicals, and nitric ox-
ide. The end result may include wheezing,
flushing, chest pain or tightness, and gastroin-
testinal symptoms. These symptoms may also
be caused by release of bradykinin and norepi-
nephrine caused by antigen-antibody com-
plex stimulation.
Phagocytosis of IgG-coated red cells leads
to cytokine release, which plays a role in pro-
ducing the effects of acute hemolysis.8 Inter-
leukin-8 (IL-8), which activates neutrophils,
and tumor necrosis factor alpha (TNF),
which activates the coagulation cascade, have
also been found after in-vitro incubation of
incompatible group O whole blood and group
A or B red cells.9 Other cytokines involved in
the pathogenesis of AHTRs include IL-1, IL-6,
and monocyte chemoattractant protein 1
(MCP-1). In a mouse model of HTR,
transfusion of incompatible red cells resulted
in very high plasma levels of MCP-1 and IL-6
and lower levels of TNF.10
If complement activation does not pro-
ceed to completion, which typically happens
with non-ABO antibodies, the red cells can un-
dergo extravascular hemolysis where cells
coated with C3b and/or IgG are rapidly
674 䡲 AABB TECHNICAL MANUAL

removed from the circulation by phagocytes. dence of ABO HTR to be 1:80,000 and the risk
In extravascular hemolysis, the consequences of a fatal ABO HTR to be 1 in 1.8 million.11 Of
of complement activation, including release of the transfusion-related fatalities reported to
anaphylatoxins and opsonization of red cells, the Food and Drug Administration (FDA) from
may still have adverse effects. 2007 to 2011, 23% (in 50 patients) were caused
Coagulation abnormalities associated by HTRs.1
with AHTRs may be caused by various mecha-
nisms. The “intrinsic” pathway of the clotting Treatment
cascade may be activated by antigen-antibody
Prompt recognition of an AHTR and immedi-
interaction, resulting in activation of Factor
ate cessation of the transfusion are crucial.
XII, also known as “Hageman factor.” Activa-
The unit of blood should be returned to the
tion of the Hageman factor can result in hypo-
blood bank for a transfusion reaction investi-
tension through its effect on the kinin system.
gation. The infusion of saline should be con-
The kinin system produces bradykinin, which
tinued to maintain venous access, treat hypo-
in turn increases vascular permeability and
tension, and maintain renal blood flow, with a
causes vasodilation. Activated complement,
goal of a urine flow rate >1 mL/kg/hour. Saline
TNF, and IL-1 may increase the expression of
infusion alone may not be adequate therapy,
tissue factor. Tissue factor, expressed by leuko-
and the urine output must be carefully moni-
cytes and endothelial cells, can activate the
tored so as not to cause volume overload in the
“extrinsic” pathway and is associated with the
patient. Studies have concluded that low-dose
development of DIC. DIC is an often life-
dopamine may improve renal function initial-
threatening consumptive coagulopathy. Its
ly, but no solid evidence exists that it can pre-
characteristics include microvascular thrombi
vent renal failure.12 However, these studies,
formation with ischemic organ and tissue
which did not include patients with AHTRs,
damage; consumption of platelets, fibrinogen,
did show a 25% increase in urine output in the
and coagulation factors; and activation of fi-
acute setting. Thus, low-dose dopamine (1-5
brinolysis with resultant production of fibrin-
µg/kg/minute) may still have a role in treating
degradation products. The end result of these
the complications of an AHTR, but no recom-
activations can vary from generalized oozing
mendations regarding its use may be made
to uncontrolled bleeding.
based on published evidence at this time.
Shock may be a component of DIC. Hypo-
The addition of the diuretic furosemide
tension, caused by the release of vasoactive
(40-80 mg intravenously in adults, 1-2 mg/kg
amines, kinins, and other mediators, produc-
in children) promotes increased urine output
es a compensatory vasoconstrictive response
and further enhances renal cortical blood
that further aggravates organ and tissue dam-
flow.13 If urine output remains diminished af-
age. Renal failure may occur as well. Free he-
ter a liter of saline has been infused, acute tu-
moglobin impairs renal function, but compro-
bular necrosis may have occurred, and the pa-
mised renal cortical supply is thought to be the
tient may be at risk of developing pulmonary
major contributing factor in renal failure. In
edema. At this point, a nephrologist should be
addition, antigen-antibody complex deposi-
consulted for further management of the pa-
tion, vasoconstriction, and thrombi formation
tient. Oliguric renal failure may be complicat-
contribute to the development of renal vascu-
ed by hyperkalemia and subsequent cardiac
lar compromise.
arrest; therefore, these patients should be
monitored with telemetry. Metabolic acidosis
Frequency
and uremia often necessitate the institution of
The frequency of AHTRs is not easy to deter- dialysis.
mine. The authors of a review article based on DIC is an equally serious component of
data from several surveillance systems esti- an AHTR. DIC is extremely difficult to treat
mated the risk of clinical or laboratory evi- and may be the first indication that hemolysis
 CHAPTER 27 Noninfectious Complications of Blood Transfusion
has occurred in an anuric or anesthetized pa-
tient. Traditional therapy for DIC includes
treating or removing the underlying cause and
providing supportive care via the administra-
tion of platelets, frozen plasma, and cryopre-
cipitated antihemophilic factor (AHF).
The administration of heparin to treat
DIC associated with HTRs is controversial.
First, the underlying condition for which the
patient required transfusion may be a contra-
indication to heparin administration. For ex-
ample, in patients with recent surgery or active
bleeding, heparin can exacerbate bleeding.
Second, there is some thought that because
the precipitating event is limited to hemolysis
of the volume of blood transfused, the risks of
administering heparin do not justify its use.
Those in favor of heparin point out, however,
that the most unstable patients (those with the
most severe reactions) are those who received
larger volumes of incompatible blood and are
thus the most likely to develop DIC.14 In the
worst cases, DIC can become a self-sustaining
vicious cycle of inflammation and consump-
tive coagulopathy.
Recent studies of optimal treatment for
DIC have evaluated the targeting of various
components of the inflammatory and coagula-
tion cascades. Activated protein C has shown
some benefit in patients with sepsis and DIC;
however, no new therapeutic agents have been
added to the arsenal to treat DIC associated
with AHTRs in recent years.15,16
Unconscious or anesthetized patients
may receive multiple units of incompatible
blood before acute hemolysis is recognized.
Because the severity of an AHTR is related to
the amount of incompatible red cells trans-
fused, red cell exchange transfusion may be
considered. Some severe reactions to a single
unit of strongly incompatible blood may re-
quire exchange transfusion as well. Antigen-
negative blood must be used for the red cell
exchange. In the case of acute hemolysis
caused by a non-ABO antibody, the blood
bank must be given adequate time to identify
the appropriate units for further simple red
cell transfusion or planned red cell exchange.
Likewise, plasma and platelets that will not
contribute to hemolysis should be chosen.
䡲 675
Prompt initiation of therapy to aggres-
sively manage hypotension, renal blood flow,
and DIC provides the greatest chance of a suc-
cessful outcome. Furthermore, consultation
with appropriate medical specialists early in
the course of treatment will ensure that the pa-
tient receives hemodialysis, cardiac monitor-
ing, and mechanical ventilation when needed.
Prevention
The most common events leading to the trans-
fusion of a component in error are the very
things that the laboratory evaluates when an
AHTR is suspected. Clerical and human errors
involving patient, sample, and blood unit
identification are the most common causes of
mistransfusion and, therefore, AHTRs. The risk
of a near miss is 1:1,000, wrong blood given is
1:15,000, and ABO-incompatible transfusion is
1:40,000.11 Institutional policies and proce-
dures should be in place to minimize the likeli-
hood of such errors, and corrective and
preventive action programs should target con-
tinual reduction of the numbers of such errors.
However, no one method for reducing the
number of errors is foolproof. Products avail-
able to increase patient safety include technol-
ogy-based solutions, such as radiofrequency
identification chips, handheld bar-code scan-
ners, and “smart” refrigerators similar to sys-
tems used for pharmacologic agents.
The prevention of potential hemolysis
from the administration of minor ABO-incom-
patible platelets remains a challenge in pa-
tients with HLA antibodies. A number of op-
tions, including anti-A or anti-B titration of the
product, limitations in total amount of incom-
patible plasma transfused from platelets, and
volume reduction, may offer some benefit.
The use of platelet additive solutions is also
promising because these solutions replace
plasma, which contains ABO antibodies and
plasma proteins.17
Nonimmune-Mediated Hemolysis
Transfusion-associated hemolysis can also be
due to several nonimmune-mediated causes.
676 䡲 AABB TECHNICAL MANUAL
Before issue, improper shipping or storage
temperatures as well as incomplete deglycero-
lization of frozen red cells can lead to hemoly-
sis. At the time of transfusion, using a needle
with an inappropriately small bore size or em-
ploying a rapid pressure infuser can cause me-
chanical hemolysis, which may result from the
use of roller pumps as well. Improper use of
blood warmers or the use of microwave ovens
or hot waterbaths can cause temperature-
related hemolysis. Few fluids are approved by
FDA for transfusion with Red Blood Cells
(RBCs).5(p45) Infusion of RBCs simultaneously
through the same tubing with hypotonic solu-
tions or some pharmacologic agents may
cause osmotic hemolysis; for safe administra-
tion, RBCs and these solutions or agents
should be given via alternate venous access lo-
cations. In rare cases, hemolysis may be
caused by bacterial contamination of the RBC
unit. Not infrequently, patients may also expe-
rience hemolysis as part of their underlying
disease process. Of note is that although a neg-
ative DAT result usually indicates no evidence
of an immune-mediated cause of hemolysis,
complete destruction of incompatible trans-
fused red cells may be associated with a nega-
tive DAT result.
When both immune and nonimmune
causes of hemolysis have been excluded, the
possibility of an intrinsic red cell membrane
defect in the recipient or even in the trans-
fused cells should be considered. Cells with
these defects, such as G6PD deficiency, have
increased fragility when challenged with par-
ticular stressors and may undergo coinciden-
tal hemolysis.
Treatment
Hemolysis of nonimmune etiology may cause
symptoms whose severity depends on the de-
gree of hemolysis and amount of component
transfused. In all cases, the transfusion should
be discontinued and appropriate care should
be administered. (See the earlier section on
the treatment of AHTRs for details on manag-
ing hypotension and declining renal function.)
Prevention
As is true for the mitigation of any type of
transfusion reaction, written procedures for all
aspects of the manufacture and transfusion of
blood and components should always be fol-
lowed. Prompt recognition of nonimmune he-
molysis and robust root cause analysis may
prevent additional occurrences.18
Transfusion-Related Sepsis
Presentation
Fever (particularly a temperature of 38.5 C or
101 F) and shaking chills and hypotension
during or shortly after transfusion are the most
frequent presenting symptoms of transfusion-
related sepsis. In severe cases, the patient may
develop shock with accompanying renal fail-
ure and DIC.
Differential Diagnosis
The abruptness of onset and severity of the
signs and symptoms associated with transfu-
sion-related sepsis may be very similar to
those of AHTRs. Mild cases may be confused
with FNHTRs. The key to diagnosing transfu-
sion-related sepsis is culturing the same
organism from both the patient and the re-
mainder of the component. The returned
component should be visually examined in
suspected cases of posttransfusion sepsis. Par-
ticular attention should be paid to any color
changes, especially brown or purple discolor-
ation in an RBC component and bubbles/
frothiness in a platelet component. A Gram’s
stain should be performed on the returned
component.
Treatment
If transfusion-related sepsis is suspected, the
transfusion should be stopped immediately
and supportive care of the patient should be
initiated. Broad-spectrum antibiotics may be
indicated. (See Chapter 8 for a more detailed
discussion of bacterial contamination of
transfused blood, including frequency data
and prevention strategies.)
 CHAPTER 27 Noninfectious Complications of Blood Transfusion
FNHTRs
Presentation
An FNHTR is usually defined as the occur-
rence of 1 C rise in temperature above 37 C
that is associated with transfusion and for
which no other cause is identifiable. Accompa-
nying symptoms may include shaking, chills,
an increased respiratory rate, a change in
blood pressure, and anxiety. In some instanc-
es, the patient may be afebrile but have the re-
maining constellation of symptoms. Symp-
toms usually occur during transfusion but may
occur up to 4 hours after. Most FNHTRs are be-
nign, although they may cause significant dis-
comfort and even hemodynamic or respirato-
ry effects.
Differential Diagnosis
The symptoms associated with an FNHTR may
be present in several other types of transfusion
reactions, the most serious of which are HTRs,
sepsis, and TRALI. Each of these other reac-
tions has signs, symptoms, and associated lab-
oratory results that help distinguish them from
an FNHTR once an investigation is begun; rec-
ognition of FNHTR requires diagnosis by ex-
clusion.
Fever may commonly occur as a compo-
nent of a patient’s underlying illness. In a pa-
tient who has been experiencing spiking fevers
during the course of admission, it may be diffi-
cult to rule out an FNHTR. Hemolysis along
with any other signs or symptoms of a serious
reaction must be ruled out in a patient who ex-
periences fever associated with transfusion.
Pathophysiology
Recipient leukocyte antibodies may cause fe-
brile transfusion reactions. As few as 0.25 × 109
residual leukocytes in a blood component can
produce a temperature elevation in the recipi-
ent. FNHTRs may also be the result of accu-
mulated cytokines in a cellular blood compo-
nent.19 This mechanism may be particularly
relevant in reactions that occur after the trans-
fusion of platelets. Some FNHTRs are attribut-
able to recipient antibodies, particularly HLA
䡲 677
antibodies that react with antigens on trans-
fused lymphocytes, granulocytes, or platelets.
Cytokine release in the recipient in response to
these antigen-antibody reactions may contrib-
ute to the severity of the reaction. Whatever
the initiating cause, cytokine release is the
common event leading to symptoms of
FNHTR.
Treatment
When an FNHTR is suspected, the transfusion
should be discontinued and a transfusion re-
action work-up initiated. Antipyretics (ie, acet-
aminophen) should be administered, and the
patient may be safely transfused once the
symptoms subside. For more severe reactions
that include rigors, meperidine may be neces-
sary where it is not contraindicated.
In general, the remainder of the implicat-
ed component and donor-related co-compo-
nents should not be transfused. Many times,
an FNHTR does not develop until after the
transfusion has been completed. If a portion of
the component remains, the laboratory work-
up to exclude hemolysis must be completed
before the transfusion is resumed. This may be
difficult to accomplish within an acceptable
amount of time. Among the few valid situa-
tions in which transfusion of the remainder of
the component should be considered is when
the component is a medically indicated rare
unit and a significant volume remains un-
transfused. In that circumstance, the blood
bank’s medical director should be consulted
before proceeding with caution because bac-
terial contamination of the component might
have been the underlying cause of the reac-
tion.20,21
Prevention
Prestorage leukocyte reduction, especially if
performed at the time of collection, signifi-
cantly decreases the frequency of FNHTRs.22
Premedication with acetaminophen may be
beneficial in further reducing the residual rate
of FNHTRs and has not been shown to impair
the ability to detect serious complications of
transfusion.21
678 䡲 AABB TECHNICAL MANUAL
Allergic Reactions
Presentation
Most allergic transfusion reactions (ATRs) are
mild, but their spectrum can range from a sim-
ple allergic reaction (urticaria) to life-threaten-
ing anaphylaxis. Symptoms generally occur
within seconds or minutes of the start of the
transfusion. In rare cases, the symptoms may
take several hours to develop. If symptoms do
not appear until more than 4 hours later, they
may represent an allergic reaction that is unre-
lated to the blood transfusion. An ATR is diag-
nosed in the same way as any other allergic re-
action.
The mildest form of ATR is urticaria, or
hives. An outbreak of swollen, raised, red ar-
eas (wheals) on the skin appears suddenly as a
result of the body’s adverse reaction to an al-
lergen. Hives usually cause itching (pruritus)
but may also burn or sting. Hives can appear
anywhere on the body and vary greatly in size.
They can last from hours to several days before
fading but often respond quickly to treatment
with antihistamines. More extensive cases may
be accompanied by angioedema, where the
swelling is caused by fluid accumulation be-
neath the skin instead of on the surface. It is a
deep swelling, often around the eyes and lips,
and generally lasts longer than urticaria. An-
gioedema can, rarely, involve the throat,
tongue, or lungs, causing respiratory distress.
More serious are anaphylactic transfusion
reactions, which include the symptoms of urti-
caria and angioedema in the majority of cas-
es.23 Severe hypotension, shock, and loss of
consciousness may also occur. In addition, the
respiratory system is often involved, with pa-
tients experiencing dyspnea, wheezing, and
stridor. Gastrointestinal disturbances such as
nausea, vomiting, diarrhea, and cramping, af-
fect approximately 30% of these patients. Car-
diovascular manifestations in addition to
hypotension may include tachycardia, ar-
rhythmia, or cardiac arrest.
Differential Diagnosis
It is important to distinguish anaphylaxis from
other reactions characterized by hypotension,
dyspnea, and/or loss of consciousness. The
most common reaction that may be mistaken
for anaphylaxis is a vasovagal reaction, which
is characterized by hypotension, diaphoresis,
nausea/vomiting, weakness, bradycardia, and
sometimes loss of consciousness. Urticaria,
angioedema, pruritus, and respiratory symp-
toms, such as wheezing or stridor, are symp-
toms of anaphylaxis but do not occur in vaso-
vagal reactions. The respiratory symptoms of
anaphylaxis may be suggestive of an acute
asthma attack or TRALI. However, the classic
symptoms of allergy, including urticaria, an-
gioedema, and pruritus, do not occur in asth-
ma attacks or TRALI. Fever, a prominent
symptom of HTR and bacterial contamina-
tion, is not a feature of anaphylaxis.
Patients who take angiotensin-convert-
ing enzyme (ACE) inhibitors and undergo
plasma exchange sometimes develop hypo-
tensive reactions that mimic anaphylaxis
when albumin is used as a replacement fluid.
Pathophysiology
ATRs are attributed to hypersensitivity reac-
tions to allergens in the component caused by
preformed IgE antibody in the recipient. In
most cases, the causative plasma protein or
antigen is not identified. Mast cell activation
(Type I hypersensitivity), resulting in degranu-
lation, with the release of allergic mediators
occurs. Secondary mediators—including cyto-
kines and lipid mediators—are also generated
and released. Recent studies suggest that the
mechanisms underlying most ATRs have not
been fully elucidated. A combination of recipi-
ent, donor, and component factors is likely in-
volved, which is supported by the incidence of
these reactions compared to the prevalence of
IgA, haptoglobin, or C4 deficiency. Recipients
with an atopic predisposition appear to have a
higher rate of ATRs, and certain donors’ plate-
lets are associated with an increased risk. In-
creased understanding of the underlying
mechanisms of ATRs is crucial to improving
prevention.24,25
ATRs that progress beyond urticaria may
occur in IgA-deficient patients. These anaphy-
lactic reactions are caused by anti-IgA in the
 CHAPTER 27 Noninfectious Complications of Blood Transfusion
recipient. Although IgA deficiency is present in
approximately 1:700 people of European an-
cestry, only a small percentage of these people
ever make antibodies against IgA. Those who
do are divided into two groups based on IgA
levels and the type of antibody formed. People
with absolute IgA deficiency (<0.05 mg/dL)
may form class-specific antibodies that are of-
ten associated with anaphylactic reactions.
Those with decreased but detectable amounts
of IgA, or relative IgA deficiency, can form sub-
class-specific antibodies (eg, anti-IgA1 or anti-
IgA2) that typically result in less severe reac-
tions.26
Although precautions should be taken
when transfusing an IgA-deficient patient, it
must be kept in mind that the majority of ATRs
are caused by allergens to substances other
than IgA.26 Other well-known triggers include
patient antibodies against haptoglobin,27 peni-
cillin, and the C4 determinant of comple-
ment.28 Patients taking ACE inhibitors can ex-
perience transfusion reactions that are thought
to result from dual actions on bradykinin: inhi-
bition 1) of its catabolism by the ACE inhibitor
and 2) of bradykinin by low levels of prekalli-
krein activity in plasma protein fraction.
Frequency
ATRs are quite common, with an overall fre-
quency of approximately 1% to 3% of transfu-
sions. ATRs also represent 12% to 33% of all
transfusion reactions.29,30 Urticaria is relatively
common, whereas anaphylactic reactions oc-
cur much less often. As with any ATR, anaphy-
laxis occurs most commonly during the trans-
fusion of plasma or platelets; it has an
incidence of 1:20,000 to 1:50,000 transfu-
sions.29,31 Of the transfusion-associated fatali-
ties reported to the FDA from 2007 to 2011, 6%
(in 12 patients) were caused by anaphylaxis.1
Treatment
Urticaria is the only transfusion reaction in
which the administration of the component
may be routinely resumed after prompt treat-
ment. When a patient develops symptoms, the
transfusion should be paused so that an anti-
histamine, typically 25 to 50 mg diphenhydr-
䡲 679
amine, may be administered. Once the symp-
toms have dissipated, the transfusion may be
resumed and a laboratory work-up need not
be initiated.30
If the symptoms do not subside—or, in
the case of severe urticaria or urticaria accom-
panied by hypotension if dyspnea, significant
edema, or gastrointestinal symptoms do not
subside—the transfusion must be stopped and
the reaction promptly treated. Severe urticari-
al reactions may require treatment with meth-
ylprednisolone (125 mg intravenously) or
prednisone (50 mg orally). Once a severe reac-
tion or developing anaphylaxis is identified,
action should be promptly initiated to main-
tain oxygenation and stabilize hypotension.
Epinephrine (1:1000) may be administered in-
tramuscularly or subcutaneously at an adult
dose ranging from 0.2 to 0.5 mL or a pediatric
dose of 0.01 mL/kg. If symptoms persist, the
dose may be repeated every 5 to 15 minutes up
to three times, unless palpitations, extreme
anxiousness, or tremors occur. If the patient is
unconscious or in shock, epinephrine may be
given intravenously at a dilution of 1:10,000
(100 µg/mL) and an initial rate of 1 µg/minute.
Such patients ideally receive cardiac monitor-
ing because of the epinephrine’s arrhythmic
potential.
Supplemental oxygen should be adminis-
tered, and the airway should be maintained.
Hypotensive patients should be placed in the
Trendelenburg position and supported with
crystalloids. If bronchospasm is present, respi-
ratory symptoms may not respond to the epi-
nephrine, and addition of a beta-2 agonist or
aminophylline may be required. Patients who
do not respond, possibly because of the use of
a beta-adrenergic blocker or an ACE inhibitor,
may respond to the addition of glucagon as a
1-mg bolus intravenously or by continuous in-
fusion.23,32
Prevention
Premedication with an antihistamine (25 to 50
mg diphenhydramine) 30 minutes before
transfusion may be helpful in patients with
a history of multiple or severe urticarial trans-
fusion reactions. Routine prophylaxis may also
680 䡲 AABB TECHNICAL MANUAL
be beneficial for patients undergoing multiple
transfusions, as in plasma exchange; however,
routine antihistamine premedication of all pa-
tients receiving a transfusion has not been
shown to decrease the risk of ATR.33 If antihis-
tamines are not sufficient, prednisone (20-50
mg orally) or parenteral steroids may be
of benefit. For patients whose reactions are se-
vere, unrelenting, and unresponsive to pre-
medication, washing red cells or platelets may
be considered. Plasma reduction or replace-
ment in platelet products may also be benefi-
cial. Alternatively, the administration of de-
glycerolized RBCs has met with some success
when reactions have occurred even after RBCs
were washed with 2 L of saline.
The plasma used for transfusions for pa-
tients with diagnosed IgA deficiency who pro-
duce anti-IgA should be from IgA-deficient do-
nors (<0.05 mg/dL). If the local blood center
cannot provide these components, they may
be available through the American Rare Donor
Program (see Method 3-13). Other cellular
components (RBCs and platelets) can be de-
pleted of plasma proteins through washing.
IgA deficiency without the presence of anti-IgA
or without a history of an anaphylactoid/
anaphylactic reaction does not warrant the
use of IgA-deficient or plasma-depleted com-
ponents. Intravenous immune globulin (IVIG)
products from various manufacturers have
different IgA amounts. The manufacturer
should be contacted to obtain detailed infor-
mation about a product and lot number.34 Au-
tologous donation programs may also be con-
sidered for patients with IgA deficiency once
they have recovered.
TRALI
Presentation
Clinical signs and symptoms of TRALI typical-
ly include fever, chills, dyspnea, cyanosis, hy-
potension, and new-onset bilateral pulmonary
edema.35 An increase in blood pressure fol-
lowed by hypotension is not uncommon.
TRALI can be life threatening or fatal. Symp-
toms arise within 6 hours of transfusion, with
most cases becoming evident within 1 to 2
hours after the end of transfusion. In addition
to the signs and symptoms traditionally asso-
ciated with TRALI, there is a growing apprecia-
tion that the disorder can be associated with a
dramatic transient neutropenia or leukope-
nia.36
All plasma-containing components, in-
cluding whole blood, RBCs, platelets, cryopre-
cipitated AHF, and fresh frozen plasma (FFP),
have been implicated in TRALI. Transfusion
volumes as small as 15 mL have led to TRALI.
TRALI is a form of acute lung injury (ALI).
The American-European Consensus Conference37
defined ALI as acute hypoxemia with a PaO2/
FiO2 ratio of 300 mm Hg and bilateral pulmo-
nary edema on frontal chest radiograph. The
Canadian Consensus Conference38 relied on
this definition of ALI when creating its diag-
nostic criteria for TRALI: 1) ALI with hypox-
emia and PaO2/FiO2 300 or SpO2 <90% on
room air, 2) no preexisting ALI before transfu-
sion, 3) onset of symptoms within 6 hours of
transfusion, and 4) no temporal relationship
with an alternative risk factor for ALI. The pan-
el also defined “possible TRALI” using the
same criteria as for TRALI, with the exception
that possible TRALI occurs in the setting of an
alternative risk factor for ALI.
Although the lung injury in ALI is usually
irreversible, the lung injury in TRALI is most
often transient. Approximately 80% of pa-
tients with TRALI improve within 48 to 96
hours. The remaining 20% of patients who do
not improve rapidly have either a protracted
clinical course or a fatal outcome. In one of the
largest studies of TRALI, 100% of the patients
required oxygen support and 72% required
mechanical ventilation.39
Differential Diagnosis
The three main conditions that need to be dis-
tinguished from TRALI are 1) anaphylactic
transfusion reactions, 2) TACO, and 3) transfu-
sion-related sepsis. In anaphylactic transfu-
sion reactions, bronchospasm, laryngeal ede-
ma, severe hypotension, erythema (often
confluent), and urticaria are prominent symp-
toms. Fever and pulmonary edema are not as-
sociated with anaphylactic reactions. The clin-
ical presentation of TACO is very similar to that
 CHAPTER 27 Noninfectious Complications of Blood Transfusion
of TRALI, with respiratory distress, tachypnea,
and cyanosis as the most prominent features.
The key distinction between TACO and TRALI
is that the pulmonary edema in TACO is car-
diogenic, whereas it is noncardiogenic in
TRALI. High fever with hypotension and vas-
cular collapse are prominent features of trans-
fusion-related sepsis. Respiratory distress is
infrequently associated with these reactions.
With rapid onset of respiratory distress, in ad-
dition to TACO and TRALI, coincident myocar-
dial infarction and pulmonary embolus as well
as other possible causes of ALI should be con-
sidered.
Pathophysiology
The precise mechanism of lung injury in
TRALI has not been determined. TRALI has
been associated with the infusion of antibod-
ies to leukocyte antigens and of biologic re-
sponse modifiers (BRMs).39 Infusions of these
antibodies or BRMs are thought to initiate a
sequence of events that results in cellular acti-
vation and damage of the basement mem-
brane. Pulmonary edema occurs secondary to
leakage of protein-rich fluid into the alveolar
space.
BRMs accumulate in some cellular com-
ponents during storage. BRMs enhance the
polymorphonuclear cell oxidative burst, are
soluble in chloroform, and consist of a mixture
of lysophosphatidylcholines.40
Antibodies to HLA Class I antigens, HLA
Class II antigens, and human neutrophil anti-
gens (HNAs) have been associated with TRALI.
These antibodies can be formed after exposure
to foreign antigens via pregnancy, transfusion,
or transplantation. In the majority of TRALI
cases with antibodies, the source of the anti-
body is the donor, not the recipient.
A two-event model of the mechanism of
TRALI has been hypothesized.40 In the first
event, generation of biologically active com-
pounds activates pulmonary vascular endo-
thelial cells and primes neutrophils, resulting
in sequestration of neutrophils in the pulmo-
nary microvasculature. This first event can
result from a variety of physiologic stressors,
including sepsis, surgery, and massive transfu-
䡲 681
sion, and it predisposes the recipient to devel-
op TRALI if a second event is experienced. The
infusion of BRMs or antibodies is the second
event. These stimuli, which ordinarily do not
activate neutrophils, activate the primed neu-
trophils in the pulmonary microvasculature,
which results in pulmonary endothelial dam-
age, capillary leakage, and pulmonary edema.
Frequency
Although the true incidence of TRALI is un-
known, TRALI is the leading cause of transfu-
sion-related mortality reported to the FDA,
with more than 34 cases reported in 2007.41 Re-
cent efforts to decrease the amount of trans-
fusable plasma collected from female donors
appear to be decreasing the number of TRALI
fatalities in the United States.1
In 2006, the year before many blood cen-
ters implemented measures to reduce the risk
of TRALI from plasma transfusions, 35 fatal
cases of TRALI, 22 of which were associated
with transfusion of FFP, were reported to the
FDA. Since 2008, the year after many blood
centers implemented such measures, the
numbers of TRALI fatalities and of TRALI fatal-
ities associated with FFP transfusions reported
to the FDA have decreased each year.
Treatment
Treatment of TRALI consists of respiratory and
circulatory support. Treatment should be as
intensive as dictated by the clinical picture.
Oxygen supplementation with or without me-
chanical ventilation is required in almost all
cases. Pressor agents may be needed to sup-
port blood pressure. Diuretics are not indicat-
ed because TRALI is not related to volume
overload. Administration of corticosteroids
has not been shown to improve the clinical
outcome in TRALI or acute respiratory distress
syndrome.42
Prevention
Strategies to reduce the risk of TRALI are com-
plicated by a number of factors. Although
approximately 10% of blood donations con-
tain HLA and/or HNA antibodies, TRALI
682 䡲 AABB TECHNICAL MANUAL
occurs in fewer than 1:1000 transfusions.39,40
There is no mechanism to identify which pa-
tients are at risk for developing TRALI. In 2013,
AABB announced that a new standard in the
29th edition of Standards for Blood Banks and
Transfusion Services would require that plas-
ma products and whole blood collected for
transfusion be from male donors, never-preg-
nant female donors, or females who have been
tested since their last pregnancy and found to
be negative for HLA antibodies.5
In 2014 the AABB issued Association Bul-
letin 14-02, providing guidance on how to im-
plement the new TRALI risk reduction require-
ment. In this bulletin, AABB outlined a
number of considerations for implementation
of TRALI risk reduction requirements in plas-
ma components and whole blood intended for
transfusion.43
Although the measures required by AABB
and described in Association Bulletin 14-02
are expected to reduce the risk of TRALI, it is
important to recognize they do not eliminate
TRALI because they do not decrease the risk of
TRALI from RBCs, platelet concentrates, or
cryoprecipitate. HLA antibody screening ad-
dresses only the risk of TRALI from HLA anti-
bodies. A practical test to screen the blood
supply for HNA antibodies is not available. In
addition, none of these risk-reduction mea-
sures addresses the risk of TRALI from BRMs.
TACO
Presentation
It is well known that transfusion can precipi-
tate acute pulmonary edema caused by vol-
ume overload, but only recently has this prob-
lem been recognized as an important
complication. Patients older than 70 years and
infants are at greatest risk, although all trans-
fusion recipients are susceptible to some de-
gree.44 Whereas large volumes of components
and nonblood fluids are most frequently im-
plicated, modest volumes can also precipitate
TACO. High flow rates are frequently cofactors.
TACO has no diagnostic signs or symp-
toms. Within 1 to 2 hours of transfusion, pa-
tients may develop any or all of the following:
gallop; jugular venous distension; elevated
central venous pressure; dyspnea; orthopnea;
new ST-segment and T-wave changes on elec-
trocardiogram; elevated serum troponin; and
likely elevated brain natriuretic peptide (BNP),
a cardiac marker.45 Increased blood pressure
characterized by a widening of the pulse pres-
sure is characteristic of TACO. Radiographs
show a widened cardiothoracic ratio.
Differential Diagnosis
TACO is frequently confused with TRALI be-
cause both types of reactions produce pulmo-
nary edema. It is possible for TACO and TRALI
to occur concurrently in the same patient. The
timelines and the clinical presentation are
similar, but hypertension is a constant feature
of TACO, whereas it is only an infrequent and
transient manifestation of TRALI. Further-
more, rapid improvement with diuresis or ino-
tropic agents is consistent with TACO.
In congestive heart failure, BNP levels are
elevated. Several studies have shown that a
posttransfusion-to-pretransfusion BNP ratio
of 1.5 with a posttransfusion level at least 100
pg/mL as a cutoff yielded a sensitivity and
specificity greater than 80% in TACO.46 Howev-
er, a recent study in the intensive care setting
found that BNP was only of moderate value for
distinguishing TACO from TRALI.47 With rapid
onset of respiratory distress, possible causes of
ALI, such as coincident myocardial infarction,
pulmonary embolus, and others, should be
considered in addition to TACO and TRALI.
Frequency
The incidence of TACO is unknown, but sever-
al studies suggest that TACO is much more
common than previously known. In the gener-
al population, one group found TACO in 1:707
recipients of RBCs, and 20% of the affected pa-
tients received a single unit of RBCs. In studies
of older orthopedic patients undergoing hip or
knee replacement, TACO was found in 1% and
8%, respectively.48,49 In one report from the
Quebec hemovigilance network, the incidence
was 1:5000 components, and 1.3% of the cases
resulted in death.50 In the critical care setting,
1:356 units transfused resulted in TACO.51
From 2007 to 2011, 15% of the transfusion-
 CHAPTER 27 Noninfectious Complications of Blood Transfusion
associated fatalities reported to the FDA (in 32
patients) were a consequence of TACO.1
Although RBCs are most commonly asso-
ciated with TACO, a recent prospective surveil-
lance study found that FFP was a frequent
cause of this complication.52 In this study, 4.8%
of patients developed TACO with a prevalence
rate of 1.5%. Of the 24 reactions, 14 (58%) oc-
curred in the intensive care unit.
Treatment
As soon as symptoms suggest TACO, the trans-
fusion should be stopped. The symptoms
should be treated by placing the patient in a
seated position, providing supplementary oxy-
gen, and reducing the intravascular volume
with diuretics. If symptoms persist in con-
firmed TACO, administration of additional di-
uretics or therapeutic phlebotomy in 250-mL
increments is appropriate.
Prevention
In the absence of ongoing and rapid blood
loss, components should be administered
slowly, particularly in patients at risk of TACO
(ie, pediatric patients, patients with severe
anemia, and patients with congestive heart
failure). Rates of 2 to 4 mL/minute and 1 mL/
kg of body weight per hour are the most fre-
quently cited, despite a paucity of data on ap-
propriate infusion rates. Total fluid input and
output must be monitored.
Complications of Massive
Transfusion
The potential complications of massive trans-
fusion, usually defined as the receipt of more
than 10 RBC units within 24 hours, include
metabolic and hemostatic abnormalities, im-
mune hemolysis, and air embolism. Metabolic
abnormalities can depress ventricular func-
tion. Hypothermia from refrigerated blood, ci-
trate toxicity, and lactic acidosis from under-
perfusion and tissue ischemia, which are often
complicated by hyperkalemia, can contribute
to this effect. Although metabolic alkalosis
caused by citrate metabolism may occur, it is
not likely to be clinically significant. Patients
䡲 683
who lose blood rapidly may have preexisting
or coexisting hemostatic abnormalities or de-
velop them during resuscitation. Hemostatic
abnormalities may include dilutional coagu-
lopathy, DIC, and liver and platelet dysfunc-
tion.
Citrate Toxicity
PATHOPHYSI OLOGY AND MA NIFESTA-
TIONS. Plasma, whole blood, and platelets
contain citrate as an anticoagulant. When
large volumes of these components are trans-
fused rapidly, particularly in the presence of
liver disease, plasma citrate levels may rise,
binding calcium and ionized calcium and re-
sulting in hypocalcemia. In patients with a
normally functioning liver, citrate is rapidly
metabolized; thus, these symptoms are tran-
sient.53 Hypocalcemia is more likely to cause
manifestations in patients who are hypother-
mic or in shock.
A decrease in ionized calcium levels in-
creases neuronal excitability, which in the con-
scious patient leads to symptoms of perioral
and peripheral tingling, shivering, and light-
headedness, followed by a diffuse sense of vi-
bration, muscle cramps, fasciculations, spasm,
and nausea. In the central nervous system, hy-
pocalcemia is thought to increase the respira-
tory center’s sensitivity to carbon dioxide,
causing hyperventilation. Because myocardial
contraction is dependent on the intracellular
movement of ionized calcium, hypocalcemia
depresses cardiac function.54
TREATMENT AND PREVENTI ON. Unless the
patient has a predisposing condition that hin-
ders citrate metabolism, hypocalcemia caused
by citrate overload during massive transfusion
can usually be treated by slowing the infusion.
Calcium replacement should be considered
when the calcium concentration falls to below
50% of its normal value or the symptoms of
hypocalcemia are evident.55
Hyperkalemia and Hypokalemia
PATHOPHYSIOLOGY. When RBCs are stored
at 1 to 6 C, the intracellular potassium gradual-
ly leaks into the supernatant plasma or addi-
684 䡲 AABB TECHNICAL MANUAL
tive solution. Although the concentration in
the supernatant may be high (see Chapter 6)
because of the small volume, the total extra-
cellular potassium load is less than 0.5 mEq for
fresh RBC units and only 5 to 7 mEq for units at
expiration. These potassium concentrations
rarely cause problems in the recipient because
rapid dilution, redistribution into cells, and ex-
cretion blunt the effect. However, hyperkale-
mia can be a problem in patients with renal
failure, premature infants, and newborns re-
ceiving large transfusions, such as in cardiac
surgery or exchange transfusion; otherwise,
hyperkalemia is typically a transient effect
during very rapid transfusions.
Hypokalemia occurs more frequently
than hyperkalemia after transfusion because
potassium-depleted donor red cells reaccu-
mulate this ion intracellularly, and citrate me-
tabolism causes further movement of potassi-
um into the cells in response to the
consumption of protons. Catecholamine re-
lease and aldosterone-induced urinary loss
can also trigger hypokalemia in the setting of
massive transfusion.56
TREATMENT A ND PRE VENTION. No treat-
ment or preventive strategy for hypokalemia
and hyperkalemia is usually necessary, provid-
ed that the patient is adequately resuscitated
from the underlying condition that required
massive transfusion.57 For infants receiving
small-volume transfusions infused slowly,
units may be used safely until the expiration
date.58 Although washing of RBC units results
in very low levels of potassium, there is no evi-
dence that this is indicated for routine RBC
transfusions, even in patients with impaired
renal function.59
Hemostatic Abnormalities in Massive
Transfusion
PATHOPHYSIOLOGY. Coagulopathy can oc-
cur in massive transfusion, particularly when
the lost blood is initially replaced with RBCs
and asanguineous fluids. Coagulopathy in
massive transfusion is frequently ascribed to
the dilution of platelets and clotting factors as
patients lose hemostatically active blood, and
enzymatic activity is reduced as the core body
temperature lowers if a blood warmer is not
used. Mortality rates associated with hemo-
static abnormalities range from 20% to 50%.60
The high rate of mortality results from hypo-
thermia, metabolic acidosis, and coagulopa-
thy.61
Studies of military and civilian trauma pa-
tients demonstrated a progressive increase in
the incidence of microvascular bleeding
(MVB) characteristic of a coagulopathy with
increasing transfusion volumes that typically
occurs after replacement of two to three blood
volumes (20 to 30 units).62,63 Although platelet
counts, coagulation parameters, and levels of
selected clotting parameters correlate with the
volume transfused, contrary to expectations
from a simple dilutional model, the relation-
ship is marked by tremendous variability.
Moreover, there is frequently discordance be-
tween the laboratory assessment and the clini-
cal evidence of bleeding. It has been suggested
that the platelet deficits play a more important
role in causing the bleeding than the coagula-
tion deficiencies.
MVB typically occurs when the platelet
count falls below 50,000 to 60,000/µL. Howev-
er, no simple relationship can be determined
between a patient’s coagulation test results
and the onset of bleeding. The etiology of
bleeding (elective surgery vs massive trauma)
may play a role as well.64
Subsequent studies have refined these
observations. Significant platelet dysfunction
has been demonstrated in massively trans-
fused patients.65,66 Low fibrinogen and platelet
counts are better predictors of hemostatic fail-
ure than elevations of prothrombin time (PT)
and partial thromboplastin time (PTT), sug-
gesting that consumptive coagulopathy is an
important factor in MVB in addition to dilu-
tion.63 Similarly, Harke and Rahman67 showed
that the degree of platelet and clotting abnor-
malities correlates with the length of time that
the patient is hypotensive, suggesting that
shock is the most important cause of DIC. In
aggregate, Collins68 concluded that “coagulop-
athy in heavily transfused patients was due to
hypoperfusion, not transfusion.” More recent-
ly, more powerful hemostatic assays have been
 CHAPTER 27 Noninfectious Complications of Blood Transfusion
introduced that may be more predictive of
blood component requirements.69
These data may not be generalizable to
patients undergoing massive transfusion in
the controlled setting of the operating room,
where hypotension caused by volume loss is
prevented. In this context, coagulation factor
levels may have priority over platelet prob-
lems. Murray and colleagues64 documented
that excessive bleeding in elective surgery pa-
tients transfused with more than one blood
volume (RBCs and crystalloid) corresponded
to a prolongation in PT and PPT compared to
patients with normal hemostasis.
TREATMENT AND PREVENTION. The dilu-
tional model of coagulopathy in massive
transfusion suggests that prophylactic re-
placement of hemostatic components based
on the volume of RBCs or whole blood trans-
fused prevents the development of a bleeding
diathesis. No specific regimen has yet been
shown to be superior to any other in prospec-
tive studies, and this is a controversial topic.70
According to AABB guidelines, there are insuf-
ficient data to recommend for or against a 1:1
RBC to plasma transfusion ratio in massive
transfusion.71
Previously, the predominant thinking was
that the replacement of platelets and coagula-
tion factors in the massively transfused surgi-
cal and trauma patient should be based on the
identification of a specific abnormality using
platelet counts, international normalized ra-
tio, activated PTT, and fibrinogen levels. Fre-
quent monitoring of these laboratory values
serves to avoid overuse of platelets and plasma
products (FFP and Cryoprecipitated AHF) by
anticipating the specific components needed
while avoiding dilutional coagulopathy. It is
imperative that the laboratory provide results
of these tests rapidly. Intraoperative and post-
operative laboratory testing, such as thrombo-
elastography, may be useful.
USE OF ADJUNCT THER APIES IN MASSIVE
TRANSFUSIONS. Recombinant Factor VIIa
(rFVIIa), a 50-kDa analog of Factor VIIa, is li-
censed in the United States for the treatment
with inhibitors of bleeding in patients with he-
䡲 685
mophilia A or B. However, the off-label use of
rFVIIa is growing in several areas, including to
treat hemorrhagic bleeding in trauma and sur-
gery.72,73 The recombinant product works by
targeting the site of tissue damage, where it
binds to tissue factor. This complex activates
Factor X to Factor Xa and, ultimately, Factor
IXa. In patients with hemophilia, rFVIIa by-
passes the need for Factor VIII or Factor IX
through the activation of the small amounts of
Factor X on activated platelet surfaces at the
site of injury. Studies of the efficacy of rFVIIa
have produced mixed results.74 In the absence
of definitive data, transfusion services should
establish guidelines on the reasonable use of
rFVIIa. In contrast, antifibrinlytics may have a
role in controlling massive bleeding from trau-
ma. The 2010 CRASH-2 study concluded that
tranexamic acid should be given as early as
possible in the trauma patient.75
Air Embolism
Air embolism can occur if blood in an open
system is infused under pressure or if air en-
ters a central catheter while containers or
blood administration sets are being changed.
Air embolism has been reported in association
with intraoperative and perioperative blood
recovery systems that allow air into the blood
infusion bag. The minimum volume of air em-
bolism that is potentially fatal for an adult is
approximately 100 mL.76 Symptoms include
cough, dyspnea, chest pain, and shock.
If air embolism is suspected, the patient
should be placed on the left side with the head
down to displace the air bubble from the pul-
monic valve. Aspiration of the air is sometimes
attempted. However, proper use and inspec-
tion of infusion pumps, equipment for blood
recovery or apheresis, and tubing couplers are
still essential to prevent this complication.
Hypothermia
Blood warmers may be used to prevent hypo-
thermia. Proper procedures for the use of
blood warmers should be followed because
686 䡲 AABB TECHNICAL MANUAL
overheating may damage or destroy red cells,
causing hemolysis and serious transfusion re-
actions, including fatalities. Blood warmers
must have a temperature monitor and
warning system to decrease the risk of over-
heating.5(p6)
DE LAYE D TR ANSFU SION
REA CTI O N S
Delayed transfusion reactions occur days to
months or even years after transfusion. As with
acute transfusion reactions, the consequences
may be severe but are often treatable.
DHTRs
Presentation
Fever and anemia occurring days to weeks af-
ter transfusion of an RBC component are char-
acteristic of a DHTR. The hemolysis associated
with a DHTR is usually not brisk, but some pa-
tients may develop jaundice and leukocytosis.
In a DHTR, the hemolysis is primarily extra-
vascular, so although hemoglobinuria may oc-
cur in rare cases, acute renal failure and DIC
are not generally present. In some cases, the
hemolysis occurs without causing clinical
symptoms. These patients present with unex-
plained anemia or do not experience an in-
crease in hemoglobin concentrations follow-
ing transfusion.
Differential Diagnosis
Fever with hemolysis may also occur well after
transfusion when the component has been
contaminated with an intracellular red cell
parasite, such as malaria or babesiosis. Fever
without hemolysis may be an indication of
graft-vs-host disease (GVHD) [described in the
transfusion-associated (TA)-GVHD section be-
low] or transfusion-transmitted viral disease
(see Chapter 8). Hemolysis resulting from anti-
body production by donor passenger lympho-
cytes may occur after transplantation of a
minor ABO-incompatible organ (eg, trans-
plantation of a group O liver in a group A pa-
tient).
Pathophysiology
After transfusion, transplantation, or pregnan-
cy, a patient may make an antibody to a red
cell antigen that he or she lacks. Red cell anti-
bodies may cause a delayed transfusion reac-
tion if the patient subsequently receives a unit
of blood expressing the corresponding red cell
antigen. Primary alloimmunization occurs
anywhere from days to months after a transfu-
sion of antigen-positive RBCs, depending on
the immunogenicity and dose of the antigen.
Approximately 1% to 1.6% of RBC transfu-
sions are associated with antibody formation,
excluding antibodies to antigens in the Rh sys-
tem. D-negative blood is usually transfused to
D-negative patients, so the frequency attribut-
able to anti-D is relatively low. Newly formed
alloantibodies are routinely detected during
pretransfusion screening (see Chapters 15 and
16). Recently transfused or pregnant patients
must have samples drawn for compatibility
testing within 3 days of the scheduled transfu-
sion to ensure identification of any potential
new alloantibodies.5(p36) A 5-year retrospective
study of alloimmunization showed that 11 of
2932 patients (0.4%) had developed new anti-
bodies, including anti-E, anti-K, and anti-Jka,
within 3 days of their transfusion.77
DHTRs and delayed serologic transfusion
reactions (DSTRs; rapid development of allo-
antibody in the absence of laboratory evi-
dence of hemolysis) rarely, if ever, occur as a
result of primary immunization and are gener-
ally associated with subsequent transfusions.
Antibody titers may slowly decrease after the
initial immune response, with as many as 30%
to 40% of alloantibodies becoming undetect-
able over months to years. Antibodies against
antigens of some blood group systems, such as
the Kidd system, frequently exhibit this behav-
ior. Subsequent transfusion of an antigen-pos-
itive unit triggers an anamnestic response,
with production of antibody occurring over
the next several days to weeks after the trans-
fusion. The rapidity of antibody production
and hemolytic potential of the antibody com-
bine to influence the clinical presentation.
Blood group antibodies associated with
DHTRs/DSTRs include those of the Kidd,
 CHAPTER 27 Noninfectious Complications of Blood Transfusion
Duffy, Kell, and MNS systems, in order of de-
creasing frequency.78(pp358-89)
In a DHTR/DSTR, antibodies may be
found in the serum, on transfused red cells, or
both. Routine antibody screening and anti-
body identification should be possible. If
transfused red cells are still present, the DAT
result may be positive. When the DAT result is
positive, an eluate should be performed and
the antibody identified. If a segment from the
unit is available, antigen typing may confirm
the diagnosis.
Frequency
As with AHTRs, the estimated rate of DHTRs
varies widely from study to study. Some of this
variation is the result of the practice of consid-
ering DSTRs and DHTRs as one category. Also,
improvements in laboratory techniques have
contributed to the increased number of DSTRs
detected. It is known that these reactions oc-
cur much more frequently than AHTRs, with
estimates of approximately 1:2500 for either
type of delayed reaction and a frequency that
is twice as high for DSTRs.79 These reactions
are likely to be greatly underrecognized be-
cause most patients do not undergo red cell
antibody screening following transfusion.80
Treatment
The treatment of DHTRs consists of monitor-
ing the patient and providing appropriate sup-
portive care. The most frequent therapy is
correction of the anemia by transfusing anti-
gen-negative RBCs as needed. When a DSTR is
identified by the laboratory, the patient’s phy-
sician and the transfusion service director
should be notified so that unrecognized he-
molysis may be appropriately identified and
treated.
Prevention
DHTRs/DSTRs caused by known antibody
specificities can be prevented by the transfu-
sion of antigen-negative RBCs. It is essential to
obtain prior transfusion records for the recipi-
ent because alloantibodies may have been
identified that are no longer detectable but re-
䡲 687
quire transfusion of antigen-negative RBCs.
Many institutions have programs to provide at
least partially phenotypically matched blood
for patients with sickle cell disease and some
other patients who have developed multiple
alloantibodies. Patients with sickle cell disease
may develop a complication known as “sickle
cell HTR,” where autologous and allogeneic
cells are destroyed (see Chapter 23).
Refractoriness to Platelet Transfusions
See Chapter 18.
TA-GVHD
Presentation
The clinical manifestations of TA-GVHD typi-
cally begin 8 to 10 days after transfusion, al-
though symptoms can occur as early as 3 days
and as late as 30 days. Signs and symptoms in-
clude a maculopapular rash, fever, enterocoli-
tis with watery diarrhea, elevated liver func-
tion test results, and pancytopenia. The rash
begins on the trunk and progresses to the
extremities. In severe cases, bullae may
develop.81
Unlike GVHD after allogeneic marrow
transplantation, TA-GVHD leads to profound
marrow aplasia, with a mortality rate higher
than 90%. The time course of the reaction is
very rapid; death typically occurs within 1 to 3
weeks of the first symptoms.
Differential Diagnosis
Because the clinical manifestations of TA-
GVHD appear several days after a transfusion,
it may be difficult to associate the patient’s
symptoms with the transfusion. The symp-
toms can easily be attributed to other condi-
tions, including drug reactions and viral ill-
ness. In cases of TA-GVHD, a skin biopsy
reveals a superficial perivascular lymphocytic
infiltrate, necrotic keratinocytes, compact or-
thokeratosis, and bullae formation. Molecular
techniques, including HLA typing, cytogenet-
ics, and chimerism assessment, can be used to
make the diagnosis.82
688 䡲 AABB TECHNICAL MANUAL
Pathophysiology
Billingham83 has proposed three requirements
for GVHD to develop in a patient. First, there
must be differences in the HLA antigens
expressed between the donor and the recipi-
ent. Second, immunocompetent cells must be
present in the graft. Finally, the host must be
incapable of rejecting the immunocompetent
cells.
In TA-GVHD, viable transfused lympho-
cytes mount an immunologic attack against
the transfusion recipient. Consistent with Bill-
ingham’s work, the three primary factors that
determine the risk of developing TA-GVHD are
the degree of recipient immunodeficiency,
number of viable T lymphocytes in the trans-
fusion, and degree of a population’s genetic di-
versity. Risk factors for developing TA-GVHD
include leukemia, lymphoma, use of immuno-
suppressive drugs administered for transplant
or myeloablative chemotherapy, congenital
immunodeficiency disorders, and the neona-
tal state.84,85
The number of viable lymphocytes in a
transfusion can be affected by the age, leuko-
cyte-reduction status, and irradiation status of
the component.86 Unfortunately, the mini-
mum number of viable lymphocytes required
to cause TA-GVHD is unknown. Although cur-
rent leukocyte-reduction technologies signifi-
cantly reduce the number of lymphocytes in a
component, leukocyte reduction does not
eliminate the risk of TA-GVHD. There are well-
documented cases in which transfusion of leu-
kocyte-reduced blood components have
caused TA-GVHD.87
Although patients who are immunocom-
promised are at risk of developing TA-GVHD,
the disorder has also been reported in transfu-
sion recipients with an intact immune sys-
tem.82 TA-GVHD can occur after a transfusion
from a donor who is homozygous for an HLA
haplotype to a heterozygous recipient (one-
way haplotype match). In this circumstance,
the recipient’s immune system is unable to
recognize the HLA-homozygous transfused
lymphocytes as foreign. In contrast, the trans-
fused lymphocytes are able to recognize the
host cells as foreign and are able to mount an
immunologic attack on the host.
The degree of genetic diversity in a popu-
lation also affects the risk of developing TA-
GVHD. For example, the estimated risk of de-
veloping TA-GVHD ranges from 1:874 in Japan
to 1:16,835 in France.82 The difference is the re-
sult of the lower diversity in HLA antigen ex-
pression in the Japanese population than in
the French population.
Treatment
Treatment of TA-GVHD has been attempted
with a variety of immunosuppressive agents.
Unfortunately, the disorder is almost uniform-
ly fatal; only rare cases of successful treatment,
many involving some form of stem cell trans-
plant, have been reported. Therefore, empha-
sis is placed on prevention of the disorder.
Prevention
The only reliable way to prevent TA-GVHD is
by irradiation of cellular blood components.
AABB Standards requires a minimum dose of
25 Gy (2500 cGy) delivered to the central por-
tion of the container and a minimum of 15 Gy
(1500 cGy) elsewhere.5(p25) AABB Standards also
requires irradiation of cellular blood compo-
nents when 1) the patient is identified as being
at risk of TA-GVHD, 2) the donor is a blood rel-
ative of the recipient, and 3) the donor is se-
lected for HLA compatibility by typing or
crossmatching.5(p40) These standards are mini-
mum requirements for irradiation of cellular
blood components, and institutions may
choose to administer irradiated components
to other categories of patients (see Table 27-2).
Posttransfusion Purpura
Presentation
Posttransfusion purpura (PTP) is a relatively
uncommon complication of transfusion, and
its true incidence is therefore difficult to esti-
mate. Nonetheless, more than 200 cases have
been reported in the literature, and data from
the Serious Hazards of Transfusion (SHOT)
program in the United Kingdom suggest that
 CHAPTER 27 Noninfectious Complications of Blood Transfusion 䡲 689

TABLE 27-2. Clinical Indications for Irradiated Components

Well-documented indications
Intrauterine transfusions
Prematurity, low birthweight, or erythroblastosis fetalis in newborns
Congenital immunodeficiencies
Hematologic malignancies or solid tumors (neuroblastoma, sarcoma, Hodgkin disease)
Peripheral blood stem cell/marrow transplantation
Components that are crossmatched, HLA matched, or directed donations (from family members or other
related donors)
Fludarabine therapy
Granulocyte components
Potential indications
Other malignancies, including those treated with cytotoxic agents
Donor-recipient pairs from genetically homogeneous populations
Usually not indicated
Patients with human immunodeficiency virus
Full-term infants
Nonimmunosuppressed patients

the disorder may be more common than previ-
ously believed.87 Hospitals that participated in
SHOT reported 44 cases of PTP in 8 years.
Patients typically present with wet purpura
and thrombocytopenia 9 days (range = 1-24),
on average, after a transfusion.88 The thrombo-
cytopenia is often profound, with platelet
counts of <10,000/µL. Bleeding from mucous
membranes and the gastrointestinal and uri-
nary tracts is common. Mortality rates in large
case series range from 0 to 12.8%, primarily
due to intracranial hemorrhage.67,68
PTP has most commonly been associated
with transfusions of RBCs or whole blood;
however, the disorder has also been associated
with transfusions of platelets or plasma.
Differential Diagnosis
The differential diagnosis of PTP includes con-
sideration of other causes of thrombocytope-
nia, such as autoimmune thrombocytopenic
purpura, thrombotic thrombocytopenic pur-
pura, heparin-induced thrombocytopenia,
DIC, and drug-induced thrombocytopenia. Al-
though the diagnosis of PTP can be obvious in
patients with previously normal platelet
counts and no other significant medical ab-
normalities, it can be a challenge in patients
with multiple medical problems. Platelet se-
rology studies may aid in the diagnosis.
Pathophysiology
The pathogenesis of PTP is related to the pres-
ence of platelet-specific alloantibodies in a pa-
tient who has previously been exposed to
platelet antigens via pregnancy or transfusion.
The female-to-male ratio of affected patients
is 5 to 1. Antibodies against human platelet an-
tigen 1a (HPA-1a), located on glycoprotein
IIIa, are identified in about 70% of PTP cases.
Antibodies to HPA-1b, other platelet antigens,
and HLA antigens have also been implicated
in PTP.88
The reason for the concomitant destruc-
tion of autologous platelets in this disorder is
unknown; three theories have been advanced.
According to the first theory, immune com-
plexes bind to platelets through the Fc recep-
tor, causing destruction of platelets.89 The sec-
ond theory posits that the patient’s platelets
690 䡲 AABB TECHNICAL MANUAL
absorb a soluble platelet antigen from donor
plasma, making them susceptible to immune
destruction. According to the third theory, the
platelet alloantibody has autoreactivity that
develops when a patient is re-exposed to a
foreign platelet-specific antigen.88 The third
theory currently has the most support.
The use of leukocyte reduction filters may
decrease the incidence of PTP. In the three
years prior to the implementation of universal
leukocyte reduction, in the United Kingdom,
there were 10.3 cases of PTP per year, com-
pared to 2.3 cases per year after universal leu-
kocyte reduction (p<0.001).87
Treatment
Because the duration of thrombocytopenia in
untreated patients is about 2 weeks, it can be
difficult to assess the effectiveness of therapies
for PTP. Steroids, whole-blood exchange, and
plasma exchange have all been used to treat
PTP. The current treatment of choice for PTP is
IVIG.88 Patients respond within 4 days, on av-
erage, and some respond within hours.
Prevention
PTP typically does not recur with subsequent
transfusions. However, there are four case re-
ports of PTP recurrence. Therefore, for pa-
tients with previously documented PTP, efforts
should be made to obtain components from
antigen-matched donors. Autologous dona-
tions and directed donations from antigen-
matched donors and family members may
also be appropriate. Because PTP has also oc-
curred after transfusions of deglycerolized re-
juvenated or washed RBCs, such manipula-
tions are not indicated for the prevention of
recurrence.88
Iron Overload
A unit of RBCs contains approximately 250 mg
of iron. The average rate of excretion of iron is
approximately 1 mg per day. As red cells are
destroyed, the majority of the released iron
cannot be excreted and is stored in the body as
hemosiderin and ferritin. Transferrin becomes
saturated after the administration of 10 to 15
RBC units to a nonbleeding patient.90 As iron
accumulates in the reticuloendothelial system,
liver, heart, spleen, and endocrine organs, tis-
sue damage leading to heart failure, liver fail-
ure, diabetes, and hypothyroidism may occur.
Patients who are chronically transfused for
diseases such as thalassemia, sickle cell dis-
ease, and other chronic anemias are at greatest
risk for iron overload. Prevention of the accu-
mulation of these toxic levels of iron by reduc-
ing the body’s iron stores through the use of
iron chelators is therefore extremely impor-
tant. A cumulative dose of 50 to 100 RBC units
can cause significantly greater morbidity and
mortality than the underlying anemia.91
Iron chelators bind to iron in the body
and tissues and help remove it through the
urine and/or feces. The development of iron-
chelating drugs, such as parenteral deferox-
amine and the oral agent deferiprone, greatly
reduced the complications of iron overload in
chronically transfused patients, leading to im-
proved quality of life. Deferiprone is more ef-
fective than deferoxamine in reducing myo-
cardial siderosis.92
The newest agent, deferasirox, has a
much longer half-life than deferoxamine and
may be administered in one daily oral dose. Al-
though in-vivo studies are in the early stages,
deferasirox has similar rapid access to intracel-
lular iron stores in cultured myocytes as defer-
iprone.93 Unlike deferiprone, which has occa-
sionally been associated with agranulocytosis,
the most frequent side effects of deferasirox
are transient gastrointestinal distress and
mildly increased creatinine levels that are rare-
ly of clinical significance. Deferasirox treat-
ment for a median of 2.7 years showed efficacy
in children and adults with sickle cell disease,
resulting in a significant dose-dependent de-
cline in their serum ferritin levels without pro-
ducing any new adverse events.94 Lower doses
of deferasirox have also been used to reduce
iron overload in non-transfusion-dependent
patients with minimal side effects.94
 CHAPTER 27 Noninfectious Complications of Blood Transfusion 䡲 691

TABLE 27-3. How to Contact the FDA

Method Contact Details

E-mail fatalities2@fda.hhs.gov
Telephone/voicemail 240-402-9160
Fax 301-827-6748, Attn: CBER Fatality Program Manger
Express mail US Food and Drug Administration
CBER Office of Compliance and Biologics Quality
Document Control Center
10903 New Hampshire Avenue
WO71, G112
Silver Spring, MD 20993-0002
FDA = Food and Drug Administration; CBER = Center for Biologics Evaluation and Research.

FAT A L IT Y REP O R T IN G
REQ UI REME N TS
When the death of a patient results from a re-
action to or complication of a transfusion, cur-
rent good manufacturing practice regulations
require that the fatality be reported to the FDA
by the facility that performed the compatibili-
ty testing.95 The director of the Office of Com-
pliance and Biologics Quality at the FDA Cen-
ter for Biologics Evaluation and Research must
be notified as soon as possible by telephone,
express mail, or facsimile or electronic mail,
followed by the submission of a written report
within 7 days. Table 27-3 lists the contact in-
formation for the FDA. The report should con-
tain the patient’s medical records, including
laboratory reports, and the autopsy results
when available. The patient’s underlying ill-
ness may make determination of the cause of
death difficult. If there is any clinical suspicion
that the transfusion may have contributed to
the patient’s death, that possibility should be
investigated. Most transfusion-associated fa-
talities are caused by acute hemolysis, TRALI,
or TACO. Investigations of these cases must at-
tempt to rule out laboratory, transfusion ser-
vice, or blood administration errors.96

KEY POINTS

1. The blood supply is safer today than at any time in history. Hemovigilance and blood man-
agement programs decrease the risk of noninfectious complications of transfusion.
2. Many transfusion reactions have signs or symptoms that may be present in more than one
type of reaction. Early recognition of the reaction, prompt cessation of the transfusion, and
further evaluation are key to the successful resolution of a reaction.
3. Acute intravascular hemolytic reactions are often caused by sample or patient misidentifi-
cation and are thus, for the most part, preventable.
4. Allergic reactions range from urticaria (hives) to anaphylaxis. Patients experiencing severe
reactions should be checked for IgA deficiency and transfused accordingly.
5. Recognition of TRALI requires diagnosis by exclusion. Most patients recover from TRALI
with supportive care.
6. TACO can be confused with TRALI because both feature pulmonary edema. TACO should
be suspected in nonbleeding patients and those with congestive heart failure.
692 䡲 AABB TECHNICAL MANUAL

7. The most common complications of massive transfusion are hemostatic abnormalities.

Each institution should develop its own massive transfusion protocol that takes into ac-
count the availability of appropriate laboratory testing.
8. TA-GVHD has a much more acute and severe course than GVHD after marrow or stem cell
transplantation. TA-GVHD is fatal in >90% of cases and can be prevented by irradiation of
blood components.
9. PTP is a serious but rare complication in which antibodies to human platelet antigens result
in destruction of autologous and allogeneic platelets.
10. Iron overload is perhaps the longest-lasting long-term complication of transfusion. Oral
iron chelators greatly increase compliance with therapy.
11. TRALI is the leading cause of transfusion-related mortality reported to the FDA.
12. Recipient fatalities must be reported to the FDA by the compatibility testing facility as soon
as possible after a fatal complication of transfusion has been confirmed.

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95. Code of federal regulations. Title 21, CFR Part
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 C h a p t e r 2 8

Approaches to Blood
Utilization Auditing

Alan Tinmouth, MD, FRCPC, MSc, and

Simon Stanworth, FRCP, FRCPath, DPhil

A U D I T I N G T H E U S E of blood trans- ical staff regarding improvements in transfu-

fusions is a required function for all hos-
pital transfusion services.1 Both The Joint
Commission and AABB require health-care in-
stitutions to monitor the use of blood compo-
nents. The Joint Commission also requires
hospitals to collect data to monitor the perfor-
mance of processes that involve risks or may
result in sentinel events, including the use of
blood and blood components (Standard
PI.3.1.1).2 The AABB requires all facilities to
have a peer-review program “that monitors
and addresses transfusion practices for all cat-
egories of blood and components” including
“usage and discard” and “appropriateness of
use.”3(p91) In addition, federal regulations per-
taining to Medicare and Medicaid require that
hospitals make recommendations to the med-
sion procedures.1
Other countries also require hospitals to
establish processes to audit and monitor
transfusion practices. For example, in Canada,
both the Canadian Standard Association and
the Canadian Society for Transfusion Medicine
require hospitals to monitor blood transfusion
use, and compliance with the Canadian Stan-
dard Association’s requirements will be man-
dated by federal law. In England, the Depart-
ment of Health’s Better Blood Transfusion
initiatives from 1997 to 2012 and the subse-
quent Patient Blood Management program
have promoted and continued to encourage
hospital participation in national audits. This
includes a specific program of National Com-
parative Audits for hospitals in transfusion.

Alan Tinmouth, MD, FRCPC, MSc, Head, General Medicine and Transfusion Medicine, The Ottawa Hospital,
and Scientist, University of Ottawa Centre for Transfusion Research, Clinical Epidemiology Program, Ottawa
Hospital Research Institute, Ottawa, Ontario, Canada, and Simon Stanworth, FRCP, FRCPath, DPhil, Consul-
tant Haematologist, Oxford University Hospitals NHS Trust, and Honorary Senior Clinical Lecturer, University
of Oxford, Oxford, United Kingdom
A. Tinmouth is supported by a Research Award from the Department of Medicine, The Ottawa Hospital; and
has disclosed a financial relationship with Amgen. S. Stanworth has disclosed no conflicts of interest.

697
698 䡲 AABB TECHNICAL MANUAL
Factors that help ensure high levels of partici-
pation in national audits in England are:
1. The audits contribute to “Quality Account”
reports required of National Health Service
(NHS) hospitals (Health Act 2009).
2. Data are used by the Care Quality Commis-
sion, an independent regulator of all health
and social care services in England.
3. NHS hospitals participating in a national
audit may receive a discount from the NHS
Litigation Authority, which manages negli-
gence and other claims against the NHS in
England.
4. The National Blood Transfusion Committee
oversees, promotes, and supports all
national audits.
In a similar vein, The Joint Commission
announced a certification program starting in
2014 to further recognize accredited hospitals
that implement system-wide measures to im-
prove blood utilization.
A number of different methods can be
employed to meet the requirements to moni-
tor or audit blood transfusion use. However,
the general objective of all blood utilization
programs is to ensure the appropriate use of
blood components. Alternative or comple-
mentary goals may include reducing inappro-
priate use and, as a result, reducing costs.
Historically, audits of blood use have concen-
trated on controlling or reducing the total
number of blood components transfused and/
or individual “overtransfusions.” This focus
was based on the assumption that inappropri-
ate use consisted primarily of overtransfusion.
Unnecessary transfusions result in unneces-
sary costs and adverse events. However, with
increased attention on limiting patients’ expo-
sure to blood components, undertransfusion
may be a concern. In addition, the dose of
blood transfusion requests, which may result
in overuse or underuse, might also need to be
assessed in audits. However, neither of these
issues has historically been a major focus of
the monitoring of blood transfusion utiliza-
tion.4
The methods used to audit blood use de-
pend on the desired objectives. Monitoring
trends in the total number of blood units
transfused, perhaps to control ordering or in-
ventory management, requires a different pro-
cess from that required to limit inappropriate
transfusion requests. In all instances, the
transfusion service (technologists and/or phy-
sicians) and the institutional transfusion med-
icine or blood utilization committee must
work together to perform the appropriate au-
dit(s) and follow-up.
R ATIONALE FOR MONITO RING
B LO OD U T I LI Z AT I O N
The primary motivation for monitoring blood
utilization is to identify instances when blood
product utilization is less than optimal. Inter-
ventions can then be implemented to change
transfusion practice. Therefore, audits or mon-
itoring must be combined with a recognized
process to effectively provide feedback on the
findings to the appropriate health-care profes-
sionals.
Improving or ensuring optimal use of
blood transfusions is important for several
reasons. Blood is a biologic agent associated
with many possible adverse events, including
both infectious and noninfectious complica-
tions.5,6 Many of these complications are well
known, others are not usually recognized by
physicians or the general population, and
some potential complications are still not fully
understood. Unnecessary complications from
inappropriate blood transfusions need to be
avoided. In addition, blood components are a
scarce and expensive resource. Transfusing a
single unit of Red Blood Cells (RBCs) has been
estimated to cost from $400 to $760 when all
the associated costs are included, and many
regions have experienced shortages.6-8
Through the careful monitoring of blood
utilization, instances of inappropriate blood
component use can be identified and correc-
tive actions can be taken. If a “real-time” or
prospective audit system is used, then a mem-
ber of the transfusion medicine service can in-
tervene, which may result in a change in the
transfusion request before the issue of a blood
unit. A retrospective review does not alter the
current transfusion episode but it does identi-
 CHAPTER 28 Approaches to Blood Utilization Auditing
fy issues that can be addressed through inter-
ventions designed to change future transfu-
sion practice.
A variety of interventions have been used
to change transfusion practice (Table 28-1).
One of the most common interventions for
change is audit and feedback, which is defined
by the Cochrane Effective Practice and Organi-
zation of Care Group as “any summary of clini-
cal performance of health care over a specified
period of time.” However, there is less under-
standing of the effective elements of audit and
feedback or how the feedback should be struc-
tured to produce changes in behavior in differ-
ent settings. Following the delivery of an
intervention, ongoing monitoring allows for
assessment of the sustained effectiveness of
the intervention and need for ongoing or addi-
tional intervention.
In summary, audits serve the dual func-
tion of 1) identifying areas of concern in the
use of blood products and 2) monitoring inter-
ventions or changes in the identified areas.
T Y PE S OF TRANSFUSION
AUDI TS
Audits of transfusion practice can examine in-
dividual blood transfusion requests and/or ag-
gregate transfusion data. Reviews of individual
requests can be performed either prospective-
ly in real time or retrospectively. Reviews of in-
dividual transfusions may also be performed
(retrospectively) very shortly after the event
TABLE 28-1. Use of Interventions to Change Transfusion Practice with Different Types of Audits
Intervention
Individual education/feedback
Group education/feedback/teaching
Guideline dissemination
Incorporation of reminders/guidelines into
transfusion request form or computerized
order entry system
++ = very complementary to audit type; + = can be complementary to audit type; – = less complementary or not complemen-
tary to audit type.
䡲 699
(usually within 24 hours); this has been termed
a “concurrent review” because it still affords
the ability to provide timely feedback on indi-
vidual transfusion episodes.9 More remote ret-
rospective audits allow for the review of aggre-
gate transfusion data, which can then be
analyzed in several ways. Reviews of individual
and aggregate transfusion data are not mutu-
ally exclusive. Because they may serve differ-
ent functions or purposes, they can, in fact, be
complementary.
Prospective (“Real-Time”) Audits
In a prospective audit, individual transfusion
requests are reviewed in “real time” (ie, before
the issue of the blood component). An elec-
tronic review using a computer-based algo-
rithm and/or a manual review by technolo-
gists is undertaken whereby the request is
compared to local transfusion audit criteria.
Clinical data (eg, hematocrit and indication
for RBC transfusion) are required to perform
the review. Ideally, this information is obtained
from the clinical staff as part of the transfusion
request process10,11 or pretransfusion blood
values are automatically retrieved from the
laboratory information system.12,13 With com-
puter provider/physician order entry (CPOE),
the institutional guidelines can be incorporat-
ed into the request process.11 Laboratory staff
can also obtain these data from the laboratory
information system,14 although this is more
labor intensive and difficult to implement
Prospective Audit Concurrent Audit Retrospective Audit
++ ++ +
– – ++
+ + +
++ ++ +
700 䡲 AABB TECHNICAL MANUAL
universally.10 Transfusions that do not meet
the audit criteria are flagged and the request-
ing physician is contacted by the transfusion
medicine service before the blood component
is issued to discuss the need for the transfu-
sion.
A prospective audit system must be orga-
nized such that undue delays in filling transfu-
sion requests do not occur. For example, when
CPOE is used to screen the appropriateness of
a transfusion request that does not comply
with the institution’s guidelines, that request
may still be completed after the ordering clini-
cian enters the rationale into the information
system.11 Delays in obtaining laboratory re-
sults and the uncertainties of clinical cases
also need to be identified and reflected in the
audit process.
Given these issues and constraints, the
criteria for undertaking a review as part of a
prospective audit may need to be less rigid
than the guidelines for the optimal utilization
of blood components because prospective au-
dits might delay some transfusions that fail to
meet audit criteria for appropriateness.13
Therefore, a mechanism must be in place to
ensure that emergency and urgent transfu-
sions are not unduly delayed. Specific clinical
areas, such as emergency departments and
operating suites, where delays in filling trans-
fusion requests might result in adverse clinical
events might be excluded from prospective
transfusion audits.
Prospective audits also have the potential
to generate animosity in the ordering physi-
cian when a transfusion request is questioned.
Engaging requesting physician groups and
broadly consulting them during the develop-
ment of the audit guidelines and process can
help reduce friction and ensure the long-term
success of a prospective audit system. In addi-
tion, interactions with the requesting physi-
cian often require the involvement of a trans-
fusion medicine physician or, at least, a senior
technologist.14,15 To reduce the potentially
onerous time requirements for technologists
and physicians in a prospective audit system,
selective audits of either specific clinical areas
(eg, obstetrics or orthopedics) and/or specific
time periods may be used.
The potential benefits of a prospective
audit include the ability to intervene directly
and to change a transfusion request before the
component is issued. As a result, an unneces-
sary transfusion may be stopped12 or a more
appropriate component or dose may be or-
dered.16 Ideally, the immediate intervention
stemming from a prospective audit also results
in long-term changes in the transfusion prac-
tice of the ordering physician. However, the
benefits of this immediate intervention must
be weighed against the additional work re-
quired by the transfusion service, including
the transfusion medicine physician.
A number of reports from single institu-
tions have demonstrated the effectiveness of
prospective audits in reducing the total num-
ber of units transfused,14,15,17 the number of
units transfused per patient,18-20 the propor-
tion of patients transfused,18,19,21,22 and the
number of inappropriate transfusions19,22,23
(Table 28-2). Many of these studies used pro-
spective audits in conjunction with other in-
terventions to change transfusion prac-
tice.14,15,17-20,22,23 Although these reports attest to
the potential utility of prospective audits to
change transfusion practice, the sustainability
of the changes is not known. In one study, the
initial reduction in inappropriate transfusions
following the implementation of a prospective
audit system did not persist when inappropri-
ate transfusion rates were reexamined 3 years
later.27
Concurrent Audits
A concurrent audit is a review of an individual
transfusion request that occurs in the 12 to 24
hours following the transfusion episode.9,13 As
such, it involves processes similar to those of
the prospective audit. However, because a
concurrent audit is a posttransfusion review, it
cannot lead to any alterations in the individual
transfusion event. Therefore, the concurrent
audit is designed only to alter future transfu-
sion practice. However, follow-up with the re-
questing physician occurs while the transfu-
sion event remains fresh in his or her memory.
It is hoped that this immediate contact im-
TABLE 28-2. Studies of the Effectiveness of Prospective and Retrospective Audits in Changing Transfusion Practice

Reduction in Reduction in Reduction in Reduction in

Blood Inappropriate Proportion of Number of Units Total Number of
Study Interventions Components Transfusions Patients Transfused per Patient Units Transfused

Cohn 201313 Concurrent audit RBCs 5%-14% per month - - -

24
Arnold 2011 Education, form, audit/feedback FFP 14% - - -
15
Tavares 2011 Education, prospective audit FFP 80.8%
14
Sarode 2010 Prospective audit, guidelines, RBCs - - - 12.6%
CHAPTER 28

education FFP - - - 59.9%

Platelets - - - 25.4%
Yeh 200617 Prospective audit, audit/ RBCs - - - 15%
feedback FFP - - - 74%
Platelets - - - 14%
Rubin 200125 Audit/feedback, education RBCs 2% - - -
26
Capraro 2001 Audit/feedback, education RBCs - 26% - -
FFP 7%
Platelets 6%
Tobin 200127 Prospective audit, guidelines RBCs +4% (increased use) - -
FFP +13% (increased use)
Platelets +14% (increased use)
Approaches to Blood Utilization Auditing

Hameedullah 200028 Audit/feedback, guidelines, edu- FFP - 21% - -

cation
Rehm 199818 Prospective audit, form RBCs - - 26% -
䡲

29
Joshi 1997 Retrospective audit, guidelines RBCs 20.7% 50% - -
(Continued)
701
TABLE 28-2. Studies of the Effectiveness of Prospective and Retrospective Audits in Changing Transfusion Practice (Continued)
702

Reduction in Reduction in Reduction in Reduction in

Blood Inappropriate Proportion of Number of Units Total Number of
䡲

Study Interventions Components Transfusions Patients Transfused per Patient Units Transfused

Tuckfield 199723 Prospective audit, form RBCs 13% - - -

FFP 10% - - -
Platelets 10% - - -
Cheng 199619 Prospective audit, form FFP 7% - 35% 31%
Platelets 10% - 22% 17%
Morrison 199330 Retrospective audit, education, RBCs - - 12% 62%
guidelines, form
Littenberg 199522 Prospective audit, guidelines RBCs - 4.1% - 1%
31
Brandis 1994 Retrospective audit, education RBCs - - 29% 19%
21
Hawkins 1994 Prospective audit FFP - - 55% -
Rosen 199332 Retrospective audit, form, RBCs - - 27% 21%
AABB TECHNICAL MANUAL

guidelines FFP - - 18% 9%

Platelets - - 23% 15%
Ayoub 198933 Retrospective audit, education, FFP - - - 46%
guidelines
Giovanetti 198834 Retrospective audit, guidelines RBCs 67% 10.9% - -
20
Solomon 1988 Prospective audit, retrospective FFP - - - 52%
audit, guidelines, education, form
Shanberge 198735 Concurrent audit, education, FFP - - - 77%
guidelines
Handler 198336 Retrospective audit, education RBCs - - - -
RBCs = Red Blood Cells; FFP = Fresh Frozen Plasma.
 CHAPTER 28 Approaches to Blood Utilization Auditing
proves the chance of changing the physician’s
future transfusion behavior.
As with the prospective audit, the concur-
rent review can be conducted by a computer
algorithm or manually using transfusion audit
criteria. The review may occur at the time of
the transfusion or shortly after the request is
filled. Requests that do not meet the audit cri-
teria are flagged for subsequent review by a se-
nior technologist or transfusion medicine phy-
sician. When performing the review, the
transfusion medicine physician may have ac-
cess to additional laboratory results that help
to determine the appropriateness of each re-
quest. However, the reviewer must also con-
sider the fact that these additional results were
not available to the ordering physician at the
time he or she made the request. Because con-
current audits do not delay the filling of trans-
fusion requests, these audits can assess urgent
transfusions and use stricter criteria for appro-
priateness than prospective audits.
Following the identification of an inap-
propriate transfusion request, the ordering
physician may be contacted by telephone or e-
mail to discuss the case. The less immediate
nature and ability to use written communica-
tion may reduce any animosity from the order-
ing physician and result in a more meaningful
dialogue. Shanberge and colleagues35 report-
ed a 77% reduction in the number of Fresh
Frozen Plasma units transfused after the intro-
duction of a concurrent audit system com-
bined with a new guideline and an education
program.
Conducting concurrent audits is time
consuming, and a transfusion medicine physi-
cian usually needs to review inappropriate
transfusion requests and follow up with the re-
questing physicians.13 For these reasons, per-
forming a concurrent audit of all transfusion
requests may not be feasible. Using selective
audits, as discussed for prospective audits, is
an option to reduce the workload involved.
Concurrent audits may be particularly relevant
for certain patient groups, such as patients
treated for trauma or others in the emergency
department, where the urgent need for blood
precludes a prospective audit, and accurate
time lines (key to determining the appropriate
䡲 703
implementation of massive transfusion proto-
cols) or communication with the transfusion
service might not be readily recalled in retro-
spective audits.
Retrospective Audits
Retrospective audits of blood utilization are
commonly performed by hospital teams or
blood transfusion services. The results should
be reviewed by the hospital’s transfusion med-
icine committee. The frequency of such audits
varies from one institution to another. Com-
puterized systems may facilitate ongoing utili-
zation review across many specialties.11 Unlike
prospective and concurrent reviews, retro-
spective audits can be used to examine aggre-
gate transfusion data and trends in transfusion
utilization. The results may be informative for
understanding wider differences in transfu-
sion practice across different specialty groups.
Individual transfusion requests can also be
reviewed for appropriateness as part of a retro-
spective audit, but doing so may be cumber-
some unless the data are collected prospec-
tively or the laboratory information system
can link data on transfusion events and labo-
ratory results.
Retrospective reviews can analyze data in
a variety of ways. The simplest analysis is an
examination of the total numbers of blood
components transfused and patients trans-
fused. However, these data might show con-
siderable temporal variability that limits
meaningful analysis. Further analysis is re-
quired to make observations regarding
differences in blood utilization. The mean or
median number of units transfused per hospi-
talized patient and/or procedure provides a
more meaningful summary of blood compo-
nent use. Simply dividing the total number of
units transfused by the total number of pa-
tients who received a transfusion is not an ap-
propriate statistical analysis; any observed
changes in total blood component use, partic-
ularly during a short period, could be skewed
by a small number of patients who required a
large number of units. The proportion of pa-
tients transfused should also be determined
and can be further examined by procedure or
704 䡲 AABB TECHNICAL MANUAL
clinical specialty. All of these analyses com-
prise the minimal set required to monitor dif-
ferences in blood utilization. Additional analy-
ses could include assessments of appropriate
and inappropriate transfusion rates, although
these analyses require collecting additional
clinical data and reviewing individual transfu-
sion requests, which may be labor-intensive
exercises.
Utilization trends by individual product,
individual physician, or clinical service can be
analyzed. For health-care institutions with
multiple sites, similar clinical services at
different sites can also be compared. These
additional layers of analysis may provide im-
portant information to identify variations in
practice and detect inappropriate transfusion
practices. These areas could then be targeted
by interventions to improve transfusion prac-
tice.
In general, a retrospective review is less
labor intensive than a prospective or concur-
rent review, particularly because it involves
less immediate attention from a technologist
or transfusion medicine physician. The
amount of time required to perform the review
depends on the amount of detail desired.
The use of retrospective audits and the
provision of dedicated feedback to clinicians
have been effective in reducing the total num-
ber of units transfused,20,24,29-33 number of units
transfused per patient,30-32,37,38 proportion of
patients transfused,26,28,36 and number of inap-
propriate transfusions24,29,34 (Table 28-2). The
long-term durability of these changes in trans-
fusion practice following the introduction of a
retrospective audit has not been evaluated.
I N TE RVE NT I O NS TO C H A N G E
TR A N SF U SI O N P R ACT I C E
As previously described, the results of transfu-
sion audits should ideally be used in conjunc-
tion with interventions to effect change and
improve transfusion practice. Thus, audits
should be considered a critical component of a
larger process to optimize the utilization of
blood components. This process can be more
widely conceived as dissemination, knowledge
transfer, or implementation work, where the
goal is to increase the uptake of research re-
sults and/or best practices into daily prac-
tice.39
The process of closing the gap between
knowledge and action involves first distilling
knowledge, usually in the form of guidelines.
National or international guidelines can sim-
ply be adopted; however, local development of
guidelines with the involvement of local stake-
holders may increase adherence to guide-
lines.40 Audits then serve the purpose of identi-
fying gaps between the guidelines (best
practices) and current practice (action). When
important gaps are identified through audits,
interventions can be developed to target prac-
tice changes. To improve the chance of achiev-
ing meaningful and lasting changes in prac-
tice, interventions should be carefully chosen
and designed.
Unfortunately, the process of identifying
the best interventions has not been well de-
fined. Ideally, the facilitators of and barriers to
change are first identified so that any selected
intervention targets these identified fac-
tors.41,42 Concurrent,13 retrospective,37 and
prospective21 audits have been effective indi-
vidual interventions in changing transfusion
practice. However, other interventions, includ-
ing dissemination of guidelines,11,20,28,30,32-35 ed-
ucation,15,20,24,30,33,36 introduction of new trans-
fusion request forms, and computer order
entry,24,30,32 have been commonly used in con-
junction with audits.
EF FE CTIVE N ES S OF
M ON I TO R I N G A N D
I NT E RV E N T IO N S TO C H A N G E
TRANSF USION PRAC TICE
Most published studies on auditing efforts
have shown that prospective and retrospective
audits reduce either the total amount of blood
transfused or the proportion of inappropriate
transfusions (Table 28-2). These reductions
occurred in studies that used a concurrent au-
dit alone,13 a prospective audit alone,21 or a ret-
rospective audit and feedback alone.37 The
prospective audit study showed a reduction in
the utilization of RBCs and frozen plasma but
 CHAPTER 28 Approaches to Blood Utilization Auditing
not platelets.21 The retrospective audit study
showed a reduction in the utilization of RBCs
but not frozen plasma or platelets.37 The re-
sults from these two studies might suggest that
prospective and retrospective audits used in
conjunction with other interventions are more
effective in improving blood utilization. How-
ever, the poor designs of the studies (almost all
of which were uncontrolled, single-center, be-
fore-and-after studies) that examined inter-
ventions to change transfusion practice and
the possibility of publication bias (eg, because
the results of studies in which an intervention
did not result in an improvement in transfu-
sion practice were not published) do not allow
the true or relative effectiveness of individual,
or combinations of, interventions to be deter-
mined.43,44
SE L E C T I N G A N AUD I T P RO C E S S
TO MO NITO R T R A N SF U S I ON S
How transfusions should be monitored de-
pends on a number of factors that are specific
to each institution.45 The first step in deter-
mining how to monitor transfusions is to de-
cide which type of audits to use. The prospec-
tive and concurrent audits serve identical
purposes and cannot be used jointly to assess
an individual transfusion episode. Prospective
audits require the greatest amount of time
from laboratory staff and transfusion medi-
cine physicians, including 24-hour coverage
and support from the laboratory information
system. These requirements may present diffi-
culties for smaller hospitals and busy transfu-
sion medicine services, respectively. In gener-
al, concurrent reviews are more practical in
smaller hospitals or hospitals with limited re-
sources for laboratory staff or transfusion
medicine physicians because transfusions can
be reviewed in the 12 to 24 hours following the
transfusion episode. If a universal prospective
audit of all blood components is not feasible,
then the prospective audit could be limited to
particular components (eg, intravenous im-
mune globulin or recombinant Factor VIIa) or
specific clinical areas. Retrospective reviews
supplement prospective or concurrent audits
䡲 705
of individual transfusions by providing data on
trends in the use of transfusions.
Lists of the steps for implementing pro-
spective, concurrent, and retrospective re-
views are provided in Tables 28-3, 28-4, and
28-5, respectively. A transfusion request form
or order entry system incorporating guidelines
and/or clinical information (see sample in Ap-
pendix 28-1) can aid in identifying inappropri-
ate transfusion requests through either pro-
spective or concurrent audits.46 Similarly
laboratory information systems and comput-
er algorithms can be used to screen transfu-
sions for appropriateness in all types of au-
dits.13,47 Reviews may be more frequent in
larger transfusion services or less frequent in
smaller hospitals. The retrospective data can
allow smaller hospitals to compare their trans-
fusion practices (eg, number of units trans-
fused per patient by diagnosis-related group)
with those of other similar institutions.
CO N CLUS IO N S
Auditing transfusion practice is a required
function for all transfusion services. Transfu-
sion audits provide information on levels of
compliance with standards and frequency of
unnecessary transfusions. Audits combined
with guidelines are clearly essential to evaluate
transfusion practices at individual institutions,
and they permit the identification of subopti-
mal transfusion practices.
In general, the findings of most audits
continue to show unnecessary use of blood
outside established guidelines. The translation
of research findings into hospital practice is
often slow and haphazard, and this applies as
much to transfusion as to other branches of
health care. Many interventions are undertak-
en by hospitals to change their transfusion
practices, but there are real uncertainties
about their effectiveness and durability.
There is a need for research that defines
the determinants of appropriate transfusion
behavior to better guide the design and selec-
tion of interventions that can produce opti-
mal changes in transfusion practice. Ideally,
the selection of audits and interventions
should be based on an assessment of local
706 䡲 AABB TECHNICAL MANUAL

TABLE 28-3. Steps for Implementing a Prospective Audit System to Monitor Blood Component
Utilization
1. The extent and frequency of the audit process is determined.
䡲 The audit may be performed on all blood components or only on specific blood products.
䡲 Areas with urgent needs for transfusion (eg, emergency or operating rooms) may be excluded from the
audit to avoid delays in transfusion for their patients.
䡲 To limit resource demands, a selective audit may be used. For example, transfusion requests may be
audited only from a single ward, selected on a rotating basis, because of its high transfusion volume or a
perceived problem in its transfusion practice.
䡲 Similarly, audits may be performed only during specific hours to limit off-hour work by laboratory staff
and transfusion medicine physicians.
2. The requirements for clinical information at the time the transfusion requests are met.
䡲 This may be part of the request form (Appendix 28-1) or computer order entry.
3. Criteria for appropriate or inappropriate indications are developed for transfusions.
䡲 Audit criteria should be less stringent than optimal transfusion guidelines to reduce audit workload and
conflicts with requesting physicians.
䡲 Audit criteria should be developed in conjunction with a transfusion medicine committee and various
medical specialists who use significant amounts of blood products to increase acceptance of transfusion
audits.
4. Transfusion requests are screened by laboratory staff, transfusion nurses, or a computer algorithm to iden-
tify inappropriate requests.
5. For all inappropriate transfusion requests, the requesting physician is contacted by the transfusion medi-
cine service.
䡲 The requesting physician is usually contacted by a transfusion medicine physician or senior technologist.
䡲 A predefined mechanism is developed to override/bypass a review if blood is required urgently and to
avoid unacceptable delays in filling a transfusion request.
6. Any decisions to change the transfusion request are made in conjunction with the ordering physician.

TABLE 28-4. Steps for Implementing a Concurrent Audit System to Monitor Blood Component
Utilization
1-4. The same as for the Prospective Audit.
5. All inappropriate transfusion requests are subsequently reviewed by senior laboratory staff or a transfu-
sion medicine physician who discusses the transfusion request with the requesting physician.
䡲 Contact with the requesting physician is made within 24 hours of the transfusion so that the physician
remembers the clinical details. This time frame is optimal for providing feedback and potentially chang-
ing future transfusion behavior.
䡲 Contact can be made by telephone or electronically.
 CHAPTER 28 Approaches to Blood Utilization Auditing 䡲 707

TABLE 28-5. Steps for Implementing a Retrospective Audit System to Monitor Blood Component
Utilization
1. Determine the extent and frequency of the audit reviews.
䡲 The frequency of the data reviews needs to be based on the volume of transfusions and resources avail-
able. Records should not be reviewed more than 6 months after transfusions were administered.
䡲 The components to be reviewed need to be determined and should include all conventional components
and frequently transfused blood derivatives.
2. Determine the outcomes.
䡲 Totals for the numbers of transfusions and patients transfused are the simplest data to collect but pro-
vide a limited understanding of, and ability to monitor, changes in transfusion utilization. Providing data
on utilization by patient (ie, mean or median number of units per patient) and proportion of patients
transfused provides a greater understanding of utilization.
䡲 The appropriateness of transfusions can also be reported if clinical and/or laboratory data are available.
These can be aggregate data from prospective or concurrent reviews, or the laboratory information sys-
tem can link laboratory data to data on transfusion events.
3. Review the audit data.
䡲 Audit data should be reviewed by the transfusion medicine service and the transfusion medicine commit-
tee.
䡲 Data may be analyzed on individual physicians, departments, or diagnoses.
䡲 Data may be compared within the institution or with data from similar institutions to evaluate overall utili-
zation rates for blood components.
䡲 Data should be shared with relevant departments and physicians.
4. Determine whether any additional interventions to optimize transfusion practice are warranted.

barriers to and facilitators of change. Further fective in certain circumstances or to compare

audits then allow the monitoring of any different interventions, including their cost-ef-
changes in transfusion practice. fectiveness. Nonetheless, audits remain a criti-
Future studies are required to determine cal part of the process to evaluate blood utili-
what interventions are likely to be the most ef- zation and improve transfusion practice.

K EY PO I NTS

1. Auditing the use of blood components is a necessary function for all transfusion medicine
services and is required by AABB and The Joint Commission. The main purpose of auditing
blood utilization is to assess the appropriate use of components and help optimize their
use.
2. Different forms of audits (prospective, concurrent, or retrospective) may be used depending
on the objectives of the audit and the resources available. In all cases, providing feedback to
the users, using other interventions, or both are necessary to effect change in transfusion
practice.
3. Prospective audits require the review of transfusion requests before issue of components,
which permits the opportunity to intervene and stop or change an inappropriate transfu-
sion request. Individual transfusion requests are reviewed using prespecified audit criteria
(eg, guidelines).
708 䡲 AABB TECHNICAL MANUAL

4. Concurrent audits review individual transfusion requests in the 12 to 24 hours following a

transfusion episode and involve similar processes as the prospective audit. The individual
transfusion events cannot be altered, as they have already occurred, but feedback to the
physician within a short period after the transfusion offers the opportunity to change future
transfusion practice.
5. Retrospective audits of transfusion offer the opportunity to review aggregate transfusion
data. Reviewing individual transfusions for appropriateness may be more difficult, given
the remoteness of the transfusion event. Feedback to individual clinicians or clinical servic-
es should be part of a retrospective audit process to help ensure the optimal use of compo-
nents.
6. All forms of audits can be used alone to effect changes in transfusion practice. Audits may
also be combined with other interventions such as dissemination of guidelines, education,
and the introduction of new transfusion request forms. Ideally the selection of interventions
includes local stakeholders and an assessment of local factors that may affect the effective-
ness of interventions.
7. Published studies evaluating the effect of prospective and retrospective audits on transfu-
sion practice have generally demonstrated a reduction in either the total amount of blood
transfused or the proportion of inappropriate transfusions. Because of the poor quality of
published studies, the true relative effectiveness of individual interventions or combina-
tions of interventions cannot be inferred.

RE FE RE N CES

1. Mintz PD. Quality Assessment and improve-
ment of blood transfusion practice. In: Mintz
PD, ed. Transfusion therapy: Clinical princi-
ples and practice. 3rd ed. Bethesda, MD: AABB
Press, 2011:813-36.
2. Comprehensive accreditation manual for hos-
pitals (CAMH): The official handbook. Oak-
Brook Terrace, IL: The Joint Commission,
2014.
3. Levitt J, ed. Standards for blood banks and
transfusion services. 29th ed. Bethesda: AABB,
2014.
4. Saxena S, Wehrli G, Makarewicz K, et al. Moni-
toring for underutilization of RBC compo-
nents and platelets. Transfusion 2001;41:587-
90.
5. Popovsky M, ed. Transfusion reactions. 4th ed.
Bethesda, MD: AABB Press, 2012.
6. Tinmouth AT, Fergusson DA, Chin-Yee IH, et
al. Clinical consequences of red cell storage in
the critically ill. Transfusion 2006;46:2014-27.
7. Amin M, Fergusson D, Wilson K, et al. The so-
cietal unit cost of allogenic red blood cells and
red blood cell transfusion in Canada. Transfu-
sion 2004;44:1479-86.
8. Shander A, Hofmann A, Ozawa S, et al. Activi-
ty-based costs of blood transfusions in surgical
patients at four hospitals. Transfusion 2010;50:
753-65.
9. Becker J, Shaz B for the Clinical Transfusion
Medicine Committee and the Transfusion
Medicine Section Coordinating Committee.
Guidelines for patient blood management and
blood utilization. Bethesda, MD: AABB, 2011.
10. Haspel RL, Uhl L. How do I audit hospital
blood product utilization? Transfusion 2012;
52:227-30.
11. Baer VL, Henry E, Lambert DK, et al. Imple-
menting a program to improve compliance
with neonatal intensive care unit transfusion
guidelines was accompanied by a reduction in
transfusion rate: A pre-post analysis within a
multihospital health care system. Transfusion
2011;51:264-9.
12. Yazer MH, Waters JH. How do I implement a
hospital-based blood management program?
Transfusion 2012;52:1640-5.
13. Cohn CS, Welbig J, Bowman R, et al. A data-
driven approach to patient blood manage-
ment. Transfusion 2014;54:316-22.
14. Sarode R, Refaai MA, Matevosyan K, et al. Pro-
spective monitoring of plasma and platelet
transfusions in a large teaching hospital re-
sults in significant cost reduction. Transfusion
2010;50:487-92.
 CHAPTER 28 Approaches to Blood Utilization Auditing
15. Tavares M, DiQuattro P, Nolette N, et al. Re-
duction in plasma transfusion after enforce-
ment of transfusion guidelines. Transfusion
2011;51:754-61.
16. Rothschild JM, McGurk S, Honour M, et al. As-
sessment of education and computerized de-
cision support interventions for improving
transfusion practice. Transfusion 2007;47:228-
39.
17. Yeh CJ, Wu CF, Hsu WT, et al. Transfusion audit
of fresh-frozen plasma in southern Taiwan.
Vox Sang 2006;91:270-4.
18. Rehm JP, Otto PS, West WW, et al. Hospital-
wide educational program decreases red
blood cell transfusions. J Surg Res 1998;75:183-
6.
19. Cheng G, Wong HF, Chan A, et al. The effects of
a self-educating blood component request
form and enforcements of transfusion guide-
lines on FFP and platelet usage. Queen Mary
Hospital, Hong Kong. British Committee for
Standards in Hematology (BCSH). Clin Lab
Haematol 1996;18:83-7.
20. Solomon RR, Clifford JS, Gutman SI. The use of
laboratory intervention to stem the flow of
fresh-frozen plasma. Am J Clin Pathol 1988;
89:518-21.
21. Hawkins TE, Carter JM, Hunter PM. Can man-
datory pretransfusion approval programmes
be improved? Transfus Med 1994;4:45-50.
22. Littenberg B, Corwin H, Gettinger A, et al. A
practice guideline and decision aid for blood
transfusion. Immunohematology 1995;11:88-
94.
23. Tuckfield A, Haeusler MN, Grigg AP, et al. Re-
duction of inappropriate use of blood prod-
ucts by prospective monitoring of transfusion
request forms. Med J Aust 1997;167:473-6.
24. Arnold DM, Lauzier F, Whittingham H, et al. A
multifaceted strategy to reduce inappropriate
use of frozen plasma transfusions in the inten-
sive care unit. J Crit Care 2011;26:636.
25. Rubin GL, Schofield WN, Dean MG, et al. Ap-
propriateness of red blood cell transfusions in
major urban hospitals and effectiveness of an
intervention. Med J Aust 2001;175:354-8.
26. Capraro L, Syrjala M. Advances in cardiac sur-
gical transfusion practices during the 1990s in
a Finnish university hospital. Vox Sang 2001;
81:176-9.
27. Tobin SN, Campbell DA, Boyce NW. Durability
of response to a targeted intervention to modi-
fy clinician transfusion practices in a major
teaching hospital. Med J Aust 2001;174:445-8.
䡲 709
28. Hameedullah, Khan FA, Kamal RS. Improve-
ment in intraoperative fresh frozen plasma
transfusion practice—Impact of medical au-
dits and provider education. J Pak Med Assoc
2000;50:253-6.
29. Joshi G, McCarroll M, O’Rourke P, et al. Role of
quality assessment in improving red blood cell
transfusion practice. Ir J Med Sci 1997;166:16-
19.
30. Morrison JC, Sumrall DD, Chevalier SP, et al.
The effect of provider education on blood uti-
lization practices. Am J Obstet Gynecol 1993;
169:1240-5.
31. Brandis K, Richards B, Ghent A, et al. A strategy
to reduce inappropriate red blood cell transfu-
sion. Med J Aust 1994;160:721-2.
32. Rosen NR, Bates LH, Herod G. Transfusion
therapy: Improved patient care and resource
utilization. Transfusion 1993;33:341-7.
33. Ayoub MM, Clark JA. Reduction of fresh frozen
plasma use with a simple education program.
Am Surg 1989;55:563-5.
34. Giovanetti AM, Parravicini A, Baroni L, et al.
Quality assessment of transfusion practice in
elective surgery. Transfusion 1988;28:166-9.
35. Shanberge JN. Reduction of fresh-frozen plas-
ma use through a daily survey and education
program. Transfusion 1987;27:226-7.
36. Handler S. Does continuing medical educa-
tion affect medical care. A study of improved
transfusion practices. Minn Med 1983;66:167-
80.
37. Lam HT, Schweitzer SO, Petz L, et al. Are retro-
spective peer-review transfusion monitoring
systems effective in reducing red blood cell
utilization? Arch Pathol Lab Med 1996;120:
810-16.
38. Lam HT, Schweitzer SO, Petz L, et al. Effective-
ness of a prospective physician self-audit
transfusion-monitoring system. Transfusion
1997;37:577-84.
39. Graham ID, Logan J, Harrison MB, et al. Lost in
knowledge translation: Time for a map? J Con-
tin Educ Health Prof 2006;26:13-24.
40. Harrison MB, Graham ID, Fervers B. Adapting
knowledge to a local context. In: Straus S, Tet-
roe J, Graham ID, eds. Knowledge translation
in health care. Oxford: Blackwell Publishing,
2009;73-82.
41. Cabana MD, Rand CS, Powe NR, et al. Why
don’t physicians follow clinical practice guide-
lines? A framework for improvement. JAMA
1999;282:1458-65.
710 䡲 AABB TECHNICAL MANUAL
42. Legare F, O’Connor AM, Graham ID, et al. Pri-
mary health care professionals’ views on barri-
ers and facilitators to the implementation of
the Ottawa Decision Support Framework in
practice. Patient Educ Couns 2006;63:380-90.
43. Wilson K, MacDougall L, Fergusson D, et al.
The effectiveness of interventions to reduce
physician’s levels of inappropriate transfusion:
What can be learned from a systematic review
of the literature. Transfusion 2002; 42:1224-9.
44. Tinmouth A, MacDougall L, Fergusson D, et al.
Reducing the amount of blood transfused: A
systematic review of behavioral interventions
to change physicians’ transfusion practices.
Arch Intern Med 2005;165:845-52.
45. Qu L, Kiss JE. Blood utilization review. In: Sax-
ena S, ed. The transfusion committee: Putting
patient safety first. 2nd ed. Bethesda, MD:
AABB Press, 2013:75-91.
46. Hannon T, Gross I. Transfusion guidelines: De-
velopment and impact on blood management.
In: Saxena S, ed. The transfusion committee:
Putting patient safety first. 2nd ed. Bethesda,
MD: AABB Press, 2013:121-44.
47. Audet AM, Goodnough LT, Parvin CA. Evaluat-
ing the appropriateness of red blood cell
transfusions: The limitations of retrospective
medical record reviews. Int J Qual Health Care
1996;8:41-9.
 CHAPTER 28 Approaches to Blood Utilization Auditing 䡲 711

䡲 APPENDIX 28-1
Transfusion Order Form in Use at St. Vincent Indianapolis Hospital Since 2001

Used with permission from Hannon T, Gross I. Transfusion guidelines: Development and impact on blood management. In:
Saxena S, ed. The transfusion committee: Putting patient safety first. 2nd ed. Bethesda, MD: AABB Press, 2013:121-44.
 C h a p t e r 2 9

The Collection and Processing of

Hematopoietic Stem Cells

Scott A. Koepsell, MD, PhD; Eapen K. Jacob, MD; and

David H. McKenna Jr, MD

HEMATOPOIETIC STEM CELLS (HSCs) poietic precursors to generate a niche that

are primitive pluripotent cells capable of
self-renewal and differentiation into any cells
of hematopoietic lineage (lymphocytes, mono-
cytes, granulocytes, erythrocytes, and plate-
lets), including committed and lineage-
restricted progenitor cells, unless otherwise
specified, regardless of tissue source [eg, mar-
row, mobilized peripheral blood, or umbilical
cord blood (UCB)].1 Clinically, HSCs are able to
fully reconstitute the functions of marrow
when transplanted into susceptible recipients.
For this reason, HSC transplantation has been
increasingly utilized to treat a diverse array of
hematologic and nonhematologic diseases
and conditions.
In vivo, HSCs are concentrated in the
marrow, where mesenchymal elements—such
as osteogenic progenitor cells, osteoblasts, ad-
ipocytes, mesenchymal stem/stromal cells,
and endothelial cells—interact with hemato-
supports and regulates hematopoiesis.2
Although the cell-surface antigen CD34 is
not specific to HSCs, CD34 is used to identify
and quantitate HSCs by flow cytometry in cel-
lular products intended for use in transplanta-
tion. Historically, CD34 quantitation by flow
cytometry suffered from significant interin-
strument and interprotocol variability, a limi-
tation that has been addressed by more rigor-
ous quality control (QC) procedures and assay
standardization.3,4
C L I NI C A L U T I L IT Y
Myriad indications exist for HSC transplanta-
tion that range from nonneoplastic immune
disorders to malignancies. An abbreviated list
is presented in Table 29-1. In general, indica-
tions vary with patient age because immuno-
deficiencies and inborn errors of metabolism

Scott A. Koepsell, MD, PhD, Assistant Professor, Department of Pathology and Microbiology, University of
Nebraska Medical Center, Omaha, Nebraska; Eapen K. Jacob, MD, Assistant Professor, Department of Labora-
tory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota; and David H. McKenna Jr, MD, Associate
Professor, Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Min-
neapolis, Minnesota
S. Koepsell and E. Jacob have disclosed no conflicts of interest. D. McKenna has disclosed a financial relation-
ship with Novartis, Inc.

713
714 䡲 AABB TECHNICAL MANUAL

TABLE 29-1. Diseases Treated with are more common in a pediatric population,
Autologous or Allogeneic Transplantation whereas the number of patients with a clonal
disorder in their marrow or a hematologic ma-
Hematologic Cancers lignancy is greater in adults. Ultimately, the
decision to perform HSC transplantation re-
Leukemia
quires a complex integration of many vari-
Lymphoma ables. These variables include patient goals,
prognosis, disease progression, previous ther-
Myeloma
apy, age, availability of a suitable HSC source
Marrow Failure States/Clonal Disorders of (ie, marrow, mobilized peripheral blood, or
Marrow UCB), and type of transplant (ie, autologous vs
allogeneic, and myeloablative vs nonmyeloab-
Aplastic anemia
lative).
Fanconi anemia
Autologous Transplantation
Pure red cell aplasia
In general, autologous HSC transplantation is
Amegakaryocytosis
used for hematopoietic rescue after high-dose
Paroxysmal nocturnal hemoglobinuria antineoplastic therapy. The antitumor effect of
the transplant comes solely from the chemo-
Myelofibrosis
therapy and radiotherapy used during the con-
Myelodysplasia ditioning phase of transplantation. For older
patients who are not traditional candidates for
Inborn Errors of Metabolism/Congenital Immuno-
autologous transplantation or patients with
deficiency
other significant morbidities, reduced-intensi-
Mucopolysaccharidoses ty induction chemotherapy can broaden the
clinical utility of HSC transplantation.
Leukodystrophies Donor requirements for autologous
Osteopetrosis transplants are based on the donor’s disease
state. The patient must be healthy enough to
Severe combined immunodeficiency syndrome undergo mobilization (as described later in
Wiskott-Aldrich syndrome this chapter) and procurement of HSCs either
by peripheral blood apheresis collection or
Pediatric Cancers marrow aspiration. Significant levels of prior
Wilms tumor chemotherapy, radiation, or ongoing marrow
disease involvement may make the mobiliza-
Neuroblastoma tion and collection of HSCs unfeasible due to
Ewing sarcoma the reduced quality or number of HSCs.
Eligibility requirements are not mandated
Medulloblastoma by the Food and Drug Administration (FDA)
Rhabdomyosarcoma for autologous HSC transplantation [Title 21,
Code of Federal Regulations (CFR) Part
Hemoglobinopathies 1271.90], so the use of screening question-
Thalassemia
naires to identify relevant communicable dis-
ease is not required. However, a general health
Sickle cell disease assessment is needed per AABB Standards for
Cellular Therapy Product Services (CT Stan-
Autoimmune Diseases
dards).1 AABB CT Standards also requires labo-
Solid Tumors ratory testing for human immunodeficiency
virus (HIV) 1/2; hepatitis B virus; hepatitis C
 CHAPTER 29
virus; syphilis; human T-cell lymphotropic vi-
rus, Types I and Type II (HTLV-I/II); and cyto-
megalovirus (CMV) because the autologous
products are cryopreserved and stored with
other products, so the presence of these virus-
es would pose a contamination risk.
Allogeneic Transplantation
Indications for allogeneic HSC transplantation
vary. However, in general, allogeneic trans-
plantation is used to treat malignant condi-
tions only when both the induction antineo-
plastic therapy and the transplanted cells, due
to the graft-vs-neoplasm (GVN) effect, are
therapeutic. For patients with an inborn error
of metabolism, congenital immunodeficiency,
or other diseases and conditions in which
germline mutations are present in the patient’s
cells, allogeneic HSC transplants offer thera-
peutic benefit by helping to replace the defi-
cient cellular machinery.
In the allogeneic setting, screening and
infectious disease testing are mandated in the
United States to determine whether the trans-
plantation of mobilized peripheral blood or
UCB poses a risk of transmission of a relevant
communicable disease to the recipient [Title
21 CFR 1271.3(r)]. This screening and testing
includes the administration of a screening
questionnaire, a physical examination, a re-
view of the relevant medical records, and ap-
plicable testing [Title 21, CFR Parts 1271.3(s)
and 21 CFR 1271 Subpart C]. Although marrow
products are administered under Sections 375
and 379 of the Public Health Services Act, mar-
row screening and testing are very similar to
mobilized peripheral blood and UCB screen-
ing and testing because the administration of
all of these products is governed by the stan-
dards of various accrediting bodies, such as
the AABB, the Foundation for the Accredita-
tion of Cellular Therapy (FACT),4 and the Na-
tional Marrow Donor Program (NMDP).5
For UCB transplantation, the screening
and testing process is performed on the moth-
er and her samples (see Chapter 30). Relevant
communicable diseases that can be transmit-
ted by transplanted HSCs to the recipient or to
the people who handle the components and
Hematopoietic Stem Cells 䡲 715
for which there is an FDA licensed screening
test [Title 21, CFR Part 1271.3(r)] include HIV,
hepatitis B and C, human transmissible spon-
giform encephalopathy, Treponema pallidum,
HTLV-I/II, and CMV. Donor questionnaires
have been developed to assist with screening6
and medical record review for these diseases
using FDA guidance documents.7
In the United States, infectious disease
testing for HSC donors must be performed in a
Clinical Laboratory Improvement Act certified
laboratory as mandated by the FDA. If screen-
ing or testing detects a risk of a relevant com-
municable disease, the potential HSC donor is
considered ineligible. All parties (the donor,
the recipient, and their physicians) are in-
formed of the donor’s eligibility status, and a
risk-benefit analysis is performed to deter-
mine whether the donor’s HSCs should be
used. If a decision is made to proceed with a
transplant of the ineligible donor’s HSCs, the
urgent medical need, as defined by the FDA
[Title 21, CFR Part 1271.3(u)], is documented.
Depending on the institution’s accrediting
body and the circumstances, such as when the
HSCs are from a related first- or second-degree
ineligible donor, the requirement for docu-
mentation of an urgent medical need may
vary. Finally, in addition to screening and test-
ing for infectious diseases, the donor’s medical
evaluation is used for determining whether
the donor’s health is adequate to allow that do-
nor to undergo the HSC mobilization and pro-
curement process (described below).
Histocompatibility
In addition to infectious diseases, donor char-
acteristics that may affect transplant out-
comes include histocompatibility with the
recipient, gender, age, parity, and ABO com-
patibility. Of these characteristics, histocom-
patibility is the most important. In general, if a
healthy HLA-matched related donor is avail-
able, that donor is selected instead of an HLA-
matched unrelated donor. However, recent
data have shown that in patients with acute
myeloid leukemia or myelodysplastic syn-
drome, an HSC transplant from an HLA-
716 䡲 AABB TECHNICAL MANUAL
matched, unrelated donor may be as effective
as a transplant from a sibling.8,9
Major histocompatibility antigens were
first studied in depth in various animal models
for skin transplantation.10(pp97-111) In humans,
these are HLA antigens and are categorized
into Class I (HLA-A, -B, and -C) and Class II
(HLA-DR, -DQ, and -DP). These highly poly-
morphic molecules are essential in determin-
ing graft survival as well as the likelihood that
the recipient will develop graft-vs-host dis-
ease (GVHD). As molecular techniques have
supplanted serologic methods, the resolution
has improved and the number of antigens that
can be compared has increased. Matching at
the antigen level has been replaced by allele-
level matching for the final selection of periph-
eral blood and marrow HSC components for a
given recipient.
HLA matching in HSC transplantation
has been reviewed in detail elsewhere and is
summarized here only briefly.11 HLA matching
has an important impact on outcomes, espe-
cially in low-risk patients. Allogeneic trans-
plantation using either mobilized peripheral
blood or marrow HSCs with high-resolution
(allele-level) mismatching at HLA-A, -B, -C,
and -DR1 is associated with a 5% to 10% de-
crease in survival with each mismatch.12 The
results are similar, although the evidence is a
bit less clear, for HSC grafts with mismatches
in Class II HLA-DQ and -DP. For mobilized
peripheral blood HSC grafts, allele-level mis-
matching is probably as detrimental to surviv-
al as antigen-level mismatching, although the
data come from smaller studies. Many centers
now match at high resolution for 8 to 10 loci
(HLA-A, -B, -C, -DR, and -DQ). High-resolu-
tion 8/8 and 10/10 matched transplants of
HSCs from unrelated donors are at least in part
responsible for the improvement in outcomes
of matched unrelated transplants compared to
matched related donors.12
Not surprisingly, HSC transplants in re-
cipients who have antibodies against donor
HLA [known as donor-specific antibodies
(DSAs)] have adverse outcomes.13 The level at
which DSAs have a significant impact is less
clear, as is the appropriate course of action
when DSAs are detected. Despite the difficulty
of predicting graft failure based on the pres-
ence of DSAs, screening HSC transplant candi-
dates for DSAs may become more routine as
more data emerge on this topic.14
UCB has several unique characteristics
with regard to HLA matching. The level of HLA
matching required for UCB is less stringent
than that required for marrow and mobilized
peripheral blood HSCs. Data on UCB indicate
that matching at 4 of 6 loci is sufficient for
HLA-A and -B at the antigen level and for HLA-
DRß1 at the allele level, provided that a suffi-
cient cell dose is achieved.15 As the number of
mismatched alleles increases (from one to
two), a higher total nucleated cell (TNC) dose
is needed to overcome their deleterious effect,
including the use of combined double-cord
transplants.16 In addition, early evidence indi-
cates that noninherited, maternal HLA may be
permissive when considering donor-recipient
mismatches.17 Unit-to-unit matching of 4 of 6
loci in the setting of double-cord blood trans-
plantation is also performed at various institu-
tions; however, firm evidence to support this
practice is not available at this time.
Other Donor Characteristics
For marrow and mobilized peripheral blood
HSC donors, other factors that may have a
positive effect on transplant outcomes include
male gender, younger age, nulliparity, ABO
and CMV status matching, and greater size of
the donor relative to the recipient. Other than
HLA, only donor age appears to be associated
with survival.11 ABO incompatibility has been
reviewed extensively. ABH antigens found on
red cells, platelets, and neutrophils would sug-
gest that ABO incompatibility would affect
outcomes. However, inconsistent results on
outcomes—such as survival, nonrelapse mor-
tality, GVHD, and graft failure—of both major
and minor incompatible transplants have
been observed.18 The risk of delayed red cell
engraftment, pure red cell aplasia, increased
transfusion requirements, and both immedi-
ate and delayed hemolysis is increased in ma-
jor ABO-incompatible allogeneic HSC trans-
plantation.
 CHAPTER 29
Other characteristics that are specific to
UCB HSC donation and may affect outcomes
include issues related to maternal history, unit
collection, processing, and unit storage.19
Characteristics of the UCB unit have been uti-
lized in a scoring system, the “Cord Blood Ap-
gar,” to determine the utility of the unit after
consideration of the TNC count, CD34 positiv-
ity, colony-forming-unit count, mononuclear
cell content, and volume.20
DE TE RMI N AT ION OF GRAF T
SOURCE
The choice of the source of HSCs for allogeneic
transplantation is determined by several vari-
ables, including the availability of an ade-
quately matched donor. As described above,
marrow and mobilized peripheral blood prod-
ucts in general need a higher level of HLA
matching than UCB units. For this reason, a
UCB transplant may be the best alternative
when only 1 and 2 allele-mismatched donor
units are available. In addition, for some cen-
ters, the relatively rapid availability of UCB
units compared to marrow and peripheral
blood products plays a substantial role in graft
choice in patients with an immediate need for
transplantation. A typical unrelated marrow or
peripheral blood donor search may take
months, whereas a search for UCB takes sever-
al days to a few weeks.21
GVHD and GVN Effect
GVHD and GVN effect, although linked in
terms of their cause, have opposite outcomes.
In GVHD, donor lymphocytes attack host-re-
cipient tissues in the skin, lung, liver, gastroin-
testinal tract, and other organs. GVHD may be
categorized as acute or chronic, and it occurs
in more than 40% of individuals who receive
an allogeneic HSC transplant. GVHD is associ-
ated with significant morbidity and mortality.
Not surprisingly, the risk of GVHD is related to
the graft source. Consequently, GVHD risk in-
creases as the lymphocyte content of the HSC
product increases. Various techniques have
been used to decrease the risk of GVHD, in-
Hematopoietic Stem Cells 䡲 717
cluding T-cell depletion, use of antithymocyte
globulin, and pharmacologic measures.22
The GVN effect is also thought to be driv-
en by donor lymphocytes. In recent years, mo-
bilized peripheral blood has greatly outpaced
marrow as an HSC source due to its easier col-
lection and the belief that GVN effect may be
improved. The first randomized controlled tri-
al comparing marrow and mobilized peripher-
al blood HSC grafts in unrelated donor mye-
loablative transplantations found that chronic
GVHD, but not acute GVHD, risk was higher in
patients who received a peripheral blood HSC
transplant.23
Kinetics
The kinetics of engraftment are affected by
many variables, such as degree of HLA match-
ing, dose of HSCs, and use of granulocyte
colony-stimulating factor (G-CSF). In addition,
the characteristics of the stem cell source may
play a role. For instance, stem cells in UCB ap-
pear to have a greater regenerative capacity
than those in marrow and peripheral blood.24
In general, the rate of engraftment is predicted
by the number of progenitor cells in each graft.
This is greatest in mobilized peripheral blood,
followed by marrow, UCB, and other HSC
sources.
In a meta-analysis of studies that com-
pared related transplantation with donor-mo-
bilized peripheral blood and marrow, the me-
dian time to neutrophil engraftment was 14 vs
21 days and to platelet engraftment was 14 vs
22 days, respectively.25 These findings have
been corroborated by a more recent meta-
analysis of studies of allogeneic related trans-
plantation in which mobilized peripheral
blood vs marrow HSC engraftment was 15 vs
21 days for neutrophils and 13 vs 21 days for
platelets, respectively.26 The more rapid en-
graftment of peripheral blood compared to
marrow HSCs was also seen in a study of unre-
lated allogeneic transplantation in which me-
dian neutrophil engraftment occurred at 15
and 19 days, respectively, and median platelet
engraftment occurred at 20 and 27 days, re-
spectively.27 These findings were confirmed in
the randomized controlled trial of Anasetti
718 䡲 AABB TECHNICAL MANUAL
and colleagues23 in which the relative differ-
ences in time to engraftment was 5 days short-
er for neutrophils and 7 days shorter for plate-
lets. A comparison of adult unrelated marrow
to UCB HSC grafts showed that engraftment
occurred earlier with both neutrophils (medi-
an of 18 days for matched marrow, 20 for mis-
matched marrow, and 27 for mismatched
UCB) and platelets (median of 29 days for
matched marrow and mismatched marrow,
and 60 days for mismatched UCB).28 In the set-
ting of reduced-intensity UCB transplantation,
median neutrophil engraftment times ap-
proaching those of mobilized peripheral blood
and marrow HSC transplants have been ob-
served.24
Survival
The most important outcome measure for any
transplant procedure is patient survival. For
recipients of transplants from matched related
donors, mobilized peripheral blood may offer
an advantage in overall and disease-free
survival over marrow HSC grafts, at least in re-
cipients with late-stage hematologic malig-
nancies.25 In recipients of myeloablative trans-
plants from unrelated donors for hematologic
malignancies, a recent randomized controlled
trial indicated that marrow and mobilized pe-
ripheral blood sources have equivalent effects
on survival, with marrow grafts associated
with less chronic GVHD but more graft fail-
ure.23
Based on these findings, the rapid in-
crease in the use of mobilized peripheral blood
relative to marrow in recent years may change.
This may not be true, however, in nonmye-
loablative patients or transplant recipients at
high risk of infection or graft failure. For pedi-
atric transplant recipients, marrow may actu-
ally be preferred over mobilized peripheral
blood, although reports differ.29,30 In addition,
when mismatched marrow and mismatched
UCB were compared, there was no difference
in the rate of overall mortality in adults with
leukemia; however, recipients of matched
marrow HSC grafts did have a lower overall
mortality rate.28
Transplant physicians face a complex
choice when determining which donor and
stem cell source is best for their patients based
on numerous factors, including the patient’s
disease, disease stage, age, and comorbidities.
As more data are collected, the choice of HSC
graft may evolve.
CO L L E C T I ON / S O U RC E S O F
H SC S
Regardless of the source of HSCs, standards of
both FACT and AABB require all institutions
that collect HSCs to have a procedure in place
to obtain informed consent from the donor or
the donor’s representative, as dictated by local
laws.1(pp16-17,20-21),4 The informed consent pro-
cess should include providing information to
the donor regarding the risks/benefits of the
procedure, the tests performed on the donor
that are designed to protect the recipient, al-
ternative collection methods, and protection
of their health information. In addition, the
donor should be given the opportunity to ask
questions as well as to refuse donation. The
risks that are specific to each collection proce-
dure are discussed below.
Another requirement for all facilities that
collect HSCs is to provide donor access to
medical care based on the risks and clinical
situation associated with each type of dona-
tion. Specifically, procedures should be in
place to provide medical or emergency care to
donors who experience adverse effects. Clear-
ance from the donor’s physician for HSC col-
lection should be documented.
Marrow HSC Collection
In addition to undergoing relevant donor
screening, infectious disease testing, and HLA
compatibility testing, marrow donors must
also be physically suitable for donation. Mar-
row harvest is an invasive procedure per-
formed under sterile conditions in the operat-
ing room under anesthesia. Therefore, the
donor must be able to tolerate the type of an-
esthesia required to successfully perform the
harvest. Another consideration is the donor’s
medical history. Autologous donors and some
 CHAPTER 29
allogeneic donors may have had previous radi-
ation therapy to the pelvis, which may limit
the amount of marrow available for harvest in
the posterior iliac crest. Similarly, previous
chemotherapy may limit the number of nucle-
ated cells that can be aspirated from the mar-
row space. For autologous donors, a signifi-
cant tumor burden in the marrow space is a
contraindication for collection of HSCs by
marrow harvest because the graft would be
contaminated by tumor cells.
Physically, the donor must be able to tol-
erate the volume loss associated with marrow
harvest, which means that young or small do-
nors may not be suitable. The Be The Match
Registry limits the volume collected from a
marrow donor to 20 mL/kg.5 Typically, the vol-
ume of the harvest requested is dictated by the
recipient’s weight, with a minimum of 2.0 to
3.0 × 108 nucleated cells/kg needed to facilitate
efficient engraftment. Thus, during harvest,
checking the TNC count midway through the
procedure can help with estimating the total
volume needed. Alternatively, CD34 quantita-
tion midway through the procedure or on the
final product for QC may also be performed,
depending on the institution’s policies.
The marrow harvest technique varies
considerably depending on institutional prac-
tice. In general, an 11- to 14-gauge needle on a
syringe flushed with anticoagulant is inserted
into the posterior iliac crest, and approximate-
ly 5 mL of marrow is aspirated. The needle and
syringe are then rotated to a different vector,
and the aspiration is repeated. Vigorous aspi-
ration is avoided to prevent significant periph-
eral blood contamination of the product. The
aspirated marrow is collected into a large col-
lection bag containing anticoagulant and me-
dia and/or an infusible-grade electrolyte solu-
tion. The process is repeated utilizing different
bone sites until the collection volume target,
based on TNC count or donor volume limit, is
reached.
Serious complications of marrow harvest
are rare. However, minor complications, such
as pain at the site of harvest, fatigue, insomnia,
nausea, dizziness, and anorexia, occur fre-
quently but resolve in most donors by 1 month
after the procedure.31 Marrow donors often
Hematopoietic Stem Cells 䡲 719
have significant decreases in their hemoglobin
concentrations after the procedure. As a result,
almost all marrow donors donate autologous
Red Blood Cells (RBCs) before the procedure,
and 76% of donors receive at least 1 autolo-
gous RBC unit during or shortly after marrow
harvest.31 If the donor requires an allogeneic
RBC or platelet transfusion before or during
the procedure, the units should be irradiated
to prevent viable leukocytes from these blood
products from contaminating the graft. Mar-
row donors should be made aware that they
might need to undergo a transfusion as part of
the informed consent process.
Peripheral Blood HSC Collection
Pharmacologic methods for mobilizing HSCs
from the marrow into the peripheral circula-
tion combined with apheresis technology have
made peripheral HSC collection the most
common procedure for HSC donation.32 Be-
cause peripheral collection of HSCs requires
only vascular access, most apheresis proce-
dures used to collect HSCs are performed on
an outpatient basis, with minimal side effects.
However, donors with poor vascular access or
who may need a number of apheresis proce-
dures for the collection of a sufficient number
of HSCs may require the placement of a cen-
tral line, which imposes an additional risk.
AABB CT Standards requires that the place-
ment of any central line be confirmed before
HSC collection is initiated.1(p48)
HSCs can be mobilized into the peripher-
al circulation using a variety of chemothera-
peutic agents, hematopoietic growth factors,
or receptor antagonists. For most healthy allo-
geneic HSC donors, sufficient numbers of HSCs
can be mobilized with the administration of
hematopoietic growth factor, often G-CSF,
alone. G-CSF is administered once per day at a
dose of 5 to 20 µg/kg, and doses are often
rounded to the nearest vial size.33 Total white
cell count and CD34 percentage can be moni-
tored to determine the optimal collection time,
which is usually 3 to 4 days after initiation of
G-CSF treatment. The side effects of G-CSF,
which are common and mild, include bone
pain, myalgia, headache, insomnia, flu-like
720 䡲 AABB TECHNICAL MANUAL
symptoms, sweating, anorexia, fever, chills,
and nausea.33 Potentially serious complica-
tions, such as splenic rupture, are rare. Other
growth factor preparations are available, in-
cluding a pegylated form of G-CSF that offers
the advantage of a one-time dose in a majority
of donors.
For some autologous donors and rare al-
logeneic donors, mobilization of HSCs can be
challenging, potentially requiring additional
pharmacotherapy to mobilize an adequate
number of cells and facilitate efficient engraft-
ment. The minimum number of cells needed
for transplantation is commonly cited as
2 × 106 CD34+ cells/kg, although 5 × 106 CD34+
cells/kg is more desirable.34 In autologous do-
nors, a chemotherapy drug, such as cyclo-
phosphamide, can be added to the G-CSF regi-
men. Although the number of HSCs collected
can be increased by using a combination of
chemotherapy and G-CSF, complications such
as cytopenias and additional apheresis may
outweigh the potential benefits of this strate-
gy.35 If the benefits of adding chemotherapy to
G-CSF to stimulate peripheral HSC mobiliza-
tion outweigh the risks, this mobilization strat-
egy may be utilized successfully in patients
with significant tumor burden.
Patients who are poor mobilizers may
benefit from the addition of plerixafor, a che-
mokine (C-X-C motif) receptor 4 antagonist,
in combination with G-CSF. Various clinical
studies have been published demonstrating
that plerixafor in combination with G-CSF can
increase HSC collection yields. A number of
clinical scenarios where plerixafor may be uti-
lized have been published, and patients with
multiple myeloma or lymphoma who have dif-
ficulty mobilizing HSCs may benefit from
plerixafor therapy to collect a sufficient quan-
tity of HSCs during donation.34
Peripheral collection of the HSCs is per-
formed using an apheresis device according to
the manufacturer’s instructions. For most allo-
geneic donors, sufficient numbers of HSCs can
be obtained with one to two collection proce-
dures. Up to 20% of donors experience minor
apheresis/collection-related adverse events,
such as citrate toxicity, nausea, fatigue, chills,
hypertension, hypotension, allergic reactions,
or syncope.32 Autologous donors experience
similar collection-related side effects, which
can be problematic in donors who require
multiple apheresis procedures due to poor
mobilization. Depending on the donor scenar-
io, large-volume apheresis techniques may be
used to limit the number of total procedures.36
AABB CT Standards requires a complete blood
count to be performed within 24 hours before
the procedure begins for all mobilized donors,
and this is especially important for autologous
donors because the apheresis procedure can
deplete platelets.1(p47)
UCB HSC Collection
UCB collection is discussed in detail in Chap-
ter 30 and is not addressed here.
PROC ESSING OF HSCS
Processing methods for HSCs can be divided
into routine methods, which are usually cen-
trifuge based, and specialized methods that
involve a variety of technologies. Routine
methods include volume (plasma) reduction,
red cell reduction, buffy coat preparation,
thawing/washing, and filtration. Volume re-
duction is performed in the settings of minor
ABO-mismatched allograft (marrow or periph-
eral blood) transplantation to reduce the
amount of incompatible plasma and prevent
fluid balance/overload issues in small patients
and/or patients with renal disease or cardiac
failure. Volume reduction may also be per-
formed before cryopreservation (eg, for UCB
banking where storage space is limited or dur-
ing cell concentration optimization).
Classically, red cell reduction employs
sedimenting agents (eg, hydroxyethyl starch)
to reduce red cell content. These agents are
used to prevent hemolytic transfusion reac-
tions when major ABO incompatible marrow
HSC allografts and allografts with other clini-
cally relevant red cell antigens (eg, Kell, Kidd)
are transplanted. Red cell reduction before
freezing also limits the amount of lysed red cell
fragments and free hemoglobin on infusion
and may be particularly important for patients
with renal failure. Red cell reduction may also
 CHAPTER 29
be useful when storage space is limited. Be-
cause apheresis instruments collect mononu-
clear cells efficiently, with very little red cell
content, peripheral blood-derived HSCs gen-
erally do not require red cell depletion.
Buffy coat concentration of marrow in-
volves centrifugation and harvesting of the
white cell fraction and can be performed with
an apheresis or cell-washing device. Manual
centrifugation may be used when product vol-
ume is too low for apheresis or cell washing
devices. Buffy coat preparation is usually used
to reduce the unit volume for cryopreservation
or as a method of red cell reduction before fur-
ther manipulation (eg, immunomagnetic se-
lection).
The thawing procedure for all HSCs, re-
gardless of source, is similar. Although this
procedure is straightforward, it should be
done carefully because frozen plastic contain-
ers are prone to break for a variety of reasons.37
The product should be handled with care
while it is verified to determine the product’s
identity and ensure the integrity of the bag.
The product is then placed into a clean or ster-
ile plastic bag and submerged in a 37 C water-
bath. If the freezing bag breaks, the product
may be recovered using this approach, but a
risk-benefit discussion with the patient’s phy-
sician should take place to determine the
course of the patient’s care.
Gentle kneading allows the thaw proce-
dure to proceed relatively quickly while pre-
venting recrystallization and consequent cell
damage/death. A hemostat should be used to
prevent loss of the product if the bag breaks,
and the contents should be aseptically divert-
ed into a transfer bag. A sample should also be
sent for culture.
Washing the HSCs removes lysed red
cells, hemoglobin, and cryoprotectant [di-
methyl sulfoxide (DMSO)]. Although UCB is
typically red-cell depleted before cryopreser-
vation, it remains the primary HSC product
that is routinely washed. This practice is
changing, however, and Chapter 30 discusses
alternative approaches to UCB preparation for
infusion. Historically, most institutions based
their UCB processing methodology, including
the thawing/washing procedure, on the proce-
Hematopoietic Stem Cells 䡲 721
dure originally described by Pablo Rubinstein
of the New York Placental Blood Program.38
Briefly, the thawing process involves slow, se-
quential addition of a wash solution (eg, 10%
dextran followed by 5% albumin), transfer into
an appropriately sized bag for centrifugation,
and resuspension of cell pellet(s) before deliv-
ery to the patient care unit for infusion. Many
laboratories perform two centrifugation steps,
removing the supernatant from the first spin
and centrifuging that portion a second time
before combining the two cell pellets. This ap-
proach optimizes cell recovery.39
Marrow harvest typically involves se-
quential filtration in the operating room or the
laboratory to remove bone spicules, aggre-
gates, and debris. Opinions regarding the use
of standard blood filters upon infusion of
HSCs vary, however. Whether to use a standard
blood filter (>170 microns) is up to the individ-
ual cell processing laboratory and/or trans-
plant center. If an institution opts to use a
standard blood filter, the laboratory should
validate its filtration process.
SPECIALIZED CELL-
PROC ESSING METHODS
Specialized cell-processing methods are used
to optimize product purity and potency be-
yond levels obtained through routine meth-
ods. Several of these methods, which require
unique reagents and instrumentation, are dis-
cussed in other chapters. For this reason, the
descriptions of these methods in this chapter
are brief and focus on their application to
HSCs.
Elutriation
Counter-flow centrifugal elutriation is a spe-
cialized method that separates cell popula-
tions based on two physical characteristics—
size and density (sedimentation coefficient). A
centrifuge is used to separate the cell popula-
tions of a cell product based on density alone.
However, if fluid/media is passed through the
chamber housing the cells in the direction that
is opposite (counterflow) to the centrifugal
force, adjustment of flow rate and/or centrifu-
722 䡲 AABB TECHNICAL MANUAL
gation speed allows the separation of cell pop-
ulations based on size as well. Through this
process, cells with “signature” size/density
profiles can be separated from the rest of the
cells. Historically, this method was used for
T-cell depletion of HSC grafts. In more recent
years, the method has been used to enrich
monocytes for preparation of dendritic-cell
vaccines.
Cell-Selection Systems
Immunomagnetic cell selection systems
incorporating monoclonal-antibody-based
technologies to target cell-surface antigens
(eg, CliniMACS system, Miltenyi Biotec Ber-
gisch, Gladbach, Germany) have become a
widely used method of cell depletion/enrich-
ment at many institutions. These methods in-
volve the isolation of the cell type of interest by
either positive selection (target cells retained)
or negative selection (target cells depleted).
Monoclonal antibodies (eg, anti-CD34 for HSC
isolation) are coupled to 50-nm ferromagnetic
particles. Magnetically labeled target cells are
retained in the process as the cell suspension
passes through a column in which a magnetic
field is generated. Unlabeled cells pass
through the column and are collected in a neg-
ative-fraction bag. Target cells are then re-
leased from the column by removal of the
magnetic field from the column, which allows
passage of the cells into a separate collection
bag.
Cell Expansion
Because the dose of nucleated, CD34+, and
colony-forming cells has a positive correlation
with patient outcomes, much effort has been
focused on ex-vivo expansion of HSCs and
progenitors. It is thought that successful ex-
pansion enhances hematopoietic engraftment
while reducing transfusion dependence, risk
of infection, and duration of hospitalization.
In recent years, UCB has become the focus of
expansion trials due both to the higher prolif-
erative and self-renewal capacity of HSCs from
UCB and the limit on cell quantity in a UCB
collection. Most expansion cultures contain a
cytokine cocktail that includes stem cell factor,
FLT-3 ligand, and thrombopoietin along with
novel and/or proprietary ingredients. The me-
dia, culture vessels, and culture duration used
vary from protocol to protocol.
CRYO PRESERVATION
Methods for cryopreservation must be used
because HSCs may need to be stored for weeks
to years prior to being transplanted.40 Most
cell-processing laboratories use the cryopro-
tectant DMSO, usually at 10% final concentra-
tion, and a source of plasma protein for cryo-
preservation of HSCs. DMSO is a colligative
cryoprotectant; it diffuses rapidly into the cell,
reducing the osmotic stress on the cell mem-
brane. DMSO prevents dehydration injury by
moderating the nonpenetrating extracellular
solutes that form during ice formation. It also
slows extracellular ice crystal formation. Some
laboratories add hydroxyethyl starch (HES),
which allows the use of a decreased concentra-
tion of DMSO (eg, 5% DMSO and 6% HES).
HES is a nonpenetrating (extracellular),
macromolecular cryoprotectant. This high-
molecular-weight polymer likely protects the
cell by forming a glassy shell, or membrane,
around the cell, retarding the movement of
water out of the cell and into the extracellular
ice crystals.
The HSCs may be frozen at a controlled
rate or a noncontrolled rate, in which the HSC
product is simply transferred into a freezer bag
and placed in a –80 C mechanical freezer. Con-
trolled-rate freezing is favored in the clinical
laboratory setting, and it utilizes computer
programming to incrementally decrease HSC
product temperature in a closely monitored
fashion. The controlled rate freezing protocols
used vary from institution to institution.
In general, the HSC product is placed in
the chamber and initially cooled at a rate of
1 C/minute. When the temperature decreases
to approximately –14 C to –24 C, the HSC
product begins to transition from a liquid to a
solid. At this time, the freezer undergoes a pe-
riod of supercooling to counteract the latent
heat of fusion that is released by the phase
change. Following solidification of the HSC
product, cooling proceeds at the rate of 1 C/
 CHAPTER 29
minute until the product has reached –60 C. At
this point, the product is cooled at a controlled
rate determined by the institution until it
reaches –100 C. Following both controlled-rate
and noncontrolled-rate freezing, the HSC
product is transferred to a storage freezer. An
increasing number of laboratories store HSCs
in the vapor phase of liquid nitrogen (LN2) at
temperatures below –150 C; however, some
laboratories do store cells in the liquid phase
of LN2.
QC
QC testing in the clinical cell therapy laborato-
ry serves two purposes: determine the suit-
ability and safety of the cellular product for the
patient and monitor overall laboratory practic-
es. QC testing is aimed at characterizing the
safety, purity, identity, potency, and stability of
the cellular product. The extent of QC testing
is primarily dependent on the complexity of
manufacturing the product and the nature of
the clinical experience (ie, standard practice vs
a clinical trial).
Common QC tests for HSCs include cell
count and differential, viability, CD34+ cell
enumeration, sterility testing, and colony-
forming unit assays. Cell count and differential
are performed on a hematology analyzer. Cell
viability may be determined using a variety of
methods, including trypan blue, acridine or-
ange, and 7-aminoactinomycin D (flow cy-
tometry). Microscope-based methods utilizing
vital dyes or fluorescent stains may be particu-
larly useful for a quick assessment of overall
nucleated cell viability. Flow cytometry-based
analysis can be useful when cell population-
specific viability needs to be determined. Most
CD34+ cell-enumeration strategies are based
on guidelines of the International Society for
Cellular Therapy.41 Sterility testing at most in-
stitutions is performed using an automated
microbial detection system.
The clonogenic assay (most commonly
used to count colony-forming units) is the
only truly functional assessment of HSCs rou-
tinely performed in clinical laboratories. The
results of this assay correlate with the speed
and likelihood of engraftment of HSCs from
Hematopoietic Stem Cells 䡲 723
marrow, peripheral blood, or UCB.42-45 Howev-
er, the similar correlation between results and
engraftment speed and likelihood as well as
the more rapid availability of results have
made CD34+ cell enumeration the accepted,
albeit surrogate, QC test for graft potency. The
clonogenic assay is still useful, despite difficul-
ties in standardization, especially for HSCs
that are stored for a long time (eg, UCB bank-
ing).46
SHIPPI NG AND TRANSP ORT OF
HSC CELLUL AR PRODUCTS
Shipping and transport of cellular therapy
products allow the geographic separation of
donors and recipients. The two terms are given
specific definitions by accreditation organiza-
tions.1(pp35-36),4(pp13-14) With shipping, the product
leaves the control of trained personnel in the
facilities involved in the distribution and re-
ceipt of the product.4(pp13-14) Conversely, with
transport the transfer of a product between or
within facilities occurs under the control of
trained personnel.
Three issues are particularly important to
ensure the safe delivery of HSC products:
product integrity, safety of the personnel in-
volved in the transport, and compliance with
applicable regulations and standards. The
necessary conditions for shipping and trans-
port vary depending on the type of product, its
state (fresh or cryopreserved), and the dis-
tance involved. These issues are reviewed in
depth elsewhere.47
During shipment, the product must be
placed in a secondary container that can pre-
vent leakage and is validated for the tempera-
ture range required for the product and the
anticipated duration of shipping. This tem-
perature range may be defined in standards
(eg, <–150 C for cryopreserved cord blood) or
by an institutional protocol.4 For fresh prod-
ucts, several studies have shown that ship-
ment at 2 C to 8 C can maintain CD34+ cell via-
bility more effectively than shipment at room
temperature particularly for shipping times of
between 24 and 72 hours.48-50 This effect ap-
pears to be more pronounced for peripheral
724 䡲 AABB TECHNICAL MANUAL
blood products than marrow products and for
products with higher concentrations of HSCs.
Cryopreserved products are shipped in
dry shippers charged with LN2. These contain-
ers maintain temperatures below –150 C for up
to 2 weeks and their temperatures are continu-
ously monitored.1(p35) If the products are
shipped to a noncontiguous facility or on pub-
lic roads, an appropriately labeled outer con-
tainer must be used to further protect the
product during transport and shipping.4 De-
pending on the mode of transport (eg, air or
ground), additional federal government re-
quirements must be met. If products are
shipped abroad, international requirements
must be met. If high-dose conditioning thera-
py has been given to the recipient, shipment
using a qualified courier is required.4 The
product should not be x-rayed; instead, it
should be manually inspected, if necessary.
Appropriate records must accompany the
product.
The receiving institution must have pro-
tocols in place for receipt and inspection of the
product for acceptability for transplant.1(pp36-37),4
PATIENT CARE
Once an HSC product is ready for infusion, it
should be delivered to the patient care unit
without delay. After the physician approves the
product for infusion and proper identification
procedures are carried out, the product is in-
fused by intravenous (IV) drip directly into a
central line, typically without a needle or
pump. Some institutions use a standard blood
filter at the bedside. To maximize cell dose, the
product bag and IV tubing may be flushed
with sterile saline after the bag empties. Sterile
saline also may be added directly to the bag if
the flow rate becomes too slow.
HSC products are usually infused as
quickly as the patient can tolerate, particularly
for thawed cells that have not been washed or
diluted, to lessen the DMSO toxicity to the
cells. Although Rowley and Anderson51 con-
cluded that DMSO is not toxic to HSCs at clini-
cally relevant concentrations (ie, 5% or 10%) at
either 4 C or 37 C for up to 1 hour of incuba-
tion, they also noted that the addition of 1%
DMSO to culture dishes suppressed colony-
forming units. However, these studies were
performed on fresh cells, and studies of the ef-
fects of DMSO on HSCs that have been previ-
ously cryopreserved are limited. The possible
functional defect due to DMSO coupled with
the not-infrequent need to hold clinical prod-
ucts (because of patient-care-related issues)
raise concern about the possibility of cell inju-
ry from the infusion of thawed, unwashed HSC
products.
The patient’s vital signs should be
checked before infusion, immediately after in-
fusion, and 1 hour after infusion, at a mini-
mum. All of the monitoring information
should be captured on the accompanying in-
fusion form. When completed, this form
should be returned to the laboratory. If an ad-
verse reaction occurs, more frequent monitor-
ing is required.
Reactions associated with HSC infusion
may be very similar to those that occasionally
occur with blood transfusion (ie, allergic, he-
molytic, and febrile reactions and those due to
microbial contamination). However, some re-
actions may be less likely depending on the
cell-processing technique used (eg, red cell re-
duction, plasma reduction, postthaw wash-
ing, or dilution). Reactions often attributed to
DMSO (eg, nausea, vomiting, cough, and
headache) are less common with infusions of
smaller volumes and/or washed/diluted prod-
ucts.52,53 HSC products are usually well tolerat-
ed, however. Because the possibility of a severe
reaction does exist, aggressive IV hydration
(eg, 2 to 6 hours before and 6 hours after infu-
sion, with the use of diuretics as needed) and
use of prophylactic antiemetics, antipyretics,
and antihistamines may be warranted.
The transplant physician and the medical
director of the cell therapy laboratory should
be notified immediately of an unexpected or
moderate-to-severe reaction. An investigation
should begin and include laboratory testing
(eg, direct antiglobulin test, antibody titer,
Gram’s stain, or culture) that targets the signs/
symptoms of the patient.
Data on clinical outcomes (eg, engraft-
ment) and adverse events should be reviewed
regularly and discussed with the institutional
 CHAPTER 29 Hematopoietic Stem Cells 䡲 725

quality management group. Quarterly reviews
are reasonable for engraftment analysis. The
medical director’s review should include as-
sessment of the HSC product’s quality indica-
tors (eg, dose, viability, and colony-forming
units), associated deviations, and presence of
infusion reactions, with a focus on potential
laboratory-related component affecting any
less-than-optimal outcome.
OTH E R R E G UL ATO RY
CO NS I DE RAT IO N S
The relevant regulations with regard to HSC
collection are discussed earlier in this chapter.
In general, HSCs that are minimally manipu-
lated and collected for transplantation in an
autologous fashion or transplanted to a first-
or second-degree relative are regulated solely
under Section 361 of the Public Health Service
Act and are subject to the jurisdiction of the
Center for Biologics Evaluation and Research
of the FDA. If HSCs are manufactured in a way
that alters their relevant biological characteris-
tics (eg, if they are genetically modified, ex-
panded ex vivo, or combined with a drug) or
the cells are intended for transplantation into
a non-first- or second-degree relative, then the
HSC product is subject to regulation under Ti-
tle 21, CFR Part 1271, as a drug and/or biologic
product and requires licensing or an exemp-
tion from licensing from the FDA as part of an
investigational new drug (IND) application.
HSCs from unrelated donors facilitated
through the Be The Match Registry may be ad-
ministered under their IND (BB-IND 6821) or
an institutionally held IND. Similarly, HSCs
can now be obtained from FDA-licensed UCB
sources or administered under an institutional
IND.
CO N CLUS IO N
The indispensable, lifesaving role of HSCs in
medicine has been established, especially for
patients with hematologic disorders. As the
understanding of HSC biology increases and
the ability to engineer HSC grafts expands, the
clinical applications of HSCs will likely contin-
ue to grow. Along with the fast-paced growth
in the use of HSCs, emerging novel technolo-
gies and approaches to manipulate HSCs are
adding to the challenge of ensuring that HSCs
continue to serve as a safe and effective cellu-
lar therapy product for patient use. Addressing
this challenge will require regulatory agencies
and accrediting bodies to continue to update
and modify their applicable rules, regulations,
and standards.

KEY PO I NT S

1. Hematopoietic stem cells (HSCs) derived from the patient being treated (autologous) or a
donor (allogeneic) can be used to treat a variety of malignant and nonmalignant conditions.
2. Autologous HSCs in general are used to rescue the marrow function of a patient undergoing
high-dose chemotherapy and/or radiotherapy.
3. In addition to rescuing the marrow function of a patient undergoing high-dose chemother-
apy and/or radiotherapy, allogeneic HSCs also have graft-vs-neoplasm effect and/or the
ability to replace defective cellular machinery.
4. Regardless of the HSC donor source, AABB CT Standards requires laboratory testing for hu-
man immunodeficiency virus, Types 1/2; hepatitis B virus; hepatitis C virus; syphilis; hu-
man T-cell lymphotrophic virus, Types I and II; and cytomegalovirus.
5. Screening and testing for infectious diseases in allogeneic HSC donors is mandated by the
Food and Drug Administration (FDA), and when a risk for a communicable disease is dis-
covered, the donor is ineligible (but may still donate if there is urgent medical need).
6. Allogeneic HSC donors are chosen with regard to their histocompatibility with the recipient.
ABO-Rh compatibility between the donor and recipient is not necessary.
726 䡲 AABB TECHNICAL MANUAL

7. HSCs can be obtained by aspiration of marrow, collection of umbilical cord blood, or by pe-
ripheral blood mobilization followed by collection with an apheresis machine.
8. HSCs usually require minimal manipulation, and can be stored following cryopreservation
with the cryoprotectant, dimethyl sulfoxide.
9. Specialized HSC manipulation techniques can be used to reduce the HSC product volume,
lysed cells, red cells, and cryoprotectant depending on the recipient’s clinical needs.
10. Quality control (QC) is essential for providing a safe and efficacious HSC product. Common
QC testing includes cells counts (CD34+ cell enumeration, total nuclear cell count), micro-
bial contamination testing, and viability testing.

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43. Douay L, Gorin NC, Mary JY, et al. Recovery of
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 C h a p t e r
Umbilical Cord Blood Banking
Aleksandar M. Babic, MD, PhD, and
Donna M. Regan, MT(ASCP)SBB
O V E R T H E P A S T 25 years, umbilical
cord blood (UCB) has evolved into an
important source of hematopoietic stem cells
(HSCs) for transplantation. Classified by the
Food and Drug Administration (FDA) as a bio-
logic drug, stem cells derived from UCB are
also used in a growing number of large, multi-
institutional trials in immunotherapy and re-
generative medicine. This chapter summarizes
the practical aspects of creating an inventory
that provides UCB cells for clinical use, recent
research, economic considerations in balanc-
ing inventory size with the potential for unit
selection, and the regulatory structure that
keeps pace with product innovation.
BACKGROUND
UCB has become a widely accepted source of
HSCs for transplantation in pediatric and
adult patients lacking an HLA-matched do-
nor.1-6 The advantages of UCB-derived HSCs
over other HSCs include their higher prolifera-
tive and self-renewal capacity and their associ-
ation with a lower incidence of graft vs host
disease (GVHD), particularly acute GVHD, de-
Aleksandar M. Babic, MD, PhD, Medical Director, and Donna M. Regan, MT(ASCP)SBB, Executive Director, St.
Louis Cord Blood Bank and Cellular Therapy Laboratory, SSM Cardinal Glennon Children’s Hospital, St. Louis,
Missouri
The authors have disclosed no conflicts of interest.
3 0
spite their less stringent HLA-matching re-
quirements.4,5,7-11 In-vitro and in-vivo animal
research suggests that these attributes of UCB
transplantation are due to the primitive nature
and naïve immune system of UCB.12-14 Trans-
plant candidates, especially those with rare
HLA types, are often successful in finding an
acceptable UCB unit. Furthermore, the search
time, in general, to find a donor is markedly re-
duced for UCB transplant recipients than for
recipients of HSCs from other sources because
the cellular characteristics, donor screening/
eligibility, and HLA typing of UCB units are de-
termined at the time of banking.15
The first UCB bank, established by Dr. Hal
E. Broxmeyer, provided the donor graft for the
historic 1988 UCB transplant as well as four
additional HLA-matched sibling trans-
plants.16,17 As UCB gained initial acceptance as
an HSC source in the related transplant set-
ting, the possibilities for the use of unrelated
HSC units became apparent and supported
the rationale for the establishment of unrelat-
ed cord blood banks (CBBs). The first unrelat-
ed CBB was established by Dr. Pablo Rubin-
stein at the New York Blood Center in 1992.18
729
730 䡲 AABB TECHNICAL MANUAL
CBBs in Dusseldorf and Milan opened shortly
thereafter. Recent reports indicate that more
than 600,000 unrelated UCB units are banked
worldwide for potential clinical use, and near-
ly 30,000 unrelated UCB transplants have been
performed to date.19 In 2011 alone, the World
Marrow Donor Association reported that 4093
UCB products were shipped to hospitals for
transplantation to unrelated patients in 47
countries; in comparison, 3,743 marrow grafts
were performed in that year.19 In addition, a
number of private CBBs have been established
worldwide for families to bank the UCB of
their infants for future use by family members.
Two of the largest private CBBs have recently
reported that they have stored UCB stem cells
from 700,000 newborns, and each of these
CBBs has distributed approximately 250 UCB
products for traditional transplantation and
regenerative applications.
The sections of this chapter describe the
current generally accepted practices for UCB
banking (primarily from a public CBB’s per-
spective), including donor-related issues, col-
lection and processing methods, and storage
and shipment as well as recommended trans-
plant center-related activities for product
preparation and infusion. The chapter contin-
ues with a brief discussion of UCB banking
economics and how inventories can be creat-
ed to meet the needs of a diverse patient popu-
lation and address cell dose limitations while
continuing to serve as cost-effective means of
providing access to therapy in an “affordable
health care” environment. The chapter finish-
es with an overview of standards and regula-
tions.20
DONOR-REL ATED ISSUES
Recruitment
Most women agree to donate the UCB of their
infants when they become aware that the UCB
that is normally discarded as medical waste
can be recovered and used to save the life of
another individual. However, a woman’s ability
to donate UCB may be limited by the availabil-
ity of a CBB servicing the area in which she
gives birth.
Engaging pregnant women and making
arrangements before they give birth to donate
their infant’s UCB is critical for achieving the
desired results. Early education of pregnant
women allows for more complete medical
screening, comprehensive donor protection,
availability of adequate supplies to collect
UCB, and acquisition of truly informed con-
sent from the woman. Recruitment by a public
CBB typically begins with education of the
physician/midwife by the CBB and the distri-
bution of informational materials to obstet-
rics offices or prenatal class staff. This ap-
proach enlists support for UCB donation while
providing information to obstetric staff. This
information empowers staff to introduce the
idea of UCB donation to pregnant women and
respond to their initial inquiries while refer-
ring more detailed questions to CBB person-
nel.
The informational materials that CBBs
distribute to obstetrics office and prenatal
class staff about UCB donation address basic
information, such as the name of the CBB,
whether the CBB is public or private, the cost
(or lack thereof) of donation, names of partici-
pating hospitals, medical uses of UCB, and
how UCB is collected. The materials also dis-
cuss the risks of UCB donation to the mother
and infant and whether the donated unit will
be available for the donating family’s use.
Within an informational packet, a CBB may in-
clude the health history questionnaire, which
is used to solicit information about the preg-
nancy, risk factors of the parents, and medical
history of first-degree family members.
The CBB explains to potential donors the
need for their written permission (informed
consent) to collect and store the UCB for later
use in transplantation or research. Participat-
ing mothers must understand that their blood
will be drawn and tested for certain infections,
such as hepatitis and human immunodefi-
ciency virus, to reduce the risk of transmission
of disease through the transplantation of their
infant’s UCB. The CBB emphasizes that all in-
formation and test results obtained during the
process are held strictly confidential to pro-
tect the identity and privacy of donors and
 CHAPTER 30
that the family will not be approached to do-
nate more cells after the infant is delivered.
The role of the pregnant woman’s physi-
cian cannot be understated. During pregnan-
cy, a woman trusts her physician to provide re-
liable care and advice. Therefore, a woman’s
decision to donate or store her UCB can be as
simple as acceptance of a recommendation of-
fered by her physician. In addition, a woman’s
decision to donate may be influenced by re-
cruitment materials designed to appeal to all
ethnic groups.
In spite of broad awareness campaigns
and recruitment efforts, some women may not
be aware of UCB donation or may not have
registered to participate when they arrive at a
hospital to give birth. For this reason, informa-
tional material on UCB donation should also
be available in the labor and delivery area. The
extent to which women not previously in-
formed about UCB donation can be recruited
at this stage (ie, delivery) varies. However, in
most cases, pregnant women have been previ-
ously informed of the option of UCB donation
but simply have not registered or completed
the necessary documentation.
Pregnant women may also be solicited for
UCB donation by private CBBs. For a fee, these
banks will arrange for UCB to be collected and
held in long-term storage for use only by that
family. These CBBs provide written or video
information to pregnant women that is specif-
ic to their program.
Consent
Consent must be obtained from the pregnant
woman for UCB collection, processing, test-
ing, storage, and medical use.21-26 Although the
UCB actually belongs to the newborn infant,
consent is obtained from the mother because
her infant cannot provide it and testing of her
blood for transmissible diseases is required.
Consent from the father is not necessary and
does not increase the safety of the UCB for
transplantation.27
Although consent to collect UCB must be
obtained before delivery of the infant, CBBs
use different approaches to obtain consent for
further manufacturing, testing, and use of
Umbilical Cord Blood Banking 䡲 731
UCB. A CBB may use a single consent form
covering all activities. Presented in the prena-
tal period, a single consent process affords the
pregnant woman adequate time to seek infor-
mation and consider her options under low-
stress circumstances. Other CBBs may use a
phased consent process that permits collec-
tion and, if the resulting harvest meets criteria
for banking, allows the bank to approach the
mother later for permission to screen, process,
test, and store the product. This latter process
facilitates collection from mothers who have
not previously been introduced to UCB
banking.
Criteria have been suggested that protect
the woman’s ability to make an informed deci-
sion during childbirth. The Advisory Council
on Blood Stem Cell Transplantation (ACBSCT)
has recommended that each CBB develop a
policy on informed consent that considers the
stage of labor and stress of the woman, the
amount of counseling on UCB donation that
she has received, and the amount of time
available for an adequate discussion of UCB
donation.28,29 Final judgment regarding the
woman’s ability to give intralabor consent
should rest with the obstetric staff.
In October 2011, the FDA classified UCB
banking as a manufacturing rather than an in-
vestigational activity when a CBB distributes
products for specified indications. If not li-
censed, manufactured UCB products may still
be distributed for nonlicensed (clinical re-
search) applications as an investigational new
drug (IND).
Health History and Medical Evaluation
It is incumbent on the CBB to provide a safe
product by minimizing the transmission of ge-
netic or infectious diseases. A donor-eligibility
determination, based on donor screening and
testing for relevant communicable disease
agents and diseases, is required for all donors
of cells or tissue, including UCB cells. A robust
medical health history designed to solicit
information on exposures to infectious diseas-
es and history of symptoms indicating genetic
disorders is obtained by interviewing the
mother and reviewing her medical record. The
732 䡲 AABB TECHNICAL MANUAL
father’s medical history may be solicited to
identify any issues that might affect the quality
of the UCB, but this is not required. If not se-
cured before delivery of the infant, the history
must be obtained no later than 7 days after de-
livery.24(p73) The history questionnaire may be
self-administered with follow-up by CBB staff,
or it can be completed through direct inter-
view by CBB staff or hospital staff who are ade-
quately trained in, and capable of, answering
the woman’s questions.
The approach to screening and testing
the mother is admittedly more conservative
than the approach for the infant donor. It is the
mother’s circulation that nourishes the fetus
during pregnancy and because of this shared
physiology, her infectious exposures are rele-
vant to determining the eligibility of her in-
fant’s UCB for transplantation. Also associated
with risk are certain maternal conditions dur-
ing labor and delivery, such as fever, prolonged
time after membrane rupture, or administra-
tion of antibiotics—all of which suggest possi-
ble bacterial contamination that could be
transmitted through the UCB product.
Medical screening includes an extensive
family genetic history because the infant has
not had an opportunity to manifest many in-
herited diseases. If first-degree relatives have a
history of malignancy or parents have been
treated with chemotherapy and/or radiation,
the infant’s UCB cannot be donated. In addi-
tion, if UCB is used to compensate for defi-
ciencies associated with specific genetic disor-
ders, the product is tested for the presence of
the targeted enzyme when such testing is
available.
Infants are not required to be retested or
reexamined at 6 to 12 months of age to evalu-
ate their eligibility to donate UCB because of
the difficulty of locating some families, con-
cerns about parental and infant privacy, the
difficulty of maintaining the records required
to trace families, and cost. In spite of the diffi-
culties of reconnecting with a donor family,
however, some CBBs follow up with families at
various intervals after birth to ensure that the
infant has not developed any transmissible
diseases and instill confidence in the suitabili-
ty of the infant’s UCB for transplantation.
Donor Testing
The second step in determining donor eligibil-
ity is accomplished through laboratory testing.
The strategy for UCB donation is similar to
that used for whole blood donation except that
the tests are performed on maternal blood and
not the blood of the infant donor. This ap-
proach is used because it is assumed that in-
fectious diseases present in the UCB originat-
ed from the mother and, thus, infectivity is
likely to be detected in a maternal blood sam-
ple.
Because testing is conducted on mater-
nal blood, it is necessary to obtain the moth-
er’s consent for testing as well as for collection
of the UCB. It is also important to note that the
testing reagents used must be approved by
FDA for use in donor screening rather than di-
agnostic testing. In addition, the sample types
collected must be cleared for use in this type of
testing, and the laboratory must be autho-
rized to perform the test. In the United States,
no donor tests have been approved for UCB
samples; therefore, infectious disease testing is
performed on a maternal sample. The samples
for infectious disease testing must be collected
on the day of UCB collection or within 7 days
before or after the delivery.24(p40)
The CBB may perform testing or issue a
contract to a laboratory to conduct testing to
rule out collections from donors with hemo-
globinopathies. Certain CBBs store units from
donors who have sickle cell trait or alpha thal-
assemia trait because these units may have
unique HLA types needed for transplantation
in patients from minority populations. Units
with homozygous abnormal hemoglobin or
compound heterozygous abnormal hemoglo-
bin are unsuitable for transplant. UCB units
selected for transplant in a patient with an in-
herited disease should be tested for that dis-
ease before being used.30 Therefore, it is im-
portant to archive appropriate UCB and
maternal ancillary samples for additional and/
or confirmatory testing.
Regardless of who obtains the medical
history or tests maternal or UCB samples, the
final donor eligibility must be determined by
 CHAPTER 30
the CBB medical director and approved by its
quality control unit.22,24(pp1-2,71-72)
U C B CO L L E C T I O N
The methods to collect UCB consist of cleans-
ing of the cannulation site and aseptic collec-
tion. Typically, a sterile collection bag contain-
ing citrate-phosphate-dextrose (CPD) solution
anticoagulant is used, although acid-citrate-
dextrose and lyophilized heparin are also ac-
ceptable anticoagulants for UCB collection.
After delivery, the umbilical cord is
clamped, cut, and separated from the infant.
The clamping must be timed so as not to inter-
fere with routine delivery practice. UCB can be
collected before (in utero) or after (ex utero)
the placenta has been delivered. There are ad-
vantages and disadvantages to each method,
but neither appears to be better overall.31 A
brief discussion of the two methods is provid-
ed below.
Regardless of whether the UCB is collect-
ed in vivo vs ex vivo, the process of UCB collec-
tions is essentially identical. As in whole-blood
collection, the venipuncture needle is at-
tached to the collection bag. After an appropri-
ate umbilical vein is identified, the site is
cleansed. Typically, this involves wiping the
cord with isopropanol and then scrubbing it
with a broad-spectrum topical microbicide
(eg, povidone iodine) for at least 30 seconds.
Alternatively, a few CBBs have chosen to use a
broad-spectrum antimicrobial formulation of
2% chlorhexidine gluconate/70% isopropyl al-
cohol in place of traditional iodophors. Subse-
quently, a hemostat is placed on the tubing a
few inches from the needle. The hemostat is
opened once the vein has been accessed, al-
lowing blood to flow into the bag. Removal of
the hemostat before puncturing the vein may
allow air to contaminate the tubing. While the
UCB is being collected (a 3- to 5-minute pro-
cess), the UCB should be gently mixed with the
anticoagulant to prevent clotting. The umbili-
cal cord appears collapsed when the collection
is complete. Placement of the bag on a labora-
tory scale during collection allows the collec-
tor to monitor the volume and provides an
Umbilical Cord Blood Banking 䡲 733
additional means to assess completion of col-
lection.
Once the collection is complete, the tub-
ing is “stripped,” forcing the blood inside the
tubing into the collection bag and allowing it
to mix with the anticoagulant. This can be ac-
complished most easily with a specialized
blood-banking tool (ie, a tube stripper), al-
though newer bags include a filtered vent to
eliminate the need for additional equipment.
The UCB unit can then be packed, stored, and
transported to the cell-processing laboratory
in a temperature-controlled environment.
In-Utero Collection
In-utero collection is generally performed by
the obstetrician or nurse/midwife after the
newborn has been delivered and assessed and
the umbilical cord has been clamped and cut.
If the well-being of the newborn and/or moth-
er is in question, the collection is not attempt-
ed. If a decision to collect the UCB is made,
care must be taken to maintain sanitary condi-
tions. Unless a sterile bag or extension set is
used, the traditional supplies are not sterile
and inadvertent misplacement could contam-
inate the surgical field.
For logistical reasons, it is advisable to
have preassembled collection kits available for
in-utero collection. An instructional packet
may be included in these kits. This packet
might contain, for example, a donor informa-
tion form; description of the collection proce-
dure; list of collection kit contents; maternal
medical history form; consent form(s); and
packaging, storage, and shipping instructions.
Collection kits also include items such as one
or two collection bags, antimicrobial supplies,
tubing closures, appropriate sample tubes for
infectious disease testing, sample tube labels,
product labels, biohazard and other appropri-
ate stickers (eg, for indicating temporary stor-
age temperatures), and a secondary specimen
and product bag. In some cases, the hospital’s
maternal label may be used to identify mater-
nal tubes and UCB products because these la-
bels include two forms of identification and
precautions are taken to protect donor confi-
dentiality on these labels.
734 䡲 AABB TECHNICAL MANUAL
Validated containers and/or processes are
used to ship the UCB. Acceptable tempera-
tures during shipping can range from cold to
ambient, depending on the shipping distance
and conditions, and are defined based on sta-
bility studies. Temperature during transport
may be monitored depending on the risk to
the UCB involved in the shipping process.
The primary advantage of in-utero com-
pared to ex-utero collection is the substantial-
ly lower cost because dedicated CBB collection
personnel are not required. As a result, the
only costs are for collection and shipping sup-
plies. In addition, several studies have suggest-
ed that in-utero collection methods may result
in the collection of greater volumes of cells
and higher counts of nucleated and CD34+
cells and colony-forming units (CFUs).32,33
In addition, the initial costs and efforts
associated with education and training of the
physician/midwife collectors need to be con-
sidered. However, once obstetrics practice
group members are trained, their hands-on
experience should improve the quality of their
collections. In-service and educational ses-
sions can provide refresher courses and dem-
onstrate appreciation of clinicians’ support for
UCB collection. Subsequently, a program of
continued competency assessments, collec-
tion-site visits, and regular communication
can be utilized to maintain high-quality UCB
collections.
Ex-Utero Collection
Ex-utero collections are often performed by
dedicated UCB collection staff. The advantag-
es of this approach over in-utero collection are
its use of standardized collection methods,
limited personnel involvement, and greater
control.
After delivery, the cord is clamped at a
time that does not interfere with the physi-
cian’s routine practice. The placenta is imme-
diately taken by CBB staff to a suitable loca-
tion, where it is suspended in a device to allow
collection of blood by gravity. The collection
proceeds as described above. The placenta
and cord are more accessible in the ex-utero
setting and, therefore, the use of manipula-
tion, such as application of pressure to the
cord (“milking”), can increase the collection
volume. Caution must be used to prevent ma-
ternal contamination and lysis of cells.
Because blood clotting readily occurs
during UCB collection, which adversely affects
the volume and number of cells collected, it is
important to minimize delays during the col-
lection process. Wong and colleagues hypoth-
esized that a higher incidence of macroscopic
clots in ex-utero collections resulted in the col-
lection of lower nucleated cell and CFU
counts.34
Because it occurs outside the delivery
room with all of its activity, ex-utero collection
may inherently provide better process control
(ie, a lower risk of microbial contamination
and labeling errors), but it raises the costs as-
sociated with collection. In addition, a lack of
availability of collectors on all shifts may limit
the opportunity for a facility to participate in
UCB collection. Some investigators have ques-
tioned these points.35 Because data demon-
strate that in-utero and ex-utero collection
methods are equivalent, the choice of UCB
collection method should be based on the
needs and capabilities of each UCB collection
program.31
U C B P RO C ES S IN G
Methods
In the early 1990s, UCB units were often stored
in an unmanipulated state without volume re-
duction (ie, red cells and/or excess plasma
were not removed before storage) in an effort
to conserve the number of stem cells in the
product.18,36 Concern about infusion-related
complications associated with red cell incom-
patibility, free hemoglobin, dimethyl sulfoxide
(DMSO), and logistical issues surrounding
limited storage capacity motivated an evalua-
tion of processing methods to minimize the
red cell content and size of the final UCB unit
while limiting stem cell loss.
At present, a variety of processing tech-
niques for volume reduction and removal of
red cells are employed, and the majority of
these methods involve sedimentation, centrif-
 CHAPTER 30
ugation, and/or filtration. The most common
means of reducing red cell content is the use of
sedimenting agents, such as hydroxyethyl
starch (HES), gelatin, polygeline, and dex-
tran.37,39 One of the earliest methods for pro-
cessing UCB utilizing HES was developed by
Pablo Rubinstein.39 Modifications of his meth-
od have been commonly used by other pro-
grams.40 An alternative preparation method
involves density separation by layering UCB
cells over Percoll or Ficoll-Hypaque. This
method ultimately results in a mononuclear
cell-enriched product essentially devoid of red
cells.41 Also, utilization of commercially avail-
able leukocyte reduction filters and semiauto-
mated methods results in similar in-vitro cell
recovery to the standard HES method.37,42
Initially, UCB was processed with other
HSC products in laboratories that were part of
a research facility, hospital transfusion ser-
vice, or blood center. However, the methodol-
ogy for processing UCB products has evolved
to involve dedicated processing laboratories
designed to comply with current good manu-
facturing practice (cGMP) by using more stan-
dardized processing techniques for volume
reduction, removal of red cells, and cryo-
preservation.
An important driving force behind most
significant recent changes in UCB processing
was a set of recommendations made by FDA as
part of its process for the submission of bio-
logics license applications (BLAs). These rec-
ommendations provide guidance on how to
provide assurances of the safety, purity, poten-
cy, and effectiveness of UCB products. These
recommendations have compelled the indus-
try to standardize its manufacturing processes
by using reagents that have been approved for
human use and systems and supplies that
have been cleared by the FDA. To date, the fol-
lowing systems have been approved by the
FDA for the processing of UCB:
䡲 Automation technology incorporated into
the SEPAX Cord Blood Processing System
manufactured by Biosafe SA (Lake Geneva,
Switzerland). This technology offers a
closed and sterile processing system that
harvests nucleated cells and enriches stem
Umbilical Cord Blood Banking 䡲 735
cells from UCB. The technology is adapt-
able to a large-scale processing environ-
ment. The SEPAX machine, which is essen-
tially composed of a centrifuge with piston
position sensors surrounding the chamber
and pneumatic pump system, is operated
using computer-controlled protocols to
achieve component separation. Consum-
able kits consist of a large syringe-type bar-
rel separation chamber with bags and tub-
ing connected via a stopcock assembly.
Biosafe’s SEPAX system received FDA clear-
ance in January 2007 and a European CE
mark of approval in 2001.
䡲 The AXP (AutoXpress Cord Blood Process-
ing System), manufactured by ThermoGen-
esis (Rancho Cordova, CA), is an automat-
ed, functionally closed system that harvests
stem-cell-rich buffy coat from UCB. The
system reduces a unit of UCB to a precise
volume chosen by the operator and selec-
tively isolates mononuclear cells. The sys-
tem includes the AXP device, a docking sta-
tion, a processing set, and XpressTRAK
software. The AXP system received 510(k)
clearance from the FDA in October 2007.
䡲 PrepaCyte-CB, a UCB-processing system
manufactured by CytoMedical Design
Group (St. Paul, MN), received FDA 510(k)
clearance in January 2009. PrepaCyte-CB is
a functionally closed sterile bag system
composed of three integrally attached pro-
cessing and storage bags containing the
PrepaCyte-CB separation solution. While
isolating nucleated cells, PrepaCyte-CB re-
moves most red cells and nucleated red
cells from the final processed UCB unit.
The system requires plasma extractors and
a standard laboratory centrifuge to sedi-
ment and concentrate desired cells.
Each of these methods has advantages
and disadvantages with respect to cell recov-
ery, red cell removal, processing time, and
cost. Each laboratory must therefore validate
and determine which processing method is
most appropriate for its situation. Cellular
function, product integrity, and safety must be
considered when developing a validation plan.
Nucleated and CD34+ cell recovery, viability,
736 䡲 AABB TECHNICAL MANUAL

potency (eg, ability to form CFUs), and sterility
testing (before and after cryopreservation) are
typical validation performance measures. Fac-
tors to consider when choosing a processing
method include workload, processing time,
cost, maximization of storage time, and pro-
duction efficiency (number of UCB units pro-
cessed per staff member per day).
Quality Control Testing and
Characterization
Quality control (QC) testing to assess the ade-
quacy of the product and process is essential.
QC testing is usually performed on receipt
(prior to manipulation) of a UCB unit and be-
fore cryopreservation. Typical QC assays in-
clude counts of nucleated cells (white cells
plus nucleated red cells), mononuclear cells
(monocytes plus lymphocytes), and CD34+
cells; hematocrit; CFU assay; and viability and
sterility (aerobic, anaerobic, and fungal) test-
ing. All testing methods must be validated for
their intended use.
UCB products used for allogeneic trans-
plant must be tested for HLA Class I and Class
II antigens, including HLA-A, -B, and -DRB1
loci; HLA-C and HLA-DQB typing is recom-
mended.22 In this setting, ABO/Rh typing is
performed primarily to assist clinicians with
red cell and plasma product support after in-
fusion. Table 30-1 lists these basic tests.
Cryopreservation and Long-Term
Storage
Successful outcomes of UCB transplantation
hinge on the CBB’s ability to preserve the in-
tegrity of UCB over substantial lengths of time.
Freezing and storage methods must be robust
enough to ensure that the quality of the UCB
unit is maintained for many years.
The most common method of UCB cryo-
preservation consists of freezing the cells in a
cryogenic bag in the presence of 10% DMSO as
a cryoprotectant.43-45 One of the main advan-
tages of using cryogenic bags over freezing vi-
als is the ability to perform post-thaw confir-
matory HLA typing and QC testing using an
integrally attached tubing segment. Both
AABB and the Foundation for the Accredita-
tion of Cellular Therapy (FACT) mandate the
use of cryogenic bags with attached segments
for the freezing of UCB.22,24(p53)
It has been well established that slow
cooling in a programmable controlled-rate
freezing device at a rate of 1 C/minute results
in adequate recovery of CD34+ cells/human
progenitor cells.44,45 Such instruments com-
pensate for the heat of fusion phase, mitigate
process variability, and limit cell damage. As
with all critical processes involved in collect-
ing and storing UCB, the freezing process must
be validated and procedures must be in place
that govern the use and operation of the con-
trolled-rate freezer (or equivalent), expected

TABLE 30-1. Summary of QC Testing for UCB

Test Method(s)

Cell count Hematology analyzer, manual differential

CD34+ cell enumeration Flow cytometry (single or dual platform)
Viability assay Dye exclusion (light and/or fluorescence microscopy), flow cytometry
Clonogenic assay CFU (most commonly used in clinical laboratories), LTC-ICs
Sterility testing Aerobic, anaerobic, and fungal culture
HLA typing Molecular
ABO/Rh typing Serology
Hematocrit Standard/routine
QC = quality control, UCB = umbilical cord blood, CFU = colony-forming unit, LTC-ICs = Long-term culture-initiating cells.
 CHAPTER 30
cooling rates and freezing curve parameters,
endpoint freezing temperature, addition of
cryoprotectant, final cell and cryoprotectant
concentrations, and storage temperature. Be-
cause equipment failures can occasionally oc-
cur, simplified passive freeze methods can also
be validated to provide satisfactory cryo-
preservation of human stem cell products.46,47
Both AABB and FACT require cryopre-
served UCB to be stored at <–150 C.22,24(p54)
Once frozen, UCB units are typically trans-
ferred into a monitored liquid-nitrogen (LN2)
storage container and either overwrapped and
immersed in liquid (at –196 C) or in vapor
phase to minimize the potential for cross-
contamination during long-term storage.
To ensure the safety and stability of the
stored UCB units, control measures should be
established for product security and segrega-
tion, storage container monitoring, inventory
control, and duration of storage. Storage con-
tainers must be located in a secure area to pre-
vent unauthorized access. For units in which
transmissible disease screening and testing re-
sults are positive or the testing is incomplete,
there must be a designated quarantine storage
area or process to prevent accidental release.
To ensure that temperature and LN2 levels are
continuously maintained, a monitoring sys-
tem with local and remote alarm capabilities
must be in place. Alarm limits should be set to
allow adequate time for staff to respond to no-
tifications and react before inventory is com-
promised. All UCB products and reference
sample storage locations should be cataloged
using an inventory-management system that
allows for rapid retrieval. The duration of stor-
age and product expiration dates should be
defined in operating procedures, even when
expiry dates have yet to be determined.
Studies of the long-term effects of ultra-
low-temperature storage of UCB units have
shown that retention of viability and/or prolif-
erative function of UCB cells frozen in the liq-
uid phase of LN2 remains acceptable for at
least 23 years.48,49 However, each CBB must es-
tablish and maintain a written testing program
designed to assess the integrity, potency, safe-
ty, and stability of drug products manufac-
tured under its facility-specific manufacturing
Umbilical Cord Blood Banking 䡲 737
and storage conditions. The CBB’s UCB pro-
gram must address individual products before
distribution (lot release testing) and demon-
strate the stability of the drug’s active ingredi-
ents, used in determining expiration dates.
Post-Thaw Stability and Other Testing
Accreditation organizations require UCB
products to have integrally attached segments
that are representative of the UCB product and
used to verify the results of HLA typing.24(p78)
One of these segments can be used for confir-
matory testing—a process in which product
identity is confirmed and potency is evaluated
before the UCB unit is released to a transplant
facility. Extended HLA typing, viability, poten-
cy, or stability testing can be performed using
other replicate aliquots of the UCB unit (reten-
tion samples). Progenitor assays, CD34+ cell
enumeration, viability testing, or others tests
performed on segment material before distri-
bution may be useful in determining the prod-
uct’s potency. Traditionally performed as an
internal QC process, the results of these tests
are not released due to their lack of demon-
strated correlation with engraftment. Howev-
er, transplant centers, knowing that these re-
sults are available, are requesting them from
CBBs when determining which UCB units to
select. Accordingly, studies are under way to
assist with interpretation of the variable re-
sults obtained when testing is performed on
the limited material in these samples.
S HI P M E N T
With a number of commercial carriers avail-
able, UCB can be routinely transported to pro-
cessing laboratories or transplant facilities
within hours to a few days. It is important to
ensure that UCB units and products are prop-
erly packaged and shipped to prevent damage
or deterioration during shipment. This pro-
cess is the responsibility of the CBB. Validated
packaging and shipping procedures should
demonstrate that acceptable temperatures
and unit integrity are maintained.24(p35) Each
CBB needs to define the shipping conditions
(temperature, type of shipping container, and
738 䡲 AABB TECHNICAL MANUAL
packaging material) for the transport of its
fresh and frozen UCB products.
Shipping methods must also be designed
to protect the safety of the personnel in-
volved.22,50 The FDA, International Air Trans-
port Association (IATA), US Department of
Transportation (DOT), AABB, and FACT have
established packaging and labeling require-
ments for the shipping of biologics.22,24,51-53
IATA requires that shipping containers be able
to withstand extreme external temperature
variability and that primary outer containers
be leakproof, constructed to resist breakage,
and durable enough to withstand pressure
changes and falls. In addition, CBBs must
package UCB in a secondary inner container,
such as a plastic resealable bag, with enough
absorbent material to contain the contents of
the product in the event of a leak or break.22,52
Shipping Fresh UCB
The requirements for the transport of freshly
collected UCB are not well defined, and each
facility must establish transport temperature
criteria and acceptable limits that are com-
mensurate with the risks involved. Available
options include transport at room tempera-
ture or use of insulated, precooled stabilizing
packs. Wada and colleagues observed a 1%
drop in viability for every 4-hour increase in
transport time for recently collected UCB units
that were shipped at ambient temperature.54
However, a series of studies has shown that
UCB can be preserved in CPD for up to
48 hours before processing and retains satis-
factory cell recovery rates and progenitor con-
tent.55-58
Shipping Cryopreserved Units
An advantage of selecting a frozen UCB prod-
uct over a fresh marrow or apheresis product is
that the frozen UCB product can be shipped
and secured at the transplant facility before
the recipient is conditioned for transplant.
Cryopreserved products are shipped to trans-
plant facilities in portable LN2 “dry” shipping
containers to maintain the products in a fro-
zen state. Insulated containers are used that
allow LN2 to be absorbed into the vessel wall,
creating an ultracold environment. FACT re-
quires that LN2 dry shippers be validated to
maintain temperature of <–150 C for at least 48
hours beyond the time of delivery to the trans-
plant facility.22
For a dry shipper to be fully effective, it
must be charged or filled with LN2 24 hours be-
fore the estimated time of release from the
processing laboratory. This allows for com-
plete absorption of the LN2 into the wall of the
shipping container. Improperly prepared dry
shippers present a risk of LN2 leakage, and
CBBs may be subject to civil/criminal penal-
ties from US DOT in the event of a spill.
Dry shippers are vulnerable to incident
handling and can be compromised if they are
mistreated or positioned incorrectly. This will
result in warm conditions and thawing of the
product, rendering it unusable for the clinical
procedure. To minimize the risk of product
loss, shipping instructions must be clear and
the conditions of transport, particularly tem-
perature, must be continuously monitored.
Transport Labeling and Record
Requirements
AABB and FACT require continuous monitor-
ing of the temperature during shipment,
which is accomplished through the use of a
data logger.22,24(p35) According to AABB and
FACT, the shipping container must include the
name, address, and telephone number of the
shipping and receiving institutions; the phras-
es “medical specimen,” “do not irradiate” (if
applicable), and “do not x-ray”; and biohazard
labels (as appropriate).22,24(p68) Biological prod-
ucts must be packaged and shipped in compli-
ance with all applicable governmental regula-
tions. Transportation records that identify the
shipping facility, date and time the unit was
shipped and received, courier, and contents of
each shipping container should be main-
tained.22
R E CE I P T OF U CB F OR
T R A N SP L AN TAT I O N
UCB transplantation is a coordinated effort
potentially involving several entities—the reg-
 CHAPTER 30
istry, such as the National Marrow Donor Pro-
gram (NMDP), CBB, and cell-processing labo-
ratory/clinical team at the transplant center.
The process is initiated by the placement of a
reservation of or order for a UCB unit, usually
by the clinical program’s transplant coordina-
tor, based on the institutional algorithm or
guidelines. The coordinator may rely on the
laboratory or medical director to answer ques-
tions related to a prospective UCB unit, in-
cluding those associated with technical issues
and donor medical history. It is advisable to
initiate involvement of the cell-processing lab-
oratory early in the unit-selection process.59
Once the unit’s quality is confirmed and it
is approved for shipment, the coordinator
should notify the cell-processing laboratory of
the impending arrival of a UCB unit, including
any special handling requirements (eg, size
and dimensions of product canister or indica-
tions of risk factors requiring quarantine).
When the unit is received at the laboratory, it
should be carefully unpacked and examined to
verify its labeling and integrity before it is
placed into the LN2 storage tank. The dry ship-
per may be weighed to check for excessive LN2
loss (in general, greater than 10 pounds).
Because both AABB and FACT standards
require continuous temperature monitoring of
cryopreserved products during shipment,
temperature-monitoring devices must be in-
cluded in the shipment. Upon the unit’s arriv-
al, the technologist should confirm that the
UCB remained within the acceptable tempera-
ture range during shipment. If the tempera-
ture-monitoring device displays a digital tem-
perature, the temperature when the unit is
unpacked should be recorded. If the device
does not show a temperature or the data can-
not be downloaded, the CBB should forward a
copy of the data when they become available.
If the device for continuous temperature
monitoring cannot be located, a temperature-
measuring device from the transplant center
should be inserted for several hours to ensure
that the shipper can maintain an acceptable
temperature. The CBB should be notified that
no temperature-monitoring device was pres-
ent in the shipment, and documentation of
Umbilical Cord Blood Banking 䡲 739
the CBB’s shipper validation should be re-
quested.
The identity of the UCB unit should be
verified at the time of inspection, before the
unit is placed in storage. The product label
should indicate the appropriate minimum
partial label requirements—unique product
identifier (ie, unit number) and proper prod-
uct name. All other product information
should be attached to the product on a tie tag
and/or in the accompanying paper work.
Whether affixed or attached, the CBB paper
work that accompanies the UCB and the docu-
ments generated by the transplant program/
coordinator should be compared. Any incon-
sistencies should be immediately and appro-
priately investigated.
The unit information, including donor
medical history and infectious disease testing
results, should again be reviewed for com-
pleteness. If there are any comments not pre-
viously communicated to the receiving labora-
tory, particularly any related to the donor’s
medical history, they should be referred to the
receiving laboratory’s medical director. The
medical director should determine whether
the information is significant and requires any
further action and/or notification of the trans-
plant physician. If any infectious disease test-
ing is found to be incomplete or the results are
positive, the unit should be placed into quar-
antine storage, and the appropriate personnel
(eg, laboratory supervisor, medical/laboratory
director, coordinator, and/or transplant physi-
cian) should be notified. The product label/tie
tag must be updated accordingly (eg, to in-
clude a biohazard label) and a special medical
release form may need to be completed as
well.
Any further testing deemed necessary by
the transplant center (eg, additional HLA or
viability testing or CFU count) should be
performed as soon as possible on an integrally
attached segment, if one accompanies the
unit. If a unit’s identity is in question and an
integrally attached segment is not available, a
rapid (Class I serologic) HLA test may be per-
formed along with the standard product test-
ing on the thawed product (ie, on the day of
the transplant procedure).60
740 䡲 AABB TECHNICAL MANUAL
Instructions for handling and preparing
frozen UCB products are provided by the UCB
manufacturer in the shipment. However, the
receiving laboratory may use alternative thaw-
ing methods based on its own validated proce-
dures, clinical protocol, or physician prefer-
ence.
TH AW ING AND WAS H ING OF
UCB
Coordination of the transplant event requires
consistent communication among all mem-
bers of the clinical team. In the days before the
planned transplantation, the coordinator
should verify the infusion date with the clini-
cal team, and both the coordinator and labora-
tory should again review all relevant records.
Subsequently, the patient care unit should be
contacted at least 1 day before the day of
transplant to schedule an infusion time. Given
the wide variety of types of products received
from an increasing number of CBBs, the most
appropriate thawing procedure must be deter-
mined based on the transplant center’s vali-
dated method or CBB’s recommendations.
There may be considerations if the product
was manufactured to reduce red cell and plas-
ma content (RBC reduced) or to reduce plas-
ma content only (red cell replete) before cryo-
preservation. The choice of the thawing
procedure may be further affected by the re-
quirements of the clinical protocol in which
the patient is enrolled.
Currently, three different practices for
preparing UCB products for infusion are avail-
able: the traditional thaw-and-wash method,
the thaw-and-dilution technique, and the bed-
side thaw method.61,62
Despite the potential advantage of the
bedside product preparation method in mini-
mizing potential cell loss from postthaw ma-
nipulation, this approach is not recommended
because of the difficulty of rescuing the prod-
uct at the bedside if the bag’s integrity is com-
promised and the adverse effect of prolonged
exposure of thawed cells to DMSO if the infu-
sion is delayed. Furthermore, this method
lacks the capacity for process control and
product assessment. Finally, this method
should not be used with red cell-replete prod-
ucts according to recent FACT requirements.
The traditional washing method original-
ly described in 1995 is recommended if the
product’s exposure to DMSO, free hemoglobin,
and volume approach critical limits for recipi-
ents, particularly pediatric patients or those
with underlying cardiac, pulmonary, or sensi-
tizing conditions.39 More recently, a dilution or
simple reconstitution approach has gained
support, primarily due to concerns about cell
loss during the wash step.63,64 Both methods
are initiated in similar fashion:
䡲 The UCB product is carefully removed from
the storage tank and a thorough inspection
is performed to evaluate the container’s in-
tegrity. Verification of the product’s identity
on its label is conducted by two technolo-
gists.
䡲 The unit is sealed in a clean or sterile trans-
parent bag (ie, a plastic zipper bag) and
submerged in a 37 C waterbath of clean or
sterile water or saline. Gentle kneading of
the UCB bag during thawing helps acceler-
ate the thawing process while preventing
recrystallization and consequential cell
damage or death. If a leak is discovered af-
ter initiation of the thaw procedure, the site
of the container break is determined and a
hemostat is employed or the product is po-
sitioned to prevent further escape of the
cellular material. The contents can then be
aseptically transferred into a transfer bag
under a biological safety cabinet.40
䡲 The thaw solution is used for product dilu-
tion and should be prepared in advance. It
typically contains 10% dextran and a pro-
tein source, usually human serum albumin
in a final concentration of approximately
2.5% to 4.2%. The supplementation of the
thaw solution with protein (albumin) has
been proven to restore osmolarity and ex-
tend cell viability.39 Subsequently, a volume
of solution at least equal to the volume of
the UCB product is gradually added to the
bag while it is gently mixed. The product
and solutions are drained into a labeled
transfer bag and left to equilibrate for 5
minutes. At this stage, if the product is to be
 CHAPTER 30
reconstituted or diluted only, samples are
removed for product testing.
䡲 If the product is to be washed, the labeled
transfer bag is centrifuged at 400-600 × g for
15 minutes at 10 C. The supernatant is ex-
pressed, leaving behind a pellet of washed
UCB cells. If it is the policy of the transplant
laboratory to centrifuge the supernatant,
the second labeled transfer bag is spun at
400 × g for 15 minutes at 10 C, the superna-
tant is again expressed, and the two cell
pellets are combined into one labeled bag.
䡲 The UCB cells are resuspended in a volume
of thaw solution that is appropriate for the
weight of the patient while additional con-
sideration is given to the potential for fluid
overload.
䡲 Some laboratories filter the resuspended
product with a standard blood filter (at 170-
260 microns).
䡲 Samples are removed for QC tests, such as
total nucleated cell (TNC), CD34+, and
CD3+ cell counts; microbial culture (bacte-
ria, fungus); confirmatory ABO/Rh typing;
CFU assay; and viability testing. Final vol-
ume, cell dose, and cell recovery are deter-
mined. After completion of paper work and
labeling, the unit can be released for infu-
sion).58
It is anticipated that thawing procedures
based on the type of product processed will be
standardized through the collaboration of
UCB bankers and transplanters.
INFUSION OF UCB
To facilitate the product infusion procedure, it
is necessary to maintain good communication
between the patient and the physician over-
seeing the infusion. Hence, it is a good practice
to again describe the general process, includ-
ing graft selection, cell processing, infusion,
potential side effects/adverse reactions, and
plans for premedication. This conversation
should take place on, or shortly before, the day
of the transplant procedure. A procedure for
infusion is outlined below.50 The general ap-
proach and potential adverse reactions are
Umbilical Cord Blood Banking 䡲 741
similar to those for infusions of other HSC
products.65
Once the UCB product has been thawed
and washed, it should be delivered to the pa-
tient care unit without delay. Subsequently, a
form acknowledging the receipt of the UCB
should be signed by a nurse, who then notifies
the patient’s physician of the UCB unit’s arriv-
al. After the physician approves the infusion
and proper identification procedures are fol-
lowed, the unit is infused by intravenous (IV)
drip or syringe push directly into a central line
without a needle or a pump. Some institutions
use a standard blood filter at the bedside. If a
filter is used in the laboratory after the thaw/
wash, a second standard blood filter at the
bedside is not needed.
The thawed UCB must be transfused as
soon as clinically possible. Timely infusion is
most likely to be challenging with UCB that
has not been washed or diluted. Although
Rowley and Anderson66 concluded that DMSO
is not toxic to HSCs at clinically relevant con-
centrations (ie, 5% or 10%) at either 4 C or 37 C
for up to 1 hour of incubation, they also noted
that the addition of 1% DMSO to culture dish-
es suppresses CFU assay results. It is impor-
tant to note that these studies were performed
on fresh cells.67 Studies of the effects of DMSO
on HSCs that have been previously cryopre-
served are limited. However, some investiga-
tors have noted similar suppression of colony
formation when thawed UCB samples are not
washed and are immediately placed into CFU
culture.67 Thus, the possible functional defect
due to DMSO coupled with the not infrequent
need to hold clinical products raises the con-
cern that cell injury could occur with thawed
UCB, particularly when cells are not washed or
diluted.
The unit bag and IV tubing should be
flushed with sterile saline after the unit bag
empties to maximize cell dose. Sterile saline
may be added directly to the unit bag if the
flow rate becomes unusually slow. Because the
final volume of a UCB unit is relatively low
(roughly 60 to 100 mL with the thaw/wash
methods described above), the infusion rate is
slow (5-10 mL/minute) and the infusion pro-
cess is typically completed within 15 to 30
742 䡲 AABB TECHNICAL MANUAL
minutes of the unit’s receipt in the patient care
unit.
It is recommended that the patient’s vital
signs be checked before infusion, immediate-
ly after infusion, and 1 hour after infusion at a
minimum. Should an adverse reaction occur,
more frequent monitoring is recommended.
The transplant physician and the medical di-
rector of the cell therapy laboratory should be
notified immediately of an unexpected or
moderate to severe reaction. A prompt investi-
gation should include confirmation of product
identity, a product inspection, and the initia-
tion of any appropriate laboratory testing (eg,
direct antiglobulin test, antibody titers, Gram’s
stain, or culture). All of the monitoring results
should be captured on the accompanying in-
fusion form, which should be returned to the
laboratory. Serious adverse reactions must
then be communicated as appropriate to the
NMDP, distributing CBB, and/or the IND ap-
plication or license holder for reporting to the
FDA.
Reactions associated with UCB infusion
may be very similar to those that occasionally
occur with blood transfusion (ie, allergic, he-
molytic, or febrile reactions and those due to
microbial contamination). However, with
combinations of the various processing meth-
ods for UCB (eg, red cell depletion, plasma re-
duction, and postthaw wash step), some types
of reactions to UCB (ie, those attributed to red
cell antigens and plasma proteins) may be less
likely to occur with UCB than other blood
transfusions. Likewise, if the UCB has under-
gone red cell depletion and/or wash steps, re-
nal failure due to infusion of red cells and free
hemoglobin should occur less often than with
infusions of HSCs from other sources. Reac-
tions (ie, nausea, vomiting, coughing, and
headache) often attributed to DMSO should
be less common with UCB infusions as
well.68,69 Bacterial contamination, which oc-
curs in up to 5% of collections, is unusual in
released UCB units because CBBs exclude
these units from their inventories.70-72
UCB infusions are usually very well toler-
ated; if reactions occur, they are typically mild
and readily managed by the clinical team.65
However, because the possibility of a severe re-
action exists, aggressive IV hydration is recom-
mended (eg, for 2 to 6 hours before and 6
hours after infusion with diuretics as needed).
In addition, administration of prophylactic an-
tiemetics, antipyretics, and antihistamines is
suggested.
ECO N O M I C I S S U ES
The establishment of a diverse and sustainable
inventory from which approximately 1% of
products are distributed for clinical use is an
expensive undertaking for a CBB. One of the
main reasons for this selectivity in product uti-
lization is the strong association of successful
outcomes of UCB transplantation with the
grade of the HLA match and cell dose.73 As a
result, UCB selection has recently been direct-
ed toward utilization of products with high
TNC content. This finding further complicates
the strategy of banking products from donors
who are not well represented in registries due
to the reduced volume and cellular content in
the collections from these populations.
To limit the negative economic impact of
these trends, CBBs must expand their collec-
tion efforts to increase the proportion of units
with higher TNC counts.74 In addition, ex-
panding the number of collection sites to al-
low broader participation of donors could
support diversification efforts. Improving the
efficiency of the collection process in estab-
lished centers could increase the number of
products eligible for further manufacture and
their likelihood of selection while offsetting
the associated costs related to education, staff-
ing, and transportation. Collaborating to iden-
tify alternative uses for clinical-grade products
could also help CBBs achieve self-sufficiency.
The transition to a full cGMP setting has
been accompanied by spending money to ex-
pand UCB inventories. The facility used to
manufacture human cells, tissues, and cellu-
lar and tissue-based products (HCT/Ps) must
be maintained in a clean, sanitary, and orderly
manner to prevent the introduction, transmis-
sion, or spread of communicable diseases, as
described in Title 21, CFR Part 1271.190(b). To
minimize the risk of product contamination,
manufacturers (CBBs) may upgrade their facil-
 CHAPTER 30 Umbilical Cord Blood Banking
ities to offer International Organization for
Standardization Class 5 processing areas, de-
fine robust cleaning and disinfection plans,
enhance environmental monitoring to ensure
continued control of the manufacturing areas,
and develop comprehensive stability plans to
establish expiry dates of products manufac-
tured under these conditions. Facility retrofit-
ting, process redesign, and hiring of additional
staff to accommodate upgraded activities in-
crease the fixed cost of UCB manufacturing.
Whereas pharmaceutical drug manufac-
turers can recover the cost of research and de-
velopment along with a substantial profit mar-
gin from the consumer, it is improbable that
CBBs will be able to incorporate increasing
costs of UCB product manufacture into the
amounts invoiced to clinical programs for
product acquisition. Doing so may encourage
transplant centers to pursue alternative means
of treatment for their patients, a strategy that
would be detrimental to the survival of UCB
manufacturers. Therefore, public CBBs must
be innovative and resourceful in all aspects of
their business strategy.
The Stem Cell Therapeutic and
Research Act
The Stem Cell Therapeutic and Research Act of
2005 was passed by Congress and signed by
President George W. Bush in December 2005
as Public Law 109-129. In October 2010, Presi-
dent Obama signed the Stem Cell Therapeutic
and Research Reauthorization Act of 2010
(Public Law 111-264). The implementation of
both acts is managed by the Health Resources
and Services Administration (HRSA) of the US
Department of Health and Human Services
(DHHS). Provisions in these acts that are spe-
cific to UCB include:
䡲 The CW Bill Young Cell Transplantation
Program, intended to increase the numbers
of UCB units and establish an outcomes da-
tabase to collect data to characterize and
refine all aspects of UCB transplantation.
Through the Stem Cell Therapeutic Out-
comes Database, the Center for Interna-
tional Blood and Marrow Transplant Re-
䡲 743
search received a contract to collect data on
all allogeneic (related and unrelated) HSC
transplantations performed in the United
States and those that were completed with
products procured through the program
but performed outside the United States.
䡲 Solicitation and subsequent contractual
agreements with CBBs to collect and store
at least 150,000 new UCB units that meet
specific criteria comprise the National Cord
Blood Inventory (NCBI). CBBs with a con-
tract from the NCBI also provide UCB units
that do not meet these criteria for research
studies.
䡲 Requirement for the US Government Ac-
countability Office to report on efforts to
increase UCB unit collection for the NCBI.
䡲 Establishment of the ACBSCT to make rec-
ommendations to the Secretary of DHHS.
This legislation not only validated UCB as
a credible therapeutic option but also provid-
ed financial support to CBBs for the creation of
larger inventories. However, federal funding
does not cover the costs associated with man-
ufacturing and is limited in terms of appropri-
ations. In the 2010 reauthorization, Congress
challenged CBBs to expand their collection
while lowering costs and improving the effi-
ciency of UCB unit collection without decreas-
ing the quality of the UCB units collected. In
addition, CBBs are required to demonstrate
ongoing measurable progress toward achiev-
ing self-sufficiency in their collection and
banking operations.
R E G U L AT I O NS A ND
S TA N D A R D S
FDA
In 1997, the FDA announced a novel, risk-
based, tiered approach to the regulation of so-
matic cells and tissues.75 The approach out-
lined in the FDA’s proposed approach to regu-
lation of cellular and tissue-based products
(February 27, 1997) was followed by the good
tissue practice (GTP) regulations of 2004
“requiring cells and tissues to be handled ac-
cording to procedures designed to prevent
744 䡲 AABB TECHNICAL MANUAL

contamination and preserve [cell and] tissue
function and integrity.”51 This historic docu-
ment has set the framework for cell therapy
laboratories by identifying the agency’s expec-
tations regarding the prevention of disease
transmission and laboratory process controls.
The GTP requirements are intended to 1) pre-
vent unknowing use of contaminated tissue at
risk of transmitting infectious disease, 2) pre-
vent improper processing of tissue in ways
that could cause damage or risk of contamina-
tion, and 3) ensure the safety and efficacy of
products that are more than minimally manip-
ulated.75,76 Based on these principles, all hu-
man cellular and tissue-based products in-
tended for use in unrelated recipients,
regardless of degree of manipulation or poten-
tial risk, are required to comply with the re-
quirements for donor testing and screening,76
establishment registration,77 and the current
GTP regulations.51
Since 2004, CBBs have been required to
register with the FDA as manufacturers of
UCB51 and demonstrate compliance with
GTPs. For CBBs that manufacture and/or store
more than minimally manipulated products
(eg, UCB that is activated, expanded, or genet-
ically modified) or combine UCB with non-
tissue components, the FDA requires submis-
sion of an IND application and adherence to
licensure application requirements. Table 30-2
summarizes the FDA’s regulations governing
HCT/Ps, including UCB.

TABLE 30-2. Summary of FDA Regulations Regarding Human Cells, Tissues, and Cellular and
Tissue-Based Products (HCT/Ps) and Hematopoietic Progenitor Cells-Cord (HPC-Cs)75

Products Regulated Specific Product

HCT/Ps that are or will be regu- 䡲 All allogeneic, unrelated HCT/Ps derived from cord and peripheral blood
lated as biological products 䡲 HCT/Ps that are more than minimally manipulated (eg, expanded, acti-
vated, or genetically modified).
䡲 HCT/Ps that are combined with a drug, device, or biological product.
䡲 HCT/Ps that are intended for nonhomologous use (eg, for cardiac
repair).
HPC-Cs that are currently subject 䡲 Allogeneic, unrelated, and minimally manipulated cells intended for use
to biologics license application in unrelated donor HPC transplantation procedures in conjunction with
(BLA) requirements an appropriate preparative regimen for hematopoietic and immunologic
reconstitution in patients with disorders affecting the hematopoietic sys-
tem that are inherited, acquired, or result from myeloablative treatment.
䡲 FDA intends to grant licensure to products manufactured according to
recommended establishment and process controls and that comply with
all applicable regulatory requirements.
HPC-C that is currently subject to 䡲 When no satisfactory alternative treatment is available, HPC-Cs that are
investigational new drug require- not FDA licensed and are:
ments 1. Allogeneic, unrelated, and minimally manipulated.
2. Intended for use in unrelated donor HPC transplantation procedures
in conjunction with an appropriate preparative regimen for hemato-
poietic and immunologic reconstitution in patients with disorders
affecting the hematopoietic system that are inherited, acquired, or
result from myeloablative treatment.
3. Manufactured in a US cord blood bank and intended to be used in
the United States.
䡲 Manufactured in a US cord blood bank before BLA approval and do not
meet licensing requirements.
䡲 Prospectively manufactured in the United States and for which there is
no satisfactory alternative.
FDA = Food and Drug Administration.
 CHAPTER 30 Umbilical Cord Blood Banking
Until 2011, minimally manipulated UCB
intended for use in unrelated recipients was
not licensed. However, the FDA has long con-
sidered licensure as a way of improving the
safety and quality of UCB by instituting re-
quirements that would be legally enforce-
able.51 The FDA believed that compelling clini-
cal safety and efficacy data existed to support
the development of product standards as an
approach toward licensure. In 1998, the FDA
issued a request for the public to submit com-
ments regarding establishment controls, pro-
cess controls, and product standards for UCB
manufacturers.78 After reviewing the submit-
ted information, the FDA determined that suf-
ficient data did exist to support the develop-
ment of processing and product standards for
granting licensure.
In October 2009, the FDA published its fi-
nal guidance, which described ways to assist
UCB manufacturers in submitting a BLA.79
Originally published with indications restrict-
ed to “hematopoietic reconstitution in pa-
tients with hematologic malignancies, certain
lysosomal storage and peroxisomal enzyme
deficiency disorders, primary immunodefi-
ciency diseases, bone marrow failure, and beta
thalassemias,” the guidance was modified by
the FDA in 2011 to broaden the indications to
“use in unrelated donor hematopoietic pro-
genitor cell transplantation procedures in con-
junction with an appropriate preparative regi-
men for hematopoietic and immunologic
reconstitution in patients with disorders af-
fecting the hematopoietic system that are in-
herited, acquired, or result from myeloablative
treatment.” This guidance applies to hemato-
poietic progenitor cells (HPCs), UCB [HPCs-
cord (HPC-Cs)] manufactured by US UCB
establishments and non-US UCB establish-
ments that distribute UCB in the United
States. For establishments that manufacture
UCB products for autologous use or use by a
first- or second-degree blood relative, the FDA
encourages the same manufacturing recom-
mendations and applicable regulations be fol-
lowed.20
The FDA recognizes that there will be cir-
cumstances in which the best available UCB
unit for a patient is not a licensed unit. There
䡲 745
may be units from UCB establishments in
which a BLA is pending; units that do not meet
licensing requirements, or units that come from
non-US CBBs that have chosen not to apply
for licensure. Under such circumstances, the
FDA requires submission of an IND application.
FDA published the guidance for such applica-
tions as companion documents to the BLA.80
In March 2014, FDA updated the 2009
BLA and 2011 IND guidance documents. The
BLA guidance document was updated to ex-
pand the indications for use, clarify the type of
clinical data required in the application, and
replaced the term “HPC-C” with “HPC, Cord
Blood.” Similar revisions were made in the
IND guidance document, in addition to clari-
fying that a table of contents is required for all
IND applications.
In summary, the FDA regulates HPCs,
HPC-C blood for unrelated allogeneic use as a
drug under the Food Drug and Cosmetic Act
and as biological products under the Public
Health Services Act. Therefore, regulations ap-
plicable to HPC-Cs include:
1. Title 21, CFR Part 201 and 610 Subpart G –
Labeling.
2. Title 21, CFR Part 202: Prescription drug
advertising.
3. Title 21, CFR Parts 210 and 211: cGMP.
4. Title 21, CFR Part 600: Biological products,
general.
5. Title 21, CFR Part 601: Licensing.
6. Title 21, CFR Part 610: General biological
products standards.
7. Title 21, CFR Part 1270 (Section 351 of the
Public Health Services Act): HCT/Ps with
systemic effect intended for use in unre-
lated persons.
8. Title 21, CFR 1271 (Section 361 of the Public
Health Services Act): HCT/P registration
and listing, donor eligibility, and GTP.
In the event that one regulation is in con-
flict with another, the one that is more specifi-
cally applicable to UCB supersedes the one
that is more general.
In combination, standards regulating bio-
logic drugs are akin to those for pharmaceuti-
cal manufacturing, and their application to a
746 䡲 AABB TECHNICAL MANUAL

cellular therapy product, such as UCB, is chal-
lenging. In contrast to pharmaceutical drugs,
which are chemicals, are manufactured in tab-
let lots, and can be terminally sterilized, UCB
products are composed of viable cells, are
manufactured in single-unit lots, and must be
processed aseptically. Recognizing the value
and unique nature of each UCB product, FDA
has been very cautious in regulating UCB to
avoid preventing patients from having access
to existing or prospectively manufactured
products. At the time of this writing, the FDA
has approved the BLAs of five CBBs in the
United States (see Table 30-3).
AABB and FACT
The AABB and FACT are the two professional
organizations that have established standards
for HPCs, including UCB, in the United
States.22,24 AABB has incorporated the require-
ments for UCB processing facilities in its Stan-
dards for Cellular Therapy Product Services.24
FACT and NetCord combined their efforts to
establish a unique set of standards for UCB-re-
lated activities.22 Both AABB and FACT have
created standards that align with the FDA’s
GMP and GTP requirements and center on
quality systems essentials. These standards
cover donor suitability, collection, processing
controls, document controls, facility manage-
ment, materials, records, storage, distribution,
quality audits, quality plans, errors/deviations,
labeling, personnel, equipment, validation,
outcome reviews, and adverse event reporting.
In 2009, the ACBSCT recommended that both
AABB and FACT be recognized as accreditation
organizations for the NCBI. Both organiza-
tions incorporated specific standards to en-
sure that CBBs comply with the contracts held
with HRSA.
In a post-licensure era, it is expected that
UCB banking will continue to evolve to bal-
ance the creation of large inventories of safely
manufactured products that not only meet de-
mands for cell dose and matching from clini-
cians, but also target new populations and ap-
plications for use of this abundantly available
resource. Currently, there are several ongoing
clinical trials involving HSCs and HPC expan-
sion approaches to overcome limited per-unit
cell doses and the lengthy time required to
achieve an absolute neutrophil count of 500/µL;
UCB-derived natural killer, T-regulatory, and
dendritic cells for immunotherapeutic treat-
ments; and UCB-derived mesenchymal stem
or stromal cells to enhance engraftment and
regenerative applications. However, optimal
methods for production and clinical applica-
bility of these new products have yet to be es-
tablished.

TABLE 30-3. Approved Cord Blood Biologic License Applications*

Product Name Manufacturer

Hemacord New York Blood Center (New York, NY)

HPC-Cord Blood University of Colorado Cord Blood Bank (Denver, CO)
Ducord Carolinas Cord Blood Bank (Durham, NC)
Allocord St. Louis Cord Blood Bank (St. Louis, MO)
HPC-Cord Blood LifeSouth Community Blood Centers (Gainesville, FL)
*As of March 2014.
 CHAPTER 30 Umbilical Cord Blood Banking 䡲 747

KEY POINTS

1. UCB stem cells have a higher proliferative capacity and immune tolerance than stem cells
from adult sources, as demonstrated by the decreased incidence of GVHD in UCB recipients
despite the less stringent HLA-matching requirements for UCB transfusion. These features
allow the use of more flexible matches with UCB, which provide the ability to support pa-
tients with rare HLA types and permit matches across ethnic lines.
2. UCB has evolved from being considered biologic waste to acceptance as a viable source of
HSCs. More than 30,000 unrelated donor UCB transplant procedures have been performed,
and there are an estimated 600,000 UCB units banked worldwide for use as an HPC source.
In 2008, UCB surpassed marrow as the most frequently utilized source of donor cells for un-
related transplantation.
3. Engaging pregnant women and arranging donation of their infant’s UCB in advance is criti-
cal for achieving the best results. Early education allows for more complete medical screen-
ing, comprehensive donor protection, availability of adequate supplies, and acquisition of
thorough informed consent.
4. UCB can be collected before (in utero) or after (ex utero) the placenta has been delivered.
There are procedural and economic advantages and disadvantages to each method. When
performed properly, both are acceptable for providing high-quality collections.
5. Current methods of manufacturing UCB products involve reduction of red cell and plasma
fractions before cryopreservation. Methods generally involve sedimentation and/or centrif-
ugation and may be performed manually or with automated devices that have been cleared
for use by the FDA.
6. Transplant centers must determine the most appropriate thawing procedure for the prod-
ucts received based on the manufacturer’s instructions, red cell volume, patient-specific
variables, and their own validated method. Current practices for preparing UCB for infusion
consist of bedside thawing, the traditional thaw-and-wash method, and a thaw-and-recon-
stitution technique.
7. Following the announcement of a tiered approach to regulating UCB in 1997, the FDA pub-
lished a guidance document in 2009 defining a process by which CBBs can submit a BLA to
the FDA for UCB. Through this process, five public CBBs have been authorized to manufac-
ture HPC products from UCB for allogeneic use.
8. The clinical utility of several cell types within UCB (eg, mesenchymal stem and immune
cells) is being investigated for nonhematopoeitic applications, immunomodulation, and re-
generative medicine.
9. To ensure their economic sustainability, CBBs must adapt to recent trends in product selec-
tion, improve collection efficiency, collaborate to identify alternative uses for clinical-grade
products, and successfully transition to a full cGMP and licensure environment.

R E F E R EN C E S

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2. Rocha V, Cornish J, Sievers EL, et al. Compari-
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3. Eapen M, Rubinstein P, Zhang MJ, et al. Out-
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16. Gluckman E, Broxmeyer HA, Auerbach AD, et
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17. Broxmeyer HE. Biology of cord blood cells and
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18. Rubinstein P, Rosenfield RE, Adamson JW, Ste-
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22. Foundation for the Accreditation of Cellular
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23. Cord blood banking for potential future trans-
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 CHAPTER 30
24. Fontaine M, ed. Standards for cellular therapy
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25. Council on Ethical and Judicial Affairs, Ameri-
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26. American Congress of Obstetricians and Gy-
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27. Askari S, Miller J, Clay M, et al. The role of the
paternal health history in cord blood banking.
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28. Vawter DE, Rogers-Chrysler G, Clay M, et al. A
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29. Health Resources and Services Administra-
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30. Wick M, Clay ME, Eastlund T, et al. Genetic
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31. Lasky LC, Lane TA, Miller JP, et al. In utero or ex
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32. Surbek DV, Schonfeld B, Tichelli A, et al. Opti-
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33. Solves P, Moraga R, Saucedo E, et al. Compari-
son between two strategies for umbilical cord
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34. Wong A, Yuen PM, Li K, et al. Cord blood col-
lection before and after placental delivery:
Levels of nucleated cells, haematopoietic pro-
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macroscopic clots. Bone Marrow Transplant
2001;27:133-8.
35. Solves P, Moraga R, Mirabet V, et al. In utero or
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question. Transfusion 2003;43:1174-6.
36. Kurtzberg J, Laughlin M, Graham ML, et al.
Placental blood as a source of hematopoietic
Umbilical Cord Blood Banking 䡲 749
stem cells for transplantation into unrelated
recipients. N Engl J Med 1996;335:157-66.
37. Solves P, Mirabet V, Planelles D, et al. Red
blood cell depletion with a semiautomated
system or hydroxyethyl starch sedimentation
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38. Tsang KS, Li K, Huang DP, et al. Dextran sedi-
mentation in a semi-closed system for the
clinical banking of umbilical cord blood.
Transfusion 2001;41:344-52.
39. Rubinstein P, Dobrila L, Rosenfield RE, et al.
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1995;92:10119-22.
40. Alonso JM III, Regan DM, Johnson CE, et al. A
simple and reliable procedure for cord blood
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and Ohio Cord Blood Bank experiences. Cyto-
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41. Almici C, Carlo-Stella C, Wagner JE, Rizzoli V.
Density separation and cryopreservation of
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42. Takahashi TA, Rebulla P, Armitage S, et al.
Multi-laboratory evaluation of procedures for
reducing the volume of cord blood: Influence
on cell recoveries. Cytotherapy 2006;8:254-64.
43. Institute of Medicine. Cord blood: Establishing
a national hematopoietic stem cell bank pro-
gram. Washington, DC: The National Acade-
mies Press, 2005.
44. Fraser JK, Cairo MS, Wagner EL, et al. Cord
Blood Transplantation Study (COBLT): Cord
blood bank standard operating procedures.
J Hematother 1998;7:521-61.
45. Donaldson C, Armitage WJ, Denning-Kendall
PA, et al. Optimal cryopreservation of human
umbilical cord blood. Bone Marrow Trans-
plant 1996;18:725-31.
46. McCullough J, Haley R, Clay M, et al. Long-
term storage of peripheral blood stem cells
frozen and stored with a conventional liquid
nitrogen technique compared with cells fro-
zen and stored in a mechanical freezer. Trans-
fusion 2010;50:808-19.
47. Antoniewicz-Papis J, Lachert E, Wozniak J, et
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48. Kobylka P, Ivanyi P, Breur-Vriesendorp BS.
Preservation of immunological and colony-
forming capacities of long-term (15 years)
750 䡲 AABB TECHNICAL MANUAL
cryopreserved cord blood cells. Transplanta-
tion 1998;65:1275-8.
49. Broxmeyer HE. Will iPS cells enhance thera-
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50. Chrysler G, McKenna D, Schierman T, et al.
Umbilical cord blood banking. In: Broxmeyer
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51. Food and Drug Administration. Current good
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52. IATA. International Air Transport Association
(IATA) dangerous goods regulations. 44 ed. Ge-
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53. Code of federal regulations. Title 49 CFR, Sub-
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54. Wada RK, Bradford A, Moogk M, et al. Cord
blood units collected at a remote site: A collab-
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blood through the Hawaii Cord Blood Bank
and store the units at the Puget Sound Blood
Center. Transfusion 2004;44:111-18.
55. Hubel A, Carlquist D, Clay M, McCullough J.
Short-term liquid storage of umbilical cord
blood. Transfusion 2003;43:626-32.
56. Hubel A, Carlquist D, Clay M, McCullough J.
Cryopreservation of cord blood after liquid
storage. Cytotherapy 2003;5:370-6.
57. Hubel A, Carlquist D, Clay M, McCullough J.
Liquid storage, shipment, and cryopreserva-
tion of cord blood. Transfusion 2004;44:518-
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58. Solomon M, Wofford J, Johnson C, et al. Fac-
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ment before cryopreservation. Transfusion
2009;50:820-30.
59. McCullough J, McKenna D, Kadidlo D, et al. Is-
sues in the quality of umbilical cord blood
stem cells for transplantation. Transfusion
2005;45:832-41.
60. McCullough J, McKenna D, Kadidlo D, et al.
Mislabeled units of umbilical cord blood de-
tected by a quality assurance program at the
transplantation center. Blood 2009;114:1684-8.
61. Nagamura-Inoue T, Shioya M, Sugo M, et al.
Wash-out of DMSO does not improve the
speed of engraftment of cord blood transplan-
tation: Follow-up of 46 adult patients with
units shipped from a single cord blood bank.
Transfusion 2003;43:1285-95.
62. Hahn T, Bunworasate U, George MC, et al. Use
of nonvolume-reduced (unmanipulated after
thawing) umbilical cord blood stem cells for
allogeneic transplantation results in safe en-
graftment. Bone Marrow Transplant 2003;32:
145-50.
63. Barker JN, Abboud M, Rice RD, et al. A “no-
wash” albumin-dextran dilution strategy for
cord blood unit thaw: High rate of engraftment
and a low incidence of serious infusion reac-
tions. Biol Blood Marrow Transplant 2009;
15:1596-602.
64. Regan DM, Wofford JD, Wall DA. Comparison
of cord blood thawing methods on cell recov-
ery, potency, and infusion. Transfusion 2010;
50:2670-5.
65. McKenna D, McCullogh J. Umbilical cord
blood infusions are associated with mild reac-
tions and are overall well-tolerated. Cytothera-
py 2003;5:438.
66. Rowley SD, Anderson GL. Effect of DMSO ex-
posure without cryopreservation on hemato-
poietic progenitor cells. Bone Marrow Trans-
plant 1993;11:389-93.
67. Laroche V, McKenna DH, Moroff G, et al. Cell
loss and recovery in umbilical cord blood pro-
cessing: A comparison of postthaw and post-
wash samples. Transfusion 2005;45:1909-16.
68. Davis JM, Rowley SD, Braine HG, et al. Clinical
toxicity of cryopreserved bone marrow graft
infusion. Blood 1990;75:781-6.
69. Stroncek DF, Fautsch SK, Lasky LC, et al. Ad-
verse reactions in patients transfused with
cryopreserved marrow. Transfusion 1991;31:
521-6.
70. Bornstein R, Flores AI, Montalban MA, et al. A
modified cord blood collection method
achieves sufficient cell levels for transplanta-
tion in most adult patients. Stem Cells 2005;
23:324-34.
71. M-Reboredo N, Diaz A, Castro A, Villaescusa
RG. Collection, processing and cryopreserva-
tion of umbilical cord blood for unrelated
transplantation. Bone Marrow Transplant
2000;26:1263-70.
72. Lecchi L, Ratti I, Lazzari L, et al. Reasons for
discard of umbilical cord blood units before
cryopreservation. Transfusion 2000;40:122-4.
73. Barker JN, Scaradavou A, Stevens CE. Com-
bined effect of total nucleated cell dose and
HLA match on transplantation outcome in
 CHAPTER 30
1061 cord blood recipients with hematologic
malignancies. Blood 2010;115:1843-9.
74. Bart T, Boo M, Balabanova S, et al. Impact of
selection of cord blood units from the United
States and Swiss registries on the cost of bank-
ing operations. Transfus Med Hemother 2013;
40:14-20.
75. Food and Drug Administration. Proposed
approach to regulation of cellular and tissue-
based products. CBER Office of Communica-
tion, Outreach, and Development, 1997.
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UCM062601.pdf (accessed December 6,
2013).]
76. Food and Drug Administration. Eligibility de-
termination for donors of human cells, tissues,
and cellular and tissue-based products; Final
Rule. Fed Regist 2004;69:29786-834.
77. Food and Drug Administration. Human cells,
tissues, and cellular and tissue-based prod-
ucts; establishment registration and listing; fi-
nal rule. Fed. Regist 2001;66:5447-69.
78. Food and Drug Administration. Request for
proposed standards for unrelated allogeneic
peripheral and placental/umbilical cord blood
Umbilical Cord Blood Banking 䡲 751
hematopoietic stem/progenitor cell products;
request for comments. Fed Regist 1998;63:
2985.
79. Guidance for industry: Minimally manipulat-
ed, unrelated allogeneic placental/umbilical
cord blood intended for hematopoietic recon-
stitution for specified indications. (October
2014). Silver Spring, MD: CBER Office of Com-
munication, Outreach, and Development,
2009. [Available at http://www.fda.gov/down
loads/BiologicsBloodVaccines/Guidance
ComplianceRegulatoryInformation/Guidanc
es/Blood/UCM187144.pdf (accessed Decem-
ber 6, 2013).]
80. Food and Drug Administration. Guidance for
industry and FDA staff: IND applications for
minimally manipulated, unrelated allogeneic
placental/umbilical cord blood intended for
hematopoietic and immunologic reconstitu-
tion in patients with disorders affecting the
hematopoietic system. (March 2014) Silver
Spring, MD: CBER Office of Communication,
Outreach, and Development, 2014. [Available
at: http://www.fda.gov/BiologicsBloodVac
cines/GuidanceComplianceRegulatoryInfor
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ucm388218.htm (accessed April 8, 2014).]
 C h a p t e r 3 1

Tissue-Derived Non-Hematopoietic
Stem Cell Sources for Use in
Cell-Based Therapies

Yameena Jawed, MD; Brian Johnstone, PhD;

Sreedhar Thirumala, PhD; and Keith March, MD, PhD

REGENERATIVE MEDICINE HOLDS variety of diseases in clinical trials. Each

great promise for the treatment of some
of the most intractable diseases afflicting hu-
mankind. Each year brings new findings that
advance the science of regenerative medicine
toward becoming clinical reality. In fact,
achieving this goal seems closer now than ever
with the recent success in growing functional
human livers in the laboratory.1 However, the
reality is that, although these results are prom-
ising, their clinical translation still will require
many years, if not decades, of further research
and refinement before patients receive organs
grown in the laboratory.
Nevertheless, the use of stem cells has al-
ready shown great promise for treating a wide
month, new successes are reported in treat-
ments of even the most serious life-threaten-
ing diseases, such as amyotrophic lateral scle-
rosis (Lou Gehrig disease).2 Interestingly, there
is scant evidence that these salutary effects oc-
cur through the regeneration of degenerated
tissues by the implanted stem cells. In fact,
there is very little evidence in animal disease
models or, where possible, human patients
that transplanted cells are capable of long-
term engraftment in sufficient numbers to
account for the observed effects. Therefore, al-
ternative mechanisms have been invoked to
explain the measurable benefits of mesenchy-
mal stem cell (MSC) therapies. The prevalent

Yameena Jawed, MD, Postdoctoral (Research) Fellow, Indiana Center for Vascular Biology and Medicine, Indi-
ana University School of Medicine; Brian Johnstone, PhD, Preclinical Director, Center for Regenerative Medi-
cine, Director, Cardiovascular Ischemia and Vasculogenesis Core, and Associate Research Professor, Indiana
University School of Medicine; Sreedhar Thirumala, PhD, Senior Scientist, Cook General BioTechnology, LLC;
and Keith March, MD, PhD, Director, Indiana Center for Vascular Biology and Medicine, Director, Vascular
and Cardiac Center for Adult Stem Cell Therapy, and Professor of Medicine, Physiology, and Biomedical Engi-
neering, Indiana University School of Medicine, Indiana University, Indianapolis, Indiana
The authors have disclosed no conflicts of interest.

753
754 䡲 AABB TECHNICAL MANUAL
hypotheses of “paracrine support” are dis-
cussed in detail below. This new understand-
ing has led to a widespread adoption of the
more general phrase “cell-based therapy” to
encompass the panoply of beneficial effects of
treatment with stem cells from a variety of
sources—effects that are independent of sta-
ble tissue engraftment and tissue regenera-
tion. In practice, though, the term “regenera-
tive medicine” is still widely used, especially by
the lay public, to refer to any stem cell-based
treatment.
Strictly speaking, regenerative medicine
is simply defined as the “process of replacing
or regenerating human cells, tissues or organs
to restore or establish normal function.”3 The
origins of cellular therapy date back to 1968
with the first bone marrow transplant of he-
matopoietic stem cells to restore the hemato-
poietic system in dysfunctional or ablated
marrow.4 From these origins, there has been
great progress in identifying many different
sources of potentially therapeutic stem and
progenitor cells in the adult system. These
findings have also been translated to early-
stage clinical trials to test the effectiveness of
these approaches in a number of diseases that
are not currently addressed by traditional
pharmacologic therapies or surgery.
Treatment with both autologous and allo-
geneic stem and progenitor cells has been
evaluated. Autologous cell therapy has the ad-
vantage of involving fewer potential regulatory
hurdles and, in some cases, such as recon-
structive surgery, is arguably within the tradi-
tional scope of the practice of medicine and,
therefore, outside regulatory oversight. Autol-
ogous cell-based therapies, although attractive
and perhaps more tractable than allogeneic
cell-based therapies, are practical only when
the source of cells is abundant and their ex-
traction is without significant morbidity or en-
hanced mortality. For these reasons, autologous
therapy has been limited to cells from marrow
and adipose tissues; however, the former is
mostly composed of hematopoietic cells,
whereas the latter is made up of approximately
15% to 30% MSCs on isolation.4,5 Allogeneic
therapies, however, usually require the mass
production of stem cells originally obtained
from genetically nonidentical donor tissue in
carefully controlled environments with estab-
lished controls to ensure quality and safety.
Much research has focused on the ability
of these regenerative therapies to replace or-
gan transplants. In 2006, an article was pub-
lished on what was labeled the “first artificial-
ly grown organ.”5 In this study, scientists were
able to produce sections of a bladder and pro-
vide some degree of urinary continence to pa-
tients with spina bifida. Currently, most re-
search relates to tissue and organ progenitor
cells that either increase or support the func-
tionality of preexisting tissues or induce new
organ structures to form in situ, a process
technically termed “organ reconstruction.”
The reconstruction of bioengineered organs or
even for a regenerative therapy treatment po-
tentially requires a massive number of stem
cells. For many stem-cell-mediated therapies,
the number of cells needed to effectively treat
a patient greatly surpasses the number of cells
available from donors.
M S C S O U RC E S
Tissue sources of stem cells used for cell-based
therapies are varied and cover nearly every or-
gan system in the body. Based on the develop-
mental stage of the donor, these cells can be
classified as embryonic, fetal, or adult. Embry-
onic stem cells are primordial cells isolated
from the eight-cell blastula that has the capac-
ity to form any cell type found in the human
body. Stem cells isolated from postnatal or re-
productive tissues are known as “adult stem
cells.” Most of these cells were discovered and
defined based on such attributes as the ability
to proliferate for many generations in culture
as well as their intrinsic property of differenti-
ation in response to environmental or chemi-
cal stimuli. The ability to differentiate along
multiple lineages (eg, mesodermal, ectoder-
mal, and endodermal) is important. The hall-
marks of pluripotentiality and multipotentiali-
ty distinguish stem cells from progenitor cells;
the latter have undergone partial differentia-
tion in situ and, thus, are committed to differ-
entiate terminally into a single cell type (eg,
endothelial progenitor cells or preadipocytes,
 CHAPTER 31
which form endothelial cells and adipocytes,
respectively).
The most studied adult multipotent cells
are mesoderm-derived MSCs, alternatively
termed “multipotent stromal cells,” which
originate from the stromal compartment of
skeletal muscle, reproductive organs (umbili-
cal cord and uterus), amnion, marrow, and ad-
ipose tissues. The recent advances in tech-
niques to reprogram terminally differentiated
cells to induce pluripotency (termed “induced
pluripotent stem cells”) have opened up a vast
source of stem cells from nonembryonic tis-
sues. Induced pluripotent cells present excit-
ing new opportunities for cell-based therapies
and are covered in detail in Chapter 33. Mar-
row-derived MSCs (BM-MSCs), which are the
nonhematopoietic cells isolated from marrow
stroma, are also discussed in detail in Chapter
33 and are only briefly discussed here, mostly
to compare them with MSCs derived from oth-
er tissues and to discuss their current thera-
peutic uses.
BM-MSCs were the first nonhematopoiet-
ic stem cells discovered and, due to having
been studied in great detail, are considered the
archetypal MSCs. Because the numbers and
quality of MSCs decline with the aging of do-
nors and due to the invasiveness of the proce-
dures required to procure marrow, other
sources of MSCs were sought. Perhaps the
most primitive adult MSCs are those obtained
from umbilical cord tissue, umbilical cord
blood, amnion, and amniotic fluid.6-10 The
dental pulp of the third molar is another
source of MSCs.11 It has been recently recog-
nized that adipose tissue is a rich and abun-
dant source of cells with stem cell properties.12
Additional sources of MSCs are placenta and
uterine endometrial cells shed during men-
ses.13,14 Each of these tissue-specific MSCs is
discussed in more detail below.
MSCs from different tissue sources differ
with respect to prevalence, proliferative capac-
ity, immunophenotype, and differentiation
potential (Table 31-1), which may be impor-
tant variables governing the clinical utility of
each MSC type. In the cases of low abundance
in source tissues or difficulty of obtaining tis-
sue specimens, MSCs must be first isolated
Tissue-Derived Stem Cells 䡲 755
from the nondesirable cell types (such as leu-
kocytes and endothelial cells) and then ex-
panded under conditions that are permissive
for maintaining stem-cell-like properties. Be-
cause of the cost and time required to generate
sufficient numbers of qualified cells under
current good manufacturing practice (cGMP)
conditions, the effort is only economically
practical when culture expansion yields suffi-
cient numbers of MSCs at lower passages to
treat many hundreds, if not thousands, of pa-
tients.
Most adult tissues contain low percentag-
es of MSCs that are not available in sufficient
abundance, with the exception of adipose tis-
sues. BM-MSCs constitute only a small frac-
tion (approximately 1 in 3.4 × 104 cells) of cells
in adult marrow.28 Although the frequency of
MSCs in fetal tissues may be higher, these tis-
sues are comparatively quite small and limited
in availability, and their use is associated with
ethical concerns when their collection in-
volves fetal destruction.29 Examples of MSCs
found in low numbers in source tissues but
that are highly expandable are muscle-derived
MSCs (M-MSCs), cord blood MSCs (CB-
MSCs), umbilical cord MSCs (UC-MSCs), pla-
cental MSCs (Pl-MSCs), endometrial lining
MSCs (EL-MSCs), and dental pulp MSCs (DP-
MSCs).
Adipose-derived stem cells (ASCs), which
are phenotypically similar to BM-MSCs, are
more than 500-fold more prevalent per gram
of fat than per gram of marrow.30 Most patients
have excess fat that can be easily and safely ex-
tracted for processing to isolate ASCs. This
property makes adipose tissue an ideal source
of MSCs for either autologous or allogeneic
applications. ASCs are perhaps the only type of
MSC available in numbers that are sufficient
for immediate or “point-of care” use in thera-
peutic applications without the need for cul-
ture expansion.31-33 Extraction of adipose tissue
is accomplished with minimally invasive tech-
niques, such as lipoaspiration, with little asso-
ciated morbidity even on removal of tissue
volumes as large as 1-3 liters. Thus, the
procurement and use of ASCs is a uniquely
practical approach to point-of-care autolo-
gous applications.
TABLE 31-1. Comparison of MSCs Derived from Different Tissue Sources
756

Frequency Cell
Study (Colony- Proliferation
(Reference Forming Potential in
䡲

Number) MSC Source Units) Vitro Senescence Multilineage Differentiation Potential Immunophenotype

9 CB + +++ + CB-MSCs have less adipogenic differentia- CD105 + CD90 + CD106 ++

BM ++ + ++ tion potential. Osteogenesis and chondro- CD105 ++ CD90 +++ CD106 ++
AT +++ ++ +++ genesis are similar. CD105 ++ CD90 ++ CD106 +
15 AT BM-MSCs have superior osteogenic CD34 +++
16 BM capacity. AT-MSCs are least osteogenic. CD34 (null)
CB
17 AM AM-MSCs and AF-MSCs have neurogenic, MSCs from AM, AF, BM– CD105+, CD90+,
AF osteogenic, chondrogenic, adipogenic, CD73+
BM hepatogenic and endothelial differentia- MSC from AM, AF-CD44+, CD49e+, CD54+,
tion. AM-MSCs also differentiate into car- CD166+
diomyocyte-like cells in vivo.
18 UC +++ +++ + Neurogenesis is higher in UC-MSCs. UC- CD106 + HLA-ABC +++ CD146 +++
AABB TECHNICAL MANUAL

19 BM + + +++ MSCs have higher osteogenesis and neu- CD106 +++ HLA-ABC ++++ CD146 +
rogenesis than BM-MSCs.
20 Pl + +++ + Osteogenesis and chondrogenesis are CD49d ++
BM + + +++ comparable but Pl-MSCs do not maintain CD49d +
adipogenesis as readily as BM-MSCs.
21 SV +++ +++ + Adipogenesis is highest in SV-MSCs and VEGFR-2 ++ CD10 +
PM +++ +++ + AT-MSCs. Chondrogenesis is superior in VEGFR-2 +++ CD10 +++
SkM +++ +++ +++ SV-MSCs. BM-MSCs, SV-MSCs, and PM- VEGFR-2 ++ CD10 +
AT +++ ++ ++ MSCs have the highest osteogenic poten- VEGFR-2 + + CD10 +
BM + +++ + tial. VEGFR-2 ++ CD10 +
22 DP +++ ++ + All dental cells express higher levels of CD106 ++
23 PD ++ ++ + neuronal markers than BM-MSCs. PD- CD106 +
BM + + +++ MSCs express higher levels of tendon- CD106 +++
ED +++ +++ + specific markers, whereas DP-MSCs retain CD106 +
weaker and chondrogenic and adipogenic
potential compared with BM-MSCs.
24 AF + +++ + Osteogenesis and adipogenesis are CD44 + CD105 +
BM +++ + +++ similar. CD44 ++ CD105 ++
25 DP All MSCs undergo osteogenesis, chondro- All MSCs express CD44, CD105, and CD90. All
UC genesis, and adipogenesis. BM-MSCs and are negative for CD34 and CD45.
AT AT-MSCs show a loss of osteogenic differ-
CHAPTER 31

BM entiation potential with increasing num-

bers of passages, and DP-MSCs have an
increasing potential.
26 CB +++ +
AT + ++
BM ++ +++
27 UC ++ AT-MSCs have more efficient adipogene- No statistically significant difference
AT + sis and neurogenic differentiation.
Tissue-Derived Stem Cells

+ = expressed; ++ = strongly expressed; +++ = very strongly expressed.

MSC = mesenchymal stem cell; CB = cord blood; BM = bone marrow; AT = adipose tissue; AM = amniotic membrane; AF = amniotic fluid; UC = umbilical cord; Pl = placenta; SV = synovium;
PM = periosteum; SkM = skeletal muscle; DP = dental pulp; PD = periodontal ligament; ED = exfoliated deciduous teeth.
䡲
757
758 䡲 AABB TECHNICAL MANUAL
Periadventitial cells (called “pericytes”),
which support the integrity of microvessels
(capillaries and arterioles), have been recently
discovered to possess multilineage potential.34
Traktuev et al highlighted the perivascular lo-
cation and presumptive prepericyte or peri-
cyte function of MSCs within adipose tissue.35
The extension of this finding to recognize the
perivascular location of MSCs throughout the
body led to the current hypothesis that peri-
cytes are the source of all MSCs; this can ex-
plain both the high phenotypic similarity of
MSCs from different tissue sources as well as
the broad distribution of these cells, in associ-
ation with blood vessels, throughout the
body.35-37 It has also been proposed that the bi-
ological function of perivascular MSCs is inti-
mately related to systemic circulation in that
once activated, they either secrete trophic fac-
tors that promote endogenous repair into the
blood or may even mobilize to home to in-
jured tissues, where they provide structural
units for repair through differentiation.38
P RO PE RT I E S O F C LI N I C A L
RELEVANCE
MSCs display several key complementary
properties that are of potential clinical use:
immunomodulation, paracrine effects, and
differentiation. Immunomodulatory func-
tions include immunosuppression or immune
stimulation, depending on the signals in the
microenvironment.39 In the laboratory envi-
ronment, MSCs can be induced to differenti-
ate into endothelial cells, neural cells, astro-
cytes, cardiomyocytes, skeletal myocytes,
chondrocytes, adipocytes, osteocytes, and
other cells that are developmentally derived
from the endoderm and exoderm.40-43 The low
immunogenicity of MSCs also makes them a
relatively safe allogeneic cell source in com-
parison with certain other cell types, including
embryonic stem cells.44 There is evidence from
preclinical and, in some cases, clinical studies
to suggest that each of these properties con-
tributes to the therapeutic efficacy of MSCs.
The paracrine effects of MSCs are increas-
ingly recognized as an important, if not the
primary, mechanism of action to promote tis-
sue rescue and repair as well as to modulate
the immune system.45 The demonstrated abili-
ty of MSCs to effect change in disease models
and patients in the absence of long-term en-
graftment and differentiation in numbers re-
quired to account for damaged tissue replace-
ment can be most easily explained by
paracrine mechanisms. Thus, exogenously ad-
ministered MSCs home to the site of injury or
disease through signals that are primarily in-
flammatory and reside temporarily at the site
where they secrete paracrine factors with pro-
survival, antiinflammatory, and repair-induc-
ing properties before clearance from the sys-
tem.46
Although the evidence supports the pre-
dominant function of secreted factors in the
diminution of injury or disease, there is also
evidence that cell-mediated contact may be an
important immunomodulator during the peri-
od of residency of MSCs at the target site.47 The
concept of paracrine support by MSCs was
first directly demonstrated in diseases of pe-
ripheral tissues and the central nervous sys-
tem by treatment of disease models with the
cocktail of factors present in media condi-
tioned by the growth of BM-MSCs and ASCs.48
In another early demonstration, ablating pro-
duction of a single factor (hepatocyte growth
factor) by stable small interfering ribonucleic
acid knockdown abolished the salutary effects
of ASCs in a mouse hind-limb ischemia model
of peripheral vascular disease.46
I SO L AT I O N A N D E X PAN SI O N
Obtaining sufficient numbers of MSCs com-
monly requires their extensive expansion in
cell culture. The isolation and expansion of
MSCs from marrow aspirate is covered in de-
tail in Chapter 33. Liposuction surgery is a
well-tolerated and safe procedure for produc-
ing a large amount of lipoaspirate that can be
processed to yield ASCs.
The method of ASC isolation in different
laboratories varies subtly, but it usually in-
volves enzymatic dissociation from adipocytes
and extracellular matrix, filtration to remove
particulates, and low-speed centrifugation to
separate adipocyte and nonadipocyte cells.
 CHAPTER 31
Following surgical removal of subcutaneous
fat, lipoaspirate samples are washed exten-
sively in phosphate-buffered saline to deplete
erythrocytes and then treated by continuous
agitation with collagenase Type I solution for
30 minutes to 1 hour at 37 C. It is important
that the collagenase also contain neutral pro-
teases, presumably to liberate cells from the
extracellular matrix fragments; these cells are
removed by subsequent filtration. The solu-
tion is neutralized with an equal volume of
serum-containing medium and centrifuged at
low speed (approximately 300 × g for 10 min-
utes). The buoyant adipocytes migrate in op-
position to centrifugal force. The sedimenting
cells are a heterogenous mixture termed the
“stromal-vascular fraction” (SVF), which con-
tains variable proportions of endothelial cells,
pericytes, ASCs, and resident leukocytes (pri-
marily monocytes and macrophages).47-50 The
cell pellet can then be resuspended in an 160
mM NH4Cl solution (for 10 minutes at room
temperature) to lyse erythrocytes, and the sus-
pension is passed through a 100-micron cell
strainer to remove debris. After an additional
low-speed centrifugation, the cells are resus-
pended in growth media, such as DMEM/F12
with 10% fetal bovine serum, for counting and
are then placed in tissue culture flasks and
grown under typical conditions of 5% CO2 at
37 C.
Enrichment of ASCs occurs through plat-
ing on uncoated tissue-culture plastic in the
presence of a rich medium that is not permis-
sive for leukocyte and endothelial cell attach-
ment; thus, the latter cells are removed with a
media change after overnight culture. After an
initial lag phase, ASCs proliferate with dou-
bling times of 36 to 48 hours, depending on the
growth medium used. The cell surface marker
profile of ASCs is altered with their expansion.
For example, the CD34 marker expression of
cells in culture decreases rapidly and becomes
unmeasurable after two or three passages.34,36
Conversely, the CD105 and CD166 markers,
which are expressed at low levels in fresh iso-
lates, increase during culture.51
The umbilical cord consists of two arter-
ies and a vein supported by a surrounding ma-
trix of gelatinous connective tissue known as
Tissue-Derived Stem Cells 䡲 759
“Wharton’s jelly.” Stromal cells associated with
the matrix include specialized fibroblast-like
cells, mast cells, and MSCs.52,53 Methods for
isolation of UC-MSCs are varied and differ be-
tween laboratories; however, in all cases, the
vein and arteries are first removed, followed by
processing of the remaining cord tissue into
smaller fragments (reviewed by Can and
colleagues54). In some protocols, the interior of
the cord is scraped to remove the Wharton’s
jelly before mechanical disruption. The pieces
are placed in an enzymatic solution of collage-
nase (typically Type I) and, depending on the
protocol, other proteolytic enzymes to digest
the matrix and release the stromal cells. Vari-
ous digestion times, from 30 minutes to 16
hours, have been employed, depending on the
enzymes used and their concentration.53
Alternative protocols involve the conduct
of explant culture with the cord fragments.56
For protocols involving enzymatic digestion,
the homogenate is filtered through a 100-mi-
cron filter to remove debris. UC-MSCs from
the total cell population can be selected by ei-
ther fluorescence-activated cell sorting or cul-
ture at low density to identify colony-forming
cells. Typical mesenchymal cell surface mark-
ers, such as CD90, CD105, and CD73, are ex-
pressed by UC-MSCs; however, the level of ex-
pression varies depending on the isolation
method used.56
Isolation of amnion-derived Pl-MSCs is
accomplished by first removing the chorion,
followed by digesting them at 37 C with either
dispase II (2.4 U/mL for 1 hour) or trypsin-
EDTA (various concentrations and incubation
times have been used) to release the epithelial
cells. The Pl-MSCs are then released through
subsequent digestion with collagenase (0.75
mg/mL) and deoxyribonuclease (0.075 mg/
mL).57 Human chorionic Pl-MSCs are liberated
similarly after removal of the surrounding lay-
ers and treatment with 2.4 U/mL dispase II at
37 C, followed by 1 to 3 hours of treatment with
collagenase II (270 U/mL) or collagenase A
(0.83 mg/mL). For both chorionic and amni-
onic Pl-MSCs, the liberated cells are centri-
fuged at 200 × g for 10 minutes to obtain the
cell pellet, which is then resuspended, count-
ed, and cultured.13 An alternative to digesting
760 䡲 AABB TECHNICAL MANUAL
placental tissue is to isolate Pl-MSCs present
in the amniotic fluid. This method relies on re-
moval of amniocytes by an initial culture peri-
od and then transferal of nonadhering Pl-MSCs
to a new culture dish, where they are allowed
to adhere and proliferate.8 Each method for Pl-
MSC isolation includes culturing to expand
the cells.58
DP-MSCs are isolated from the interior
pulp chamber of extracted molars. The pulp
tissue is removed from the crown and root re-
gions and then digested with a combination
collagenase Type I (3 mg/mL) and dispase
(4 mg/mL) for 1 hour at 37 C.59 Single-cell sus-
pensions are separated from debris by passage
through a 70-micron filter. Cells proliferate
well in -MEM supplemented with 20% fetal
calf serum (FCS), L-ascorbic acid 2-phosphate
(100 µM), and L-glutamine (2 mM).60,61
Because extraction and digestion of tis-
sues are not needed to isolate cells, EL-MSCs
are a convenient therapeutic cell source. Isola-
tion of EL-MSCs is easily accomplished by
culturing menstrual-blood-containing, density-
gradient-purified mononuclear cells on un-
coated plastic vessels and selecting adherent
cells after a brief overnight incubation in com-
plete media, such as DMEM supplemented
with 20% FCS.14 Adherent EL-MNCs are ex-
panded in the same medium for multiple gen-
erations until sufficient numbers of cells are
obtained.
M-MSCs are pluripotent stem cells that
are distinct from committed progenitors (such
as satellite cells in skeletal muscle) and can be
isolated from skeletal, smooth, and cardiac
muscle tissue.62,63 The most common tech-
nique to isolate M-MSCs is based on their ad-
hesion characteristics and uses a process
termed the “modified preplate technique.” In
this method, the muscle mass is isolated by a
biopsy and dissected into pieces with razor
blades or scalpels. The tissue is dissociated us-
ing enzymes, such as collagenase II (1%) and
dispase II (2.4 U/mL).64,65 The dissociated cells
are then resuspended in culture medium,
which is then placed in collagen-coated flasks.
Within minutes to hours of seeding, the first
cells, including fibroblastic-like and myoblast
cells, attach. The supernatant containing the
nonadherent fraction is then transferred to a
new collagen-coated plate. The slowly adher-
ing cells, which contain M-MSCs, are allowed
to adhere and grow over several days to weeks,
after which the proliferating cells are expand-
ed by passaging.
STANDARDIZ ATION OF METHODS
FOR ISOL ATION AND EXPANSION
OF CLINI CAL PRODUCT
Although many countries have implemented
regulations governing the manufacture and
use of stem cells for cell therapy, there is still
significant variation in the procedures used for
isolation, expansion, preservation, and ship-
ping, which are critical components of clinical
translation.66,67 It is imperative that cGMP re-
quirements and quality-control systems be
used to ensure, to the greatest extent possible,
consistency in stem cell identity and potency.
These practices are established at most
companies that are pursuing federal regulato-
ry approval for off-the-shelf allogeneic stem
cell products. This regulatory approval re-
quires careful documentation of manufactur-
ing processes and product characterization
(termed “chemistry, manufacturing, and con-
trols”). These companies include Mesoblast
Ltd. (Melbourne, Australia), Athersys Inc.
(Cleveland, OH), Medistem Inc. (San Diego,
CA), and Pluristem Therapeutics Inc. (Haifa,
Israel).
Autologous MSC therapies are also typi-
cally required to demonstrate consistency of a
product for regulatory approval when the cells
are derived from multiple donors. Two com-
plementary models for autologous cell prepa-
ration are currently employed: the centralized
manufacturing facility and point-of-care de-
vices. The former is typified by Aastrom Biosci-
ences Inc. (Ann Arbor, MI) and RNL Bio (Seoul,
South Korea). Devices for isolation in the oper-
ating theater are in development (and, in lim-
ited cases, approved for marketing) by such
companies as Cytori Therapeutics (San Diego,
CA), Tissue Genesis Inc. (Honolulu, HI), and
Ingeneron (Houston, TX).
 CHAPTER 31
C E L L P RO D U C T B A NK I N G A N D
MANAGEMENT OF SU PPLY
CHAIN AND END USE
Manufacturing, storage/banking, and distri-
bution are all critical for ensuring a steady
supply of cellular products to clinics around
the world. Fortunately, these processes can be
modeled on those already established by the
biological drug industry, which has more than
two decades of experience with supply-chain
optimization. The biologics model must be
adapted to cellular therapeutics to address key
logistical considerations, such as maintaining
the viability of cellular therapeutics during
shipping as well as after receipt at a clinical fa-
cility. Many biologics are stored as a lyophi-
lized powder or in suspension or solution at
normal freezer or refrigeration temperatures
(ie, –20 C). Extended storage of cells requires
temperatures below –80 C, and these cells are
most commonly stored in liquid-nitrogen tank
facilities.
Defining optimal and reproducible con-
ditions for long-term storage, transport, and
revival of MSCs is critical for maintaining cell
viability and, thus, potency. Defining these
conditions is particularly important for alloge-
neic MSCs produced in a centralized facility
under cGMP controls but also needs to be ad-
dressed for autologous cells that will be used
for repetitive treatments and the avoidance of
repeated extractions and isolations of cells.
Good results with long-term storage at
–196 C (temperature of liquid nitrogen) or
<–150 C (temperature of vapor-phase nitro-
gen) have been obtained with typical cell cryo-
preservation protocols using media contain-
ing serum and dimethyl sulfoxide (DMSO),
provided that cooling and thawing rates are
carefully controlled.68 Although both DMSO
and animal serum present concerns, there are
currently few acceptable alternatives to their
use; thus, most protocols require that these
media be depleted before the cells are used for
the treatment of patients.69-73
Clinical banking requires strict adher-
ence to cGMP requirements for cell product
processing, storage, and shipment to the end
user to ensure preservation of viability and
Tissue-Derived Stem Cells 䡲 761
therapeutic potency. Consequently, closed-
system containers are required to meet phar-
maceutical quality standards for packaging,
storage, and distribution of cellular products
at low temperatures. An ideal cryopreservation
container is stable despite exposure to a wide
range of temperatures for extended periods
and allows selective access to the sample when
desired for retrieval. As the demand for cell
therapy increases, commercial-scale cell ther-
apy product manufacturing and banking will
require the containers to be compatible and
integrated into the automated fill lines tradi-
tionally used in pharmaceutical settings to en-
able lot sizes of several hundred to several
thousand doses.
Although freezers that maintain cryo-
preservation temperatures are likely to be
available in large hospitals, especially those af-
filiated with universities, these freezers are not
available within most clinical practices. Smaller-
scale solutions, such as liquid-nitrogen Dewar
flasks or dry ice in insulated coolers, can be
employed for relatively short-term storage;
however, use of these flasks or coolers may be
associated with logistical and safety issues. Al-
ternatively, a just-in-time approach may be
taken where therapeutic cells are shipped di-
rectly from centralized facilities in insulated
containers that have sufficient dry ice to main-
tain a steady temperature for a few days to a
week. The cells are thawed immediately before
use, washed to remove cryoprotective agents,
and resuspended in diluent for administra-
tion.
Another option is to use practices estab-
lished for the shipment and short-term stor-
age of tissue products. In general, viable tis-
sues are placed in biocompatible containers in
contact with nutrient-rich media and shipped
at ambient temperatures within insulated con-
tainers via overnight courier service. The shelf
life of certain tissues packaged and shipped in
this manner is approximately 1 week. An ex-
ample is Apligraf (manufactured and distribut-
ed by Organogenesis, Canton, MA), a living cell
skin graft approved by the US Food and Drug
Administration for the treatment of chronic
venous leg and diabetic foot ulcers. This prod-
uct has a shelf life of 10 days after it is shipped.
762 䡲 AABB TECHNICAL MANUAL

Regardless of the method used to transfer

cellular therapy products to the end user, there
is a critical need to manage the supply chain’s
integrity and ensure that the cell transport and
clinical site storage run efficiently while ad-
verse incidents are minimized. Particularly
with autologous or type-matched therapeu-
tics, and with downward cost pressures due to
increased competition and falling reimburse-
ment rates, it is critical to avoid errors and in-
terruptions in the delivery of these therapies to FIGURE 31-1. Number of registered clinical trials of
hospitals and private clinics. therapeutic uses of mesenchymal stem cells (as of
March 2014).
NR = not reported.
TH E R A PE U T I C A P P L ICAT I O NS
OF MSCS
The majority of these studies are early-phase
Cell therapy using BM-MSCs or ASCs is cur- trials (Fig 31-1). As can be seen in Fig 31-2, the
rently being investigated in clinical trials for a types of diseases under investigation are wide
wide variety of diseases that are not adequate- ranging and affect essentially all organs and
ly addressed by current pharmacological or systems in the body. The target populations in-
surgical approaches. These trials include some clude both pediatric (65 studies) and adult
that evaluate culture-expanded autologous (286 studies) patients.
and allogeneic MSCs and ASCs and others that
involve autologous ASCs that can be freshly Immunomodulation and Skeletal
isolated in the context of SVF using any of sev- Repair
eral point-of-care systems.
The number of clinical studies evaluating The most intensively investigated class of dis-
these therapies has steadily increased over the orders treated with MSCs is immune system
last decade, and the number of publications diseases (58 trials), which include graft-vs-
relating to stem cell preclinical as well as clini- host disease (GVHD; 23 trials) and autoim-
cal studies over time demonstrates this trend. mune disorders, such as multiple sclerosis

70
60
50
Number of Trials

40
30
20
10
0

Disease Category

FIGURE 31-2. Diseases for which mesenchymal stem cell therapy is under investigation (as of March 2014).
CNS = central nervous system.
 CHAPTER 31
(13 trials), Type 1 diabetes (10 trials), and lu-
pus (4 trials).
The potential of MSC treatment for modi-
fying these diseases is supported by a substan-
tial volume of literature demonstrating the
immunomodulatory effects of these cells in
preclinical studies of animal diseases. Admin-
istration of MSCs significantly reduced the
incidence and severity of GVHD after trans-
plantation of major-histocompatibility-com-
plex-mismatched blood and tissues in animal
models.74-77 These preclinical results have been
translated into seminal early trials and then
successful Phase II and III clinical trials with
BM-MSCs for prevention and treatment of
acute and chronic GVHD after allogeneic he-
matopoietic stem cell transplantation.78-80 The
success of the Phase II and III trials led to regu-
latory approval of the first licensed stem cell
therapy, Prochymal (Osiris Therapeutics), in
North America for treatment of refractory
GVHD. This therapy is also available in
Canada.
The discovery in 1998 that MSCs differen-
tiate into cartilage under the influence of
transforming growth factor-beta ushered in a
new era of investigations of MSC performance
in a multitude of chondrogenic conditions.81
At the time of publication, 28 clinical studies
were evaluating the use of MSCs to treat osteo-
arthritis and rheumatoid arthritis. Similarly,
clinical trials have demonstrated that MSCs
can be used successfully to repair various bone
defects, including critical size defects in the
long bone, hard palate reconstruction, and
calvarial defects.82-84
Cardiovascular and Cerebral Diseases
MSCs are being investigated for stand-alone
treatment of ischemic heart diseases as well as
in combination with coronary artery bypass
surgery and left ventricular device implanta-
tion. Early open-label, uncontrolled trials of
MSCs and ASCs have had safety-related pri-
mary endpoints. The APOLLO trial demon-
strated that the intracoronary delivery of au-
tologous SVF to patients with acute ST
segment elevation myocardial infarction was
safe.85 The complementary PRECISE trial was a
Tissue-Derived Stem Cells 䡲 763
clinical trial of SVF administration in the treat-
ment of symptomatic, nonrevascularizable
ischemic myocardium. The POSEIDON trial
evaluated both allogeneic and autologous BM-
MSCs as a therapy for ischemic cardiomyopa-
thy and found that both cell types were safe
when delivered directly into the myocardi-
um.86 Two Phase II randomized, double-blind,
placebo-controlled trials of intramyocardially
injected autologous BM-MSCs for heart failure
have been recently initiated in Europe and the
United States to confirm the positive effects of
these cell treatments observed in early open-
label Phase I/II trials.87,88
Diseases of peripheral limb vascular in-
sufficiency are also under clinical investiga-
tion as targets for MSC therapy. In six patients
with Buerger disease, 24 weeks of treatment
with multiple intramuscular ASC injections
had positive clinical outcomes, including de-
creased pain.89 Intramuscular injection of
ASCs also reduced symptoms of diabetic foot
and atherosclerotic obliterans.90 A random-
ized, placebo-controlled trial of BM-MSC in-
jected directly into the affected limb of
patients with critical limb ischemia demon-
strated safety as well as indices of efficacy.91
Central nervous system trials involving
MSC treatment have been dominated by in-
vestigations of the treatment of ischemic
stroke (nine trials).92 Additional diseases for
which MSCs are being evaluated include Al-
zheimer disease, Parkinson disease, spinal
cord injury, amyotrophic lateral sclerosis, and
multiple sclerosis.93-96 The safety and efficacy
of intravenous autologous BM-MSC infusion
in patients with severe middle cerebral artery
ischemic stroke was evaluated in a 5-year fol-
low-up study in which the mortality rate of the
treated group was lower than that of the
control group, and no major side effects were
observed during the follow-up period.97
Inflammation Modulation and Barrier
Repair
The antiinflammatory and proendothelial sur-
vival activities of MSCs suggest that they might
have therapeutic utility for diseases involving
systemic inflammation and associated endo-
764 䡲 AABB TECHNICAL MANUAL
thelial barrier dysfunction, such as sepsis,
acute lung injury, and pancreatitis. Related po-
tentially therapeutic properties in this context
include immunomodulation; differentiation
into lung epithelial cells; secretion of antimi-
crobial peptides; as well as other cytoprotec-
tive factors affecting endothelium, such as ke-
ratinocyte growth factor and angiopoietin.98-101
Preclinical studies in murine models have
shown that MSCs can be beneficial in acute
lung injury induced by lipopolysaccharide,
pneumonia, or systemic sepsis.102 In one study,
ASC therapy decreased oxidative stress and
minimized ischemia/reperfusion-induced dam-
age to the rat lung.103 Phase I trials of both BM-
MSCs and ASCs to treat acute respiratory dis-
tress syndrome are under way. Intravenous
UC-MSC therapy has also been shown to alle-
viate fibrosis, improve histologic scores, and
decrease inflammatory cytokine levels in rats
with chronic pancreatitis.104
Gastrointestinal Diseases
Intestinal diseases are an additional category
of disorders for which MSC treatment is cur-
rently undergoing investigation. The largest
number of trials (11) address Crohn’s disease.
In a Phase I trial of 10 patients with fistulizing
Crohn’s disease, local injections of BM-MSCs
resulted in complete fistula closure in seven
patients and partial closure in three.105 In 2012,
a Phase III trial investigating the safety and ef-
ficacy of allogeneic ASCs for the treatment of
complex perianal fistulas in patients with
Crohn’s disease was initiated.106
Treatment of end-stage, chronic liver dis-
eases, primarily cirrhosis, using MSCs is un-
der active investigation. A Phase I/II clinical
study, in which eight patients with end-stage
liver disease were treated with predifferentiat-
ed autologous BM-MSCs injected into either
the portal vein or peripheral vein, showed sig-
nificant improvement in liver function and
clinical parameters.107 A placebo-controlled
trial of 30 patients with decompensated liver
cirrhosis demonstrated that systemic infu-
sions of UC-MSCs were safe and improved liv-
er function at 1 year.108 An open-label trial of
peripheral venous delivery of UC-MSCs to pa-
tients with biliary cirrhosis also showed that
the treatment was safe and improved liver
function.109 Autologous BM-MSCs were ad-
ministered to 53 patients with liver failure due
to complications of hepatitis B infection, and
patient responses were evaluated at early and
late intervals.110 Within 3 weeks of the infu-
sions, there was a significant improvement in
liver function in treated patients compared to
the 105 matched patients admitted in the
same time frame. Although indices of safety at
the early and late time points (approximately
3.5 years) were no different in the treatment
and control groups, the benefit of the treat-
ment did not persist at the late time point.
CU R R E N T R E S E A RCH A N D
DEVE LOP MEN T : FO C U S O N
C E L L C U LTU R E A N D H A N D L I N G
In the context of the positive results and lack of
apparent safety concerns from numerous clin-
ical studies conducted on MSCs to date, there
are many issues that must be routinely
addressed before regulatory approval and
widespread adoption of MSCs in the clinical
setting. For many culture protocols, animal-
derived supplements, such as bovine serum
(which is also commonly included in preserva-
tion media), have historically been used as a
source of growth factors and to facilitate post-
thaw cell culture. However, the use of bovine
serum and the xenogeneic proteins it contains
could stimulate immune reactions in the host.
In addition, bovine serum must be obtained
from sources that minimize the risk of trans-
mitting prions and other as-yet-unidentified
zoonotic diseases.111,112 Furthermore, DMSO’s
toxicity at concentrations currently used to
cryopreserve MSCs along with the undesirable
osmotic shock to the cells during its addition
and removal may render DMSO undesirable to
preserve MSCs intended for clinical use.
Considerable effort has been invested in
identifying substitutes for bovine serum and
DMSO; both of these agents must be depleted
by washing the cells upon thawing and before
injection.69-73 For example, there has been con-
siderable effort directed at developing serum-
 CHAPTER 31
free, or at least animal-serum-free, cryopreser-
vation media containing cell protectants oth-
er than DMSO.113,114 Strategies for eliminating
or reducing the exposure of MSCs to DMSO
and technologies to remove DMSO from
thawed cells have been proposed in the litera-
ture but rarely implemented in clinical set-
tings.
An important consideration that has re-
strained efforts to alter the growth conditions
of MSCs is that such modifications might alter
the phenotype, genetic stability, or subsequent
biological properties of MSCs.115-118 Alterations
that could influence these properties are not
limited to serum components because micro-
nutrient levels can also influence genomic sta-
bility and biological properties.119 A particular
concern regarding the modification of media
constituents is that much of the preclinical
and clinical data that underlie our knowledge
regarding the potential benefits and safety of
MSCs may not be directly relevant to cells
grown in medium containing bovine serum
substitutes.120 The absence of continuity be-
tween the extensive amount of previous pre-
clinical data on MSC safety and efficacy may
increase the regulatory burden for obtaining
licensing to market MSCs as drugs.
In addition to addressing issues of manu-
facturing and storage, the adequate demon-
stration that exogenously administered MSCs
possess an exceedingly low potential to form
tumors or promote endogenous tumor forma-
tion has been required to obtain regulatory ap-
proval for MSCs. Published reports indicate a
lack of consensus on the tumor-formation po-
tential of multipotent MSCs. Long-term cul-
tures of MSCs have been reported to be associ-
ated with genomic instability, resulting in the
accumulation of genetic alterations that have
the potential to become manifest as malignant
transformation.121-123 Conversely, other studies
have found no indication of chromosomal ab-
errations with extended cultures of MSCs and
ASCs.124-127 These opposing findings may be
due to subtle differences in the MSC cultures
examined, culture conditions, or methods for
detecting genetic abnormalities.
Regardless, tumor formation resulting
from MSC or ASC treatment is indeed very rare
Tissue-Derived Stem Cells 䡲 765
and has been calculated to occur at a frequen-
cy of 10-9.128 There has been at least one report
of MSC transformation in vivo to form tu-
mors.19 These studies emphasize the need to
thoroughly test highly expanded MSCs to be
used in clinical trials for genetic alterations.
These results also may suggest that, whenever
possible, lower-passage MSCs, or ASCs and
umbilical MSCs, should be used because these
cells require less expansion due to their much
greater availability after their initial recovery
from tissue.
Tumorigenesis may also be promoted by
the recruitment of MSCs to the tumor stroma.
There are conflicting reports regarding wheth-
er human MSCs support tumor formation. A
number of investigations have shown that
MSCs enhance tumor growth, whereas others
have reported that MSCs are associated with
tumor suppression.130-141 These discrepant re-
sults may be due to the different experimental
systems used to assess tumor promotion. In
addition, the conditions for each MSC prepa-
ration might have differed in substantial ways
that influenced the outcomes.
Given this still-evolving understanding, it
is important to assess each MSC preparation
for tumorigenic potential and carefully con-
sider the exclusion of patients with active or
recent cancer diagnoses in the context of clini-
cal applications.
CO N C LUS IO N S A N D F U T U R E
DIRE CTIONS
It is evident that the field of cell-based thera-
pies holds great promise and is on the cusp of
widespread commercialization, which will
have an extensive impact on the health-care
field in the coming years. Today, the potential
market value of stem cell therapy has been es-
timated at $5.1 billion with adult stem cells
holding a majority share (more than 80%) of
the market.142
An important concern revolves around
the potential expense of both autologous and
allogeneic cell-based therapies. Much emphasis
over the last several years has focused on off-the-
shelf allogeneic cell therapy products, which are
considered to be more suitable for clinical
766 䡲 AABB TECHNICAL MANUAL

adoption and successful commercialization.
However, allogeneic therapies may be chal-
lenging to produce cost-effectively due to the
complexities involved in their manufacturing
and storage. For allogeneic use, cells from a sin-
gle qualified source must be massively expanded
and stored for a long time to treat large num-
bers of patients. Because successful commer-
cialization is heavily influenced by costs, the
manufacturing and banking of these massive
number of cells in a safe, robust, and cost-
effective manner while preserving key thera-
peutic properties will be critical. These param-
eters are significantly affected by the source of
material, isolation and manufacturing meth-
ods, safety, banking, shipping, and tracking.
Finally, the successful use and ultimate
potential for widespread adoption of these
therapies will be affected by regulatory re-
quirements for the production and marketing
of any cell-based therapies, including issues
ranging from product characterization to safe-
ty testing and clinical trial design. As the field
continues to mature, the regulatory pathway
and its challenges are becoming progressively
refined. It is hoped that this evolution will con-
tinue to lead to the establishment of a better-
defined path for permitting the field to test the
promise of cell therapy more efficiently and
meet the expected future demands for these
treatments.

KEY POINTS

1. Multipotent mesenchymal stem cells (MSCs) can be harvested from a diverse array of tis-
sues, including marrow, adipose tissue, reproductive organs, placenta, dental pulp, and
skeletal muscle.
2. The hallmarks of pluripotentiality and multipotentiality distinguish stem cells from progen-
itor cells.
3. The beneficial properties of these therapeutic cells appears to be more related to transitory
effects produced by secreted substances or brief contact with target cells rather than direct
tissue replacement.
4. The key complementary properties that are of potential clinical use are immunomodula-
tion, paracrine effects, and differentiation.
5. The method of MSC isolation usually involves enzymatic dissociation, filtration, and low-
speed centrifugation.
6. Early clinical experience suggests that cellular therapies are safe and effective; however, lon-
ger-term studies are required to fully assess the durability of these effects and the potential
for tumorigenicity.
7. Key to future commercial success will be developing well-characterized and consistent
therapeutic cell products through establishing strict controls for manufacturing, storage,
and supply processes.

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114. Thirumala S, Gimble JM, Devireddy RV. Cryo-
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115. Bieback K, Ha VA, Hecker A, et al. Altered gene
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116. Abdelrazik H, Spaggiari GM, Chiossone L,
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117. Goedecke A, Wobus M, Krech M, et al. Differ-
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118. Tonti GA, Mannello F. From bone marrow to
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119. Arigony AL, de Oliveira IM, Machado M, et al.
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122. Wang Y, Huso DL, Harrington J, et al. Out-
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123. Rosland GV, Svendsen A, Torsvik A, et al. Long-
term cultures of bone marrow-derived human
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125. Avanzini MA, Bernardo ME, Cometa AM, et al.
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126. Tarte K, Gaillard J, Lataillade JJ, et al. Clinical-
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 C h a p t e r
Human Allografts and the Hospital
Transfusion Service
Lance D. Trainor, MD, and Rita A. Reik, MD
T H E S U R G I C A L U S E of human tis-
sue allografts continues to expand. Every
year, member organizations of the American
Association of Tissue Banks (AATB) recover tis-
sue from more than 30,000 donors and provide
more than 2 million tissue grafts for transplan-
tation.1 Many surgical specialties, including
orthopedics, plastic surgery, urology, neuro-
surgery, sports medicine, trauma, and recon-
structive surgery, use human tissue. Increas-
ingly sophisticated grafts are being developed
by tissue suppliers to meet diverse clinical
needs.
There is no regulatory requirement for the
activities of a tissue-dispensing service to be
managed by any particular individual or de-
partment within a hospital. However, because
ordering, receiving, storing, dispensing (issu-
ing), tracking, tracing, investigating adverse
events, and managing recalls are functions
performed by both transfusion and tissue-dis-
pensing services, the AABB recommends a
centralized tissue-dispensing model located
within the transfusion service.2
Lance D. Trainor, MD, Divisional Medical Director, LabCorp, Dublin, Ohio, and Rita A. Reik, MD, Chief Medi-
cal Officer, OneBlood, Lauderhill, Florida
The authors have disclosed no conflicts of interest.
3 2
T IS S U E T R A NS P L AN TAT IO N
Allografts are selected for transplantation
based on intrinsic qualities that meet the sur-
geon’s functional requirements for the patient.
Bone, tendons, and corneas are the most fre-
quently implanted human tissues; others in-
clude cartilage, skin, veins, dura mater, fascia,
and heart valves. Most tissues are procured
from deceased donors within 24 hours of
death after appropriate consent (also known
as “authorization”) is obtained from a desig-
nated legal authority (eg, the donor’s next of
kin) or by first-person authorization if the do-
nor registered his or her wishes before death.
Strict adherence to aseptic surgical recovery
techniques minimizes contamination of tis-
sues with microorganisms from the donor’s
skin and intestinal flora as well as the sur-
rounding environment.
Responsible persons at tissue banks de-
termine donor eligibility based on an evalua-
tion of 1) answers provided by the next of kin
or other knowledgeable person to questions
773
774 䡲 AABB TECHNICAL MANUAL
about the donor’s travel and medical history
and any high-risk behaviors, 2) available medi-
cal records, 3) the autopsy report (if an autopsy
was performed), 4) a thorough physical assess-
ment of the donor to identify evidence of high-
risk behaviors or active communicable disease,
5) the suitability of any blood sample collected
for infectious disease testing, and 6) the cir-
cumstances of death. A history of high-risk ac-
tivity that increases the possibility of transmis-
sion of disease leads to the rejection of the
donor. Allografts, similar to blood products,
are released for transplantation only after the
donor has been tested for relevant communi-
cable diseases and the results are determined
to be acceptable (see Table 32-1).
Background and Definitions
Human tissue banking in the United States
started more than 60 years ago at the US Navy
Tissue Bank. The use of tissue allografts, now
common, has evolved into a highly innovative
and continuously changing field. Collabora-
tion between tissue bank scientists and end-
user surgeons has produced a wide variety of
life-saving and life-enhancing tissue grafts.
Allografts are tissues transferred between
individuals of the same species. Allografts may
be processed to remove cells or carefully pre-
served to maintain cellular viability. They can
be derived from a single tissue or multiple tis-
sues acting as a functional unit. During pro-
TABLE 32-1. Required Infectious Disease Testing of Human Allografts3
Infectious Agent
Hepatitis B
Hepatitis C (HCV)
Human immunodeficiency virus (HIV)
Human T-cell lymphotropic virus (HTLV)*
Syphilis
*Required for tissues that are rich in viable leukocytes only.
cessing, human tissue allografts may be com-
bined with other biocompatible agents to
achieve desired handling and functional char-
acteristics. Bone allografts, due to their hard
and rigid nature, can be precisely machined
for compatibility with surgical instrumenta-
tion.
Autografts are tissues implanted into the
individual from whom they were removed. For
example, bone that has been surgically re-
moved from a patient’s ilium can be shaped to
desired dimensions and implanted into the
vertebral disk space of the same patient. The
autograft bone promotes fusion of adjacent
vertebrae, providing stability and relief from
the pain of degenerative disc disease or trau-
ma. Advantages of autografts include the elim-
ination of communicable disease transmis-
sion risk and the ready availability of graft
material. Disadvantages include morbidity as-
sociated with the additional surgical proce-
dure for the patient, including pain and poten-
tial surgical-site infection. In addition, the
quality (eg, strength) and quantity of autolo-
gous tissue may not be adequate for the in-
tended use, and removal of the patient’s tissue
may adversely affect function at the site from
which it was removed.
Isografts, which are tissues transferred be-
tween genetically identical individuals, such
as identical twins, are uncommonly used.
Test Performed
Hepatitis B surface antigen
Hepatitis B core antibody (IgM and IgG)
Hepatitis C antibody
HCV nucleic acid testing
HIV-1 and HIV-2 antibodies
HIV-1 nucleic acid testing
HTLV-I and HTLV-II antibodies
Nontreponema- or treponema-specific assay
 CHAPTER 32
Xenografts are tissues transplanted from
one species to another; a growing number of
medical products are being developed from
highly processed nonhuman animal tissues.
Tissue Processing
After tissue has been procured from a de-
ceased donor, it is processed using aseptic
technique in a controlled environment in a fa-
cility designed to prevent contamination or
cross-contamination. Similar to good manu-
facturing practice, good tissue practice (GTP)
requires the facility to establish and maintain
controls over temperature, humidity, ventila-
tion, and air filtration. Thorough cleaning and
disinfection of processing rooms and equip-
ment are required to ensure a proper environ-
ment.
Tissues are debrided of extraneous soft
tissue and cut to specification. In some cases,
more than 100 grafts can be produced from a
single donor. Precision bone grafts, including a
growing array of spinal implants, are crafted
using sophisticated, computer-aided cutting
devices to meet the exacting specifications re-
quired by surgical instrumentation; precisely
milled allografts allow surgeons to operate
more quickly and efficiently.
During processing, various solutions, in-
cluding antibiotics, alcohols, and surfactants,
may be used to reduce or eliminate bacterial
contamination and remove extraneous lipids
and other biologic material. Depending on the
type of graft, graft sterilization may or may not
be possible; grafts containing viable cells or a
fragile matrix cannot be subjected to steriliza-
tion methods without destruction of cellular
or matrix integrity. Ionizing radiation is the
most frequently used sterilization technique.
Ethylene oxide and proprietary methods of tis-
sue sterilization may also be used by tissue
processors.
Several methods of tissue preservation
are available for long-term storage of human
allografts, including freezing and lyophiliza-
tion (freeze drying). Both processes destroy
cell viability. Tissue integrity can be enhanced
through cryopreservation, a process in which
tissues are frozen in a protective medium at a
Tissue Services in the Hospital 䡲 775
steady, controlled rate. The use of cryoprotec-
tants, such as glycerol or dimethyl sulfoxide,
minimizes cell damage caused by cell shrink-
age and intracellular ice formation during
freezing.
Refrigerated storage can preserve cellular
viability in osteochondral allografts for which
preservation of living chondrocytes is essen-
tial. These grafts, which consist of an intact ar-
ticular surface with associated soft tissue and
bone, are used for treating traumatic and de-
generative joint conditions. Refrigeration is
not suitable for long-term storage of allografts.
Clinical Uses of Allografts
Allografts are used in a variety of surgical pro-
cedures. Cadaveric human bone can be used
to replace bone lost to degenerative disease,
trauma, or malignancy. Allogeneic human
bone has unique healing characteristics, in-
cluding osteoconductive and osteoinductive
properties. In vivo, it acts as a scaffold that al-
lows recipient capillary growth into the graft
(osteoconductivity) and provides stimulation
for the production of new bone (osteoinduc-
tivity) by exposing the patient’s osteogenic
progenitor cells to bone morphogenetic pro-
teins (BMPs), growth factors in bone that in-
duce new bone formation. The result is creep-
ing substitution, in which bone remodeling
occurs through osteoclastic resorption of the
implanted tissue and osteoblastic generation
of new bone.
A variety of human tissues are used for
transplantation.4 Selected clinical uses are list-
ed in Table 32-2. Bone-tendon (Achilles ten-
don) or bone-ligament-bone (patellar-tibia
ligament) grafts are routinely used for anterior
cruciate ligament repair. The implanted ten-
don or ligament spans the joint space, and the
bone provides an anchor into the femur and/
or tibia, restoring joint stability. Crushed bone
subjected to acid demineralization can be
used alone or suspended in a biologically
compatible carrier and applied to exposed
bone surfaces. BMPs in demineralized bone
stimulate osteogenesis, fusion of adjacent
bones, and healing. Cadaveric skin can be
used as a temporary wound dressing for severe
776 䡲 AABB TECHNICAL MANUAL

TABLE 32-2. Clinical Uses of Selected Human Allografts

Allograft Tissue Surgical Use

Amnion Wound covering

Conjunctiva surface repair
Corneal ulcer repair
Bone (cortical, corticocancellous, and cancellous) Skeletal reconstruction
Spinal fusion
Dental implant placement
Bone-tendon (Achilles tendon) Anterior cruciate ligament repair
Bone-tendon-bone (patellar ligament) Posterior cruciate ligament repair
Tendon (semitendinosus, gracilis, and peroneus Rotator cuff restoration
longus) Biceps tendon rupture repair
Cardiac valves (aortic and pulmonary) Valvular insufficiency correction
Congenital cardiac defect repair
Cartilage (costal) Facial reconstruction
Cornea Keratoconus repair
Traumatic scarring reversal
Corneal ulcer excision
Decellularized skin Hernia repair
Soft tissue reconstruction
Gingival restoration
Demineralized bone Dental implant placement
Spinal fusion
Dura mater Dural defect/cerebrospinal leak repair
Fascia lata Soft tissue reconstruction
Pelvic floor support
Meniscus Meniscus replacement
Osteoarticular/osteochondral graft (bone and Joint restoration
joint cartilage)
Pericardium Dura patch
Eyelid reconstruction
Soft tissue reconstruction
Sclera Eye enucleation
Scleral ulcer repair
Eyelid repair
Skin Protection of underlying tissues from severe burns
Veins/arteries Coronary artery bypass grafting
Tissue revascularization
Aneurysm repair
Dialysis access shunts
 CHAPTER 32
burns, protecting underlying tissues from de-
hydration and environmental pathogens. Hu-
man skin can be processed to remove cellular
elements, producing an acellular collagen ma-
trix that provides a scaffold for revasculariza-
tion and cellular incorporation in soft tissue
reconstructive surgery. In addition, donor tis-
sues can be used to treat a variety of ocular
conditions. Thinning, scarring, or clouding of
the cornea may be treated by corneal trans-
plantation. Sclera and amnion-derived al-
lografts can be used to treat glaucoma, scleral
ulcers, and traumatic injuries.
Although bone and soft tissue allografts
may provoke a recipient immune response,
such responses are not usually clinically signif-
icant, presumably due to a lack of residual cel-
lular material in processed grafts.5 Therefore, it
is not necessary to match most allografts to the
recipient’s HLA or ABO type. Possible excep-
tions include cryopreserved veins and arteries,
for which some clinicians request ABO com-
patibility, and frozen, unprocessed bone al-
lografts containing marrow or red cells. Devel-
opment of Rh(D), Fy(a), and Jk(b) antibodies
in recipients following transplantation of un-
processed bone allografts has been document-
ed.6 If unprocessed bone is to be used for an
Rh(D)-negative female of childbearing poten-
tial, her future offspring may be at risk of he-
molytic disease of the fetus and newborn. Rh
Immune Globulin prophylaxis should be con-
sidered if the Rh type of the red cells or marrow
contained in the allograft is positive or un-
known.
Disease Transmission through Tissue
Transplantation
Rare, sporadic transmissions of infectious dis-
eases, including human immunodeficiency vi-
rus (HIV), hepatitis C virus (HCV), hepatitis B
virus (HBV), and Creutzfeldt-Jakob disease
(CJD), have been documented following tissue
implantation.7 In addition, bacterial and fun-
gal infections from allografts have resulted in
morbidity and death. Potential sources of
contaminating agents include donor flora,
premortem infection, and environmental
contaminants in recovery and processing fa-
Tissue Services in the Hospital 䡲 777
cilities. Advances in screening, testing, and
processing methods continue to improve the
safety profiles of human tissue products.
R E G U L AT I O NS A ND
S TA N D A R D S
The Food and Drug Administration (FDA) reg-
ulates the activities of tissue banks and tissue
distribution intermediaries, which are estab-
lishments or persons engaged in the recovery,
screening, testing, labeling, processing, stor-
age, and/or distribution of human tissues for
clinical use, under Title 21, Parts 1270 and
1271 of the Code of Federal Regulations
(CFR).3 These entities manufacture what the
FDA classifies as human cells, tissues, and cel-
lular and tissue-based products (HCT/Ps). Ex-
amples of common tissue products are listed
in Table 32-3.
The three rules of 21 CFR Part 1271 con-
cern 1) registration of tissue bank establish-
ments, 2) donor eligibility, and 3) GTP related
to handling HCT/Ps. Compliance with current
GTP regulations is required of tissue banks and
tissue distribution intermediaries to control
contamination and cross-contamination that
may lead to transmission of disease. Tissue-
dispensing institutions, such as hospitals, den-
tal offices, and surgical centers that provide
and use tissue within their own facility are not
subject to this oversight except under certain
circumstances (ie, redistribution of allografts
or autografts to affiliated institutions located
at a different address or attempts to sterilize
autografts).
For a hospital–based tissue-dispensing
service, regulatory oversight is governed by
voluntary accrediting organizations. Title 21,
CFR Parts 1270 and 1271 do not apply to facili-
ties that only receive, store, and dispense tis-
sue, and engage in no further manufacturing
or redistribution of the tissue product. Howev-
er, The Joint Commission, AABB, College of
American Pathologists (CAP), Association of
periOperative Registered Nurses (AORN),
AATB, and Eye Bank Association of America
(EBAA) all publish standards that apply to the
practices of tissue-dispensing services.8-13 The
778 䡲 AABB TECHNICAL MANUAL

TABLE 32-3. Examples of Common Cell and are safe, available, and of high quality. AATB’s
Tissue Products8,14 standards pertain to institutional and quality
program requirements; donor authorization/
Amniotic membrane informed consent; donor screening and test-
Bone and demineralized bone matrix ing; and tissue recovery, processing, release,
and distribution.12 EBAA’s scope encompass-
Bone marrow
es all aspects of eye banking.13 Accreditation
Bone paste, powder, or putty by these organizations is based on verified
Cancellous chips compliance with established standards and
periodic inspections. Both organizations
Cardiac (heart) valves serve as scientific and educational resources
Cartilage for the donation and transplantation com-
munities. A tissue-dispensing service may
Cornea
find AATB and EBAA accreditation of a sup-
Dermal matrix plier to be valuable in assessing that suppli-
Dura mater er’s qualifications.

Embryos
H O SPI TA L T I SS U E S E RVI C E S
Fascia
There is no requirement for a tissue-dispens-
Hematopoietic stem cells
ing service to be managed in any particular de-
Ligaments partment or by any specific individual. The
Meniscus AABB supports a tissue-dispensing service
within the transfusion service, which has ex-
Ocular tissues
pertise in providing human-derived products
Oocytes that are perishable, potentially infectious, and
sometimes in short supply and that require
Pericardium
temperature-controlled storage. The tasks of
Peripheral blood stem cells ordering, receiving, storing, distributing (issu-
Sclera ing), tracking, and tracing products as well as
investigating adverse events, including com-
Semen and sperm plaints, recalls, and look-back investigations,
Skin are activities that are common to the transfu-
sion service and tissue-dispensing service.
Tendons
Umbilical cord blood stem cells Responsibility for Hospital-Based
Vascular grafts (veins and arteries) Tissue Services
The Joint Commission requires that organiza-
tions assign oversight responsibility for their
location of a tissue-dispensing service in a tissue program, use standardized procedures
hospital or medical facility and the scope of its in tissue handling, maintain traceability of all
responsibilities dictate which standards are tissues, and have a process for investigating
applicable. All of these standards are updated and reporting adverse events. Either a central-
regularly; therefore, a periodic review of the ized or decentralized process is permitted to
most recent versions is required to guarantee manage these activities. In either model, des-
ongoing compliance. ignated oversight is required to coordinate tis-
The AATB and EBAA are voluntary accred- sue-related activities and ensure standardiza-
iting organizations dedicated to ensuring that tion of practices throughout the organization.
human tissues intended for transplantation The Joint Commission standards apply to
 CHAPTER 32
human and nonhuman cellular-based trans-
plantable and implantable products, including
both tissue allografts and certain medical de-
vices, as classified by the FDA.
Written Standard Operating
Procedures
Hospital tissue services must have written pro-
cedures (printed or electronic) for all functions
pertaining to the acquisition, receipt, storage,
issuance, and tracing of tissue grafts as well as
procedures for investigating adverse events
and handling recalls. Manufacturers’ instruc-
tions for handling tissues should be incorpo-
rated into standard operating procedures
(SOPs). When the blood bank or transfusion
service is responsible for tissue, the AABB re-
quires the medical director to approve all
medical and technical policies and procedures
pertaining to tissue.9(p2)
Tissue Supplier Qualifications and
Certification
In contrast to blood banks, tissue processors
and distributors often specialize in providing
particular types of products (grafts). As a re-
sult, a hospital tissue-dispensing service may
need to acquire human tissue products from
more vendors than the number of vendors
from which a transfusion service commonly
obtains blood products.
Tissue suppliers should be selected based
on their ability to reliably provide high-quality
tissues that meet expectations for availability,
safety, and effectiveness. The tissue service
should establish minimal criteria for the quali-
fication of prospective suppliers. According to
The Joint Commission, tissue supplier require-
ments must include evidence of current FDA
registration and state licensure if such licen-
sure is required. A written process for review
and approval of suppliers is expected and
should contain the elements listed in Table 32-
4. A list of approved suppliers that includes
documentation of each supplier’s qualifica-
tions, certifications, and appropriate licenses
or permits should be developed and main-
tained. Tissue services should establish proce-
Tissue Services in the Hospital 䡲 779
dures to receive or monitor evidence of com-
pliance or noncompliance, such as reviewing
FDA warning letters and tissue allograft recalls
or market withdrawals. Accreditation by AATB
and/or EBAA may be desirable.
Qualification information for each sup-
plier should be reviewed and approved annu-
ally by the hospital tissue service. During these
reviews, the performance of suppliers in meet-
ing the transplant facility’s needs should be
evaluated. Each year, the tissue service should
determine whether the supplier remains regis-
tered with the FDA; whether AATB and/or
EBAA accreditation is current can also be con-
firmed. FDA web postings should be reviewed
for information related to closures, recalls, or
MedWatch reports. FDA inspection reports on
tissue processors can be requested from the
FDA through the Freedom of Information Act.
Complaints from transplanting surgeons con-
cerning the supplier’s tissue should be re-
viewed along with any reports of infections
that might have been caused by transplanted
allografts. Further use of a particular tissue
supplier should depend on approval by the tis-
sue service’s director. Hospital management
may consider establishing a committee of in-
ternal stakeholders, including transplant phy-
sicians, to provide oversight in the approval of
tissue suppliers.
Inspection of Incoming Tissue
Allografts
Before being placed in storage, tissue allografts
must be inspected upon receipt from a tissue
supplier to ensure that the packaging remains
intact and the label is complete, appears to be
accurate, and is adequately affixed and legible
before acceptance into inventory. The incom-
ing inspection results should be recorded
along with the date, time, and name of the staff
person who conducted the inspection.
The Joint Commission requires that hos-
pitals verify package integrity and ensure that
the transport temperature was controlled and
acceptable. Inspection of the shipping con-
tainer for evidence of residual coolant (eg, wet
ice for refrigerated grafts or dry ice for frozen
grafts) may be useful to determine that the re-
780 䡲 AABB TECHNICAL MANUAL
TABLE 32-4. Vendor Qualification Criteria for Human Allografts
Criterion
FDA registration
FDA inspection findings
Voluntary accreditation, if available
State license and registration, if required by state law
Reliable supply of needed tissues
Transparency of the organization
Medical consultation
Quality assurance resources
New or trial tissue product support
Professionalism of sales representatives
FDA = Food and Drug Administration, AATB = American Association of Tissue Banks, EBAA = Eye Bank Association of America;
eHCTERS = Human cell and tissue establishment registration.
quired tissue-specific storage environment
was maintained during transportation.
Many distributors use “validated” ship-
ping containers that are tested to maintain re-
quired temperatures for a specified period. If
such a container was used, the receiver of the
tissue simply needs to verify that there is no
damage to the container and that it has been
received and opened within the specified time
frame.
Tissues requiring “ambient temperature”
(defined as the temperature of the immediate
environment) for storage and shipping do not
need to have the temperature verified upon re-
ceipt. However, the temperature of tissues re-
quiring “room-temperature” storage should be
verified and documented if the manufacturer
Documentation/Performance
Current Form FDA 3356 /eHCTERS Query
Form 483 findings, if any
Warning letters and responses, if any
Current AATB accreditation for tissues
Current EBAA accreditation for ocular tissues
Copy of state license and registration (to be reviewed
annually)
Adequate notification of tissue shortages
Ability to meet special requests
Suitable expiration dates for tissue products
Willingness to provide information regarding donor-
selection and tissue-processing processes
Accessibility of the tissue supplier’s medical director
Accessibility of the tissue supplier’s quality assurance
staff
Willingness to provide information on newly released
tissue products
Approval sought by representatives through desig-
nated channels before promoting or providing tissue
within the hospital
has specified a temperature storage range in
the package insert.
Tissue Storage
As with blood components, tissue grafts are
stored under various conditions (see Table 32-
5). The appropriate storage conditions depend
on the nature of the tissue, method of preser-
vation, and type of packaging.
Hospital tissue services should store tis-
sue allografts according to the processor’s
instructions in the package insert. Storage
devices can include “ambient” and/or room-
temperature cabinets, refrigerators, mechani-
cal freezers, and liquid-nitrogen storage units.
Continuous temperature monitoring of refrig-
 CHAPTER 32 Tissue Services in the Hospital 䡲 781

TABLE 32-5. Storage Conditions for Commonly Transplanted Human Tissue12

Human Tissue Storage Conditions Temperature*

Cardiac and vascular Frozen, cryopreserved –100 C or colder

Musculoskeletal Refrigerated Above freezing (0 C) to 10 C
Frozen, cryopreserved and non- –20 C to –40 C
cryopreserved (temporary storage
for 6 months or less)
Frozen, cryopreserved and non- –40 C or colder
cryopreserved (long-term storage)
Lyophilized Ambient†
Reproductive Frozen, cryopreserved Liquid nitrogen (liquid or vapor
phase)
Skin Refrigerated Above freezing (0 C) to 10 C
Frozen, cryopreserved –40 C or colder
Lyophilized Ambient†
*Warmest target temperature unless a range is listed.
†
Ambient temperature monitoring not required for lyophilized tissue.

erators and freezers is required. Room-tem-
perature storage equipment need only be
monitored if this is required by the allograft’s
package insert. Storage equipment should
have functional alarms and emergency back-
up capability. Lyophilized tissues whose pack-
age insert instructions specify ambient tem-
perature or colder storage can tolerate a very
broad range of temperatures and their temper-
atures do not require monitoring.
Storage SOPs should address steps to be
taken in case of excursions from allowable
temperature limits or in the event of equip-
ment or power failure. Emergency backup al-
ternatives, including arrangements for tempo-
rary storage, should be described in written
procedures.
Tissue Allograft Traceability and
Record Keeping
Proper management of human allografts re-
quires that the hospital tissue service docu-
ment all of the steps taken in tissue handling
as they occur and maintain comprehensive
records of these steps. The records should be
accurate, legible, and indelible. They must
identify the staff who have handled the tissue
and the dates when the tissue was handled as
well as the time when the tissue was accepted,
prepared, or processed. The records should be
detailed and provide a clear history of all ac-
tions performed. Documentation of the tissue
supplier, unique numeric or alphanumeric
identifier(s) of the allograft, its expiration date,
and the recipient’s name must be maintained
for all tissue grafts used.
Tissue service records need to permit bi-
directional traceability of all tissues from the
donor and tissue supplier to the recipient(s) or
other final disposition, including the discard
of tissue because of damage to package integ-
rity, opening of the tissue package in the oper-
ating room without use, or expiration. Records
should be retained for 10 years, or longer if re-
quired by state or federal law, after distribu-
tion, transplantation, discard, or expiration
(whichever occurs last).
782 䡲 AABB TECHNICAL MANUAL
Tissue usage information cards or other
systems supplied with the allograft by the tis-
sue bank must be completed and returned to
the tissue source facility. This information
helps maintain the traceability of the allograft
and expedites market withdrawals or recalls
when necessary. The tissue supplier may also
use this information to better understand al-
lograft utilization, obtain positive or negative
feedback, and better meet the needs and ex-
pectations of customers.
Recognizing and Reporting Adverse
Events Possibly Caused by Allografts
Use of human-derived medical products, such
as tissue allografts, carries risks that must be
balanced with clinical benefits. Human al-
lografts have been associated with bacterial,
viral, fungal, and prion transmissions. In addi-
tion, allografts may have structural defects
that sometimes lead to unsuccessful outcomes
as a result of donor selection or processing fac-
tors.
Hospital tissue services are required to
have procedures to investigate in a timely way
any infections suspected to be caused by a tis-
sue allograft. The Joint Commission requires
that allograft-transmitted infections and other
severe adverse events be immediately report-
ed by the hospital to the tissue supplier.
Transplant surgeons play a critical role in
the identification of allograft-associated ad-
verse outcomes and need to immediately noti-
fy the hospital tissue service when they sus-
pect such events. Prompt notification of
adverse events enables the hospital tissue ser-
vice to investigate the cause, report the issue
to the tissue supplier, and institute corrective
action, including sequestration of any other
suspect allografts. The investigation of infec-
tions and other adverse events requires coop-
eration between the tissue-dispensing service,
clinicians, and tissue supplier. Consultation
with the hospital infection-control depart-
ment or an infectious disease specialist may
be beneficial. Early notification can prevent an
untoward outcome for other recipients of al-
lografts from an implicated donor.
State health departments have lists of
communicable diseases that must be reported
when they are newly diagnosed. A new diagno-
sis of HIV or viral hepatitis in a tissue allograft
recipient where the allograft is suspected as a
possible source must be reported to the state
department of health by the patient’s physi-
cian or the director or medical director of the
hospital tissue service. An epidemiologic in-
vestigation may be needed to establish wheth-
er the tissue allograft was the source of the re-
cipient’s infection. Early notification of the
state department of health can result in timely
assistance with the investigation.
Recalls and Look-Back Investigations
A tissue product recall or market withdrawal
occurs when a tissue allograft is determined by
the tissue supplier to be compromised or po-
tentially infectious. The supplier may recall all
of the tissues from a specific donor or process-
ing lot, sequester tissues in inventory, and no-
tify hospitals to which the allografts were
shipped. The hospital may be instructed by
the supplier to quarantine the allografts in in-
ventory, identify recipients, and/or notify the
transplanting surgeon(s) of the recall. Sur-
geons should evaluate the circumstances of
the recall and notify the recipient as appropri-
ate that a tissue graft was recalled.
Look-back investigation can be triggered
when a tissue donor is subsequently found to
have been infected with HIV, human T-cell
lymphotropic virus Types I or II, HBV, HCV, or
other infectious disease known to be transmit-
ted by tissue grafts. Because most tissues are
procured from deceased donors, infectious
disease testing of subsequent donor blood
samples is not possible. Look-back investiga-
tions involving tissue grafts are uncommon.
Tissue Autograft Collection, Storage,
and Use
Surgical reconstruction using the patient’s
own tissues often has advantages over the use
of cadaveric tissue. These advantages include
ready availability, faster incorporation, appro-
 CHAPTER 32
priate size or shape, and relative safety from
viral disease transmission.
A bone flap removed during decompres-
sive craniectomy is a commonly used example
of tissue autograft use. In this procedure, a sec-
tion of skull is excised by the neurosurgeon to
reduce intracerebral pressure caused by brain
swelling due to trauma, stroke, or surgery. After
removal, the skull fragment is rinsed,
packaged, frozen, and stored for future reim-
plantation during a procedure known as “cra-
nioplasty.”
Written procedures should address the
collection, microbial testing, packaging, stor-
age, and issuance of tissue autografts for reim-
plantation. Bacterial testing by obtaining ap-
propriate cultures should be performed after
surgical removal and before packaging. Auto-
grafts should not be collected from patients
with systemic infections or if the tissue is in
close proximity to an infected area. Autografts
may be stored at the medical facility where
they are collected or at an off-site, FDA-regis-
tered tissue bank. Procedural recommenda-
tions have been published by the AATB and
AORN.
TR ANSF USION SERVICE
SU P P O RT F O R O RG A N
TR A N SP L AN TAT I O N
Organ transplantation relies on the coordinat-
ed activities of organ-procurement organiza-
tions (OPOs) and the hospital transplant team.
A successful transplant program requires an
interdisciplinary team that has well-defined
policies, procedures, and communication
pathways. The transfusion service provides
appropriate compatibility testing and transfu-
KEY POINTS
1. Surgical use of human allografts has grown dramatically in recent years, with more than
2 million tissue grafts used annually in the United States. The majority of these grafts are ob-
tained from deceased human donors who meet stringent screening requirements similar to
those applied to blood donors.
2. Not all tissue allografts are sterile. Depending on the type of allograft, sterilization may not
be possible because it could jeopardize the viability of the cellular elements or fragile matrix
Tissue Services in the Hospital 䡲 783
sion support before, during, and after trans-
plantation.
OPOs evaluate potential donors, obtain
authorization (consent), and prepare organs
for transportation. The United Network for Or-
gan Sharing (UNOS) coordinates the US organ
transplant system. The evolving UNOS guide-
lines for organ allocations are designed to
achieve equitable distribution of life-saving
organs. Factors such as severity of recipient ill-
ness, geographic proximity, and donor-recipi-
ent compatibility are considered. UNOS pro-
vides detailed policies and other relevant
information on its website.15
Depending on the organ, ABO and/or
HLA compatibility may be important factors
for transplantation success. ABO antigens ex-
pressed on the vascular endothelium of organs
constitute strong histocompatibility barriers.
Major incompatibility between donor ABO an-
tigens and recipient plasma can result in acute
humoral rejection of the transplanted organ
and threaten a successful outcome. As a result,
UNOS requires two separate determinations
of ABO type for both 1) transplant candidates
prior to listing their needs on the Organ Pro-
curement and Transplantation Network wait-
ing list and 2) donors prior to making an organ
available for transplant. ABO-incompatible or-
gan transplants are sometimes performed fol-
lowing a conditioning regimen that may in-
clude plasma exchange.
Services that may be required by a trans-
plant program include provision of cytomega-
lovirus-reduced-risk components, blood irra-
diation, massive transfusion support, ABO
subgroup typing, and immunohematology ref-
erence testing. Transfusion services should
understand and address such expectations of
the transplant team.
784 䡲 AABB TECHNICAL MANUAL

of the graft and adversely affect in-vivo performance. Methods of sterilization include the
use of ionizing radiation or ethylene oxide and some proprietary processes and techniques.
3. Rare examples of HIV, HCV, HBV, CJD, and bacterial and fungal disease transmission from
allografts have been documented. Therefore, like blood components, allografts are released
for use only after infectious disease testing has been completed and the results deemed ac-
ceptable.
4. In general, bone and soft tissue allografts do not need to be matched for HLA or ABO type.
5. Hospital-based tissue services are not subject to FDA regulatory oversight if their activities
are limited to receiving, storing, and dispensing tissue to a requesting surgeon. However,
The Joint Commission, AABB, CAP, AORN, AATB, and EBAA publish standards that apply to
such services.
6. Tissue banks are engaged in the recovery, screening, testing, labeling, processing, storage,
and distribution of human tissues. They are regulated by the FDA under the Title 21, CFR
Parts 1270 and 1271 as manufacturers of HCT/Ps.
7. No regulatory or standard-setting organization requires a hospital tissue-dispensing service
to be managed by a particular department or individual. However the AABB favors a cen-
tralized model for managing this activity within the transfusion service.

REFER ENCES

1. American Association of Tissue Banks. About
AATB. McLean, VA: AATB, 2013. [Available at
http://www.aatb.org/About-AATB (accessed
December 8, 2013).]
2. Eastlund DT, Eisenbrey AB for the Tissue Com-
mittee. Guidelines for managing tissue al-
lografts in hospitals. Bethesda, MD: AABB,
2006.
3. Code of federal regulations. Title 21, CFR Parts
1270 and 1271. Washington, DC: US Govern-
ment Printing Office, 2014 (revised annually).
4. Woll JE, Smith DM. Bone and connective tis-
sue. Clin Lab Med 2005;25:499-518.
5. Malinin TI. Preparation and banking of bone
and tendon allografts. In: Sherman OH,
Minkoff J, eds. Arthroscopic surgery. Balti-
more, MD: Williams and Wilkins, 1990:65-86.
6. Cheek RF, Harmon JV, Stowell CP. Red cell allo-
immunization after a bone allograft. Transfu-
sion 1995;35:507-9.
7. Eastland T, Warwick, RM. Diseases transmit-
ted by transplantation of tissue and cell al-
lografts. In: Warwick E, Brubaker SA, eds. Tis-
sue and cell clinical use: An essential guide.
West Sussex, United Kingdom: Blackwell,
2012:72-113.
8. The Joint Commission. Transplant safety. In:
Comprehensive accreditation manual for hos-
pitals: The official handbook. Oakbrook Ter-
race, IL: The Joint Commission, 2014:TS1.
9. Levitt J, ed. Standards for blood banks and
transfusion services. 29th ed. Bethesda, MD:
AABB, 2014.
10. College of American Pathologists. Standards
for laboratory accreditation. Northfield, IL:
CAP, 2012.
11. Association of Perioperative Registered Nurs-
es. Perioperative standards and recommended
practices. Denver, CO: AORN, 2013.
12. Dock N, Osborne J, Brubaker S, et al. Stan-
dards for tissue banking. 13th ed. McLean, VA:
American Association of Tissue Banks, 2012.
13. Eye Bank Association of America. Medical
standards. Washington, DC: EBAA, 2011.
14. Food and Drug Administration. FDA regula-
tion of human cells, tissues, and cellular and
tissue based products (HCT/Ps) product list.
Rockville, MD: Food and Drug Administration,
2013. [Available at www.fda.gov/Biologics
BloodVaccines/TissueTissueProducts/Regula
tionofTissues/ucm150485.htm (accessed No-
vember 13, 2013).]
15. United Network for Organ Sharing. Richmond,
VA: UNOS, 2013. [Available at www.unos.org
(accessed December 8, 2013).]
 C h a p t e r 3 3

Blood and Marrow-Derived

Nonhematopoietic Stem Cell
Sources and Immune Cells for
Clinical Applications

Mickey B. C. Koh, MD, PhD; Edward R. Samuel, PhD, MSc; and

Garnet Suck, PhD, MSc

HEMATOPOIETIC STEM CELL presenting cells” and are key to orchestrating

transplantation (HSCT) is an established
modality of treatment for a wide range of he-
matologic diseases, including leukemias and
lymphomas. One of the most powerful mecha-
nisms for cure is the presence of the graft-vs-
tumor (GVT) or graft-vs-leukemia (GVL) effect,
which is mediated largely by mature immune
effector cells, such as T and natural killer (NK)
cells.1 The immune response that is generated
is derived from a complex system of interac-
tions involving antigen recognition and pre-
sentation mediated by dendritic cells (DCs).
DCs are also known as “professional antigen-
the adaptive immune response in an antigen-
specific manner.
The success of HSCT has led to enormous
interest in the isolation, characterization, ex-
pansion, and modification of these immune
cells to augment the powerful GVT response.
Most of these cells form part of the hemato-
poietic stem cell (HSC) graft infused into pa-
tients and can be derived from blood.
In fact, the discovery of the GVT effect
came from the observation that depleting T
cells from the graft resulted in a greater risk of
leukemia relapse. Conversely, the occurrence

Mickey B. C. Koh, MD, PhD, Director, Stem Cell Transplantation, St. George’s Hospital and Medical School,
London, United Kingdom, Medical Director, Cell Therapy Facility, Health Sciences Authority, Singapore;
Edward R. Samuel, PhD, MSc, Clinical Scientist and Senior Research Associate, Department of Haematology,
University College London Medical School, Royal Free Campus, London, United Kingdom; and Garnet Suck,
PhD, MSc, Division Director, Productions, Institute for Transfusion Medicine, German Red Cross Blood Dona-
tion Service North-East nonprofit GmbH, Berlin, Germany
The authors have disclosed no conflicts of interest.

785
786 䡲 AABB TECHNICAL MANUAL
of graft-vs-host disease (GVHD), which is also
mediated by T-cells, was protective and re-
duced the chance of relapse.1
In addition to HSCs and their differentiat-
ed progeny, the marrow contains nonhemato-
poietic cells, often referred to as “stromal
cells,” which include mesenchymal stem cells
(MSCs). In addition to providing stromal sup-
port for hematopoietic cells, MSCs give rise to
structural components, such as bone, carti-
lage, tendon, and fat. Their wide-ranging and
potent differentiation properties have generat-
ed considerable clinical interest in their use for
cell therapy. MSCs also appear to exert immu-
nosuppressive effects and are therefore of in-
terest as immunomodulators.
Self-renewing pluripotent stem cells are
rare and difficult to isolate. The demonstration
that pluripotent stem cells can be derived
from differentiated cells, including skin fibro-
blasts and blood, has transformed the field of
stem cell therapy. The use of these cells has the
potential to have myriad applications in all ar-
eas of medicine.
IM MUNE CELLS FOR CLINICAL
THERAPY
Cellular immunotherapy is the exploitation of
immune cells to target and treat diseases. This
approach is most established in cancer pa-
tients, where these novel treatment modalities
are used to overcome the limitations and tox-
icities of chemotherapy and radiotherapy.
Considerable progress in cellular process-
ing technology has allowed for advanced and
complex immune-cell manipulations. It is now
possible to characterize immune cells with
high precision, isolate specific cell types with
high purity, and engineer and amplify them ex
vivo in sophisticated culture systems that
comply with good manufacturing practice
(GMP) requirements. The adoption of auto-
mation has also allowed for more reproducible
immune cell expansion.
Blood banks have been instrumental in
these advances in cellular immunotherapy
due to their preexisting infrastructure, which
includes the GMP-compliant processing of
blood units for transfusion and meticulous
quality-assurance systems embedded into the
processes. In addition, the stringent donor-
selection processes, widespread use of auto-
mation, use of mandatory release criteria for
blood units, and requirements of traceability
and hemovigilance of blood banks have been
incorporated into cellular immunotherapy.
Donor Lymphocyte Infusion: A Post-
HSCT Treatment
Although allogeneic HSCT can achieve long-
lasting cures in many patients, relapse still oc-
curs in some patients and is often associated
with a poor prognosis.2 A second HSCT is pos-
sible but is often associated with high toxicity
and morbidity risk, especially if the relapse oc-
curs within a year of the first transplant.
Clinical Efficacy of Donor Lymphocyte
Infusions
The discovery that T cells are one of the main
drivers of the GVT or GVL response led to the
development of donor leukocyte (lymphocyte)
infusions (DLIs) after HSCT. One of the key ob-
servations in the development of DLIs was that
patients with chronic myeloid leukemia (CML)
that relapsed after HSCT could achieve long-
lasting remissions with the administration of
DLIs.3
Lymphocytes for DLI are collected from a
donor via leukapheresis using a process simi-
lar to peripheral blood stem cell collections,
except that prestimulation with a colony-stim-
ulating factor or mobilizing agent is not usual-
ly required. The leukapheresis product is usu-
ally then aliquoted into doses starting from as
low as 1  106 CD3-positive cells/kg recipient
weight, followed by half- or 1-log increments.
The initial dose is often infused fresh, and ali-
quots for subsequent doses are cryopreserved.
The efficacy of DLIs is dependent on the
type and aggressiveness of the underlying dis-
ease and the disease burden at the time of re-
lapse. It has been demonstrated that patients
with relapsed CML benefit most from DLIs
with an 80% complete response rate for those
in cytogenetic relapse; patients in hematologic
relapse respond less well.3 In comparison, only
 CHAPTER 33 Blood/Marrow-Derived Non-HPC and Immune Cells
15% to 40% of patients with relapsed acute
myeloid leukemia (AML) respond to DLIs, and
patients with acute lymphocytic leukemia
(ALL) have virtually no response to DLI. The
lack of efficacy of DLI in acute leukemias com-
pared to CML is thought to be related to a lack
of antigen expression on tumor cells and the
more rapid proliferative kinetics associated
with acute leukemias.4
The starting dose for DLIs is often in the
order of 1 × 106 CD3-positive cells/kg.5 Lower
starting doses have been administered, espe-
cially in the unrelated transplant setting. Re-
sponse is usually not immediate and can take
as long as 3 months. For this reason, subse-
quent doses of DLIs are often administered as
far apart as 3 months and in incremental doses
half to 1 log apart so that responses can be ad-
equately gauged.3
There is no exact correlation between
dose and response. Patient response can be
difficult to predict because it depends on the
disease, stage of relapse, patient factors, HLA
matching, and donor characteristics.6
Complications of DLIs
The major complication of DLI is GVHD,
which is caused by alloreactive donor T cells
attacking healthy host cells. In early studies,
up to 50% to 90% of patients developed GVHD
after DLI. However, more recently, the rate of
GVHD following DLI has declined due to an
improved understanding of the biology of DLIs
and predictive risk factors for GVHD in this
setting. The GVHD that occurs after DLI can be
very severe and require systemic immune sup-
pression, which can lead to significant mor-
bidity and mortality as a result of opportunis-
tic infections.
Another major complication of DLI is the
development of marrow aplasia, which is
thought to be due to the immune-mediated
destruction of host hematopoeisis.6
The variable results in the initial studies
of DLIs in patients with frank disease relapse
together with the treatment’s potential toxici-
ties have resulted in crucial modifications to
this T-cell therapy to increase its GVT activity
while minimizing its side effects. These modi-
䡲 787
fications include the use of CD8-depleted and
alloantigen-depleted DLIs and the introduc-
tion of a “suicide” gene, such as the Herpes
simplex virus-tyrosine kinase gene, into the T
cells to selectively eliminate them if GVHD de-
velops.4
A group of researchers at University Col-
lege London Hospital has also pioneered a
strategy of using a dose-escalating DLI
regimen to treat residual or progressive dis-
ease or mixed donor chimerism after reduced-
intensity, T-cell-depleted transplantation.5 This
strategy has resulted in an impressive 70%
response rate in patients with low-grade lym-
phomas or Hodgkin disease.
New Directions in DLI Applications:
Targeted and Antigen-Specific T-Cell
Therapy
Adoptive immunotherapeutic approaches em-
ploying more targeted or antigen-specific T-
cell populations have been used successfully
in the treatment of hematologic malignancies
and solid tumors as well as viral infections af-
ter HSCT. To develop immunotherapies using
cytotoxic T lymphocytes (CTLs; T cells ex-
pressing CD8), potential target antigens on tu-
mor cells or viruses must first be identified.
These antigens must be capable of providing
epitopes for specific immune responses and
be present in sufficient quantity and duration
to engage responder T cells.4
A step forward in efficiently isolating
T cells was achieved with the development of
the major histocompatibility complex (MHC)
multimer method. In this method, high-affini-
ty binding of MHC molecules to the T-cell re-
ceptor is achieved through oligomerization of
MHC molecules (multimers) as ligands.7 This
method allows specific detection and isola-
tion of antigen-specific T cells through fluores-
cence-activated cell sorting in a flow cytome-
ter or the use of closed magnetic selection
systems, such as the automated CliniMACS in-
strument (Miltenyi Biotec, Bergisch Gladbach,
Germany). The multimer method has recently
been improved with the invention of the novel
streptamer technology, which has entered
clinical testing.8,9
788 䡲 AABB TECHNICAL MANUAL
Adoptive transfer of donor-derived, virus-
specific CTLs constitutes an important novel
treatment of life-threatening viral infections
after HSCT and an alternative to traditional
antiviral drug therapy, such as ganciclovir. Cy-
tomegalovirus (CMV) reactivation is a com-
mon complication of HSCT. Infusions of CTLs
generated against the CMV pp65 antigen have
achieved good clearance of the virus with few
side effects and limited GVHD. Similarly, do-
nor-derived Epstein-Barr virus (EBV)-specific
CTLs have been successfully used for both
prophylaxis and treatment of EBV-associated
lymphoproliferative disease (LPD). In one
study, more than 60 patients were prophylacti-
cally treated with these CTLs and none devel-
oped LPD, compared to 11.5% of patients who
developed LPD in a historical, nontreated con-
trol group.4 Significantly, gene-marked donor
CTLs have been demonstrated to persist in
DLI recipients for as long as 7 years.4
A recent major breakthrough has been
achieved with the development of engineered
T cells bearing chimeric antigen receptors
(CARs) composed of antibody-binding do-
mains connected to domains that activate T
cells. This strategy was developed to overcome
T-cell tolerance, which has been a limitation of
CTL populations that have been isolated and
expanded based on recognition of tumor-as-
sociated antigens. One such CAR construct
utilizes an antibody-binding domain with
specificity for the B-cell antigen CD19 cou-
pled with CD137 (a costimulatory receptor in
T cells) and the CD3-zeta signaling domain.10
The construct’s efficacy was recently demon-
strated in a clinical pilot trial in which autolo-
gous CAR-T cells directed against CD19-bear-
ing advanced chronic lymphocytic leukemia
(CLL) with more than 1000-fold expansion in
vivo and demonstration of trafficking to the
marrow. Significantly, memory CAR-T cells
were generated, and two out of three patients
with advanced CLL achieved complete remis-
sion with persistence of T cells for more than 6
months.10 This proof of concept has just been
extended into the treatment of B-cell ALL with
impressive results and the attainment of com-
plete remissions in some treated patients.11
NK Cells
Particularly promising candidates for cellular
immunotherapy are NK cells. These large
granular lymphocytes of innate immunity are
characterized by a CD3–CD56+ phenotype in
humans and are natural defenders against ma-
lignancies and infectious diseases. Their cyto-
toxicity is immediate and they do not require
antigen priming. Autologous NK cell attack is
prevented via recognition of HLA Class I mole-
cules by cognate NK-cell inhibitory killer im-
munoglobulin-like receptors (KIRs).
In-Vivo Expansion
Early cellular immunotherapy protocols in-
volving NK cells employed short-term stimula-
tion in vitro with high-dose interleukin (IL)-2
for 1 to 5 days, generating a heterogeneous
lymphokine-activated killer (LAK) cell product
with a relatively low representation of cytotox-
ic NK cells. A better understanding of NK cell
biology and recent advances in cell selection,
monitoring, and expansion technologies have
enabled the development of more promising
new NK-cell-therapy approaches.12
An important key finding was the obser-
vation that alloreactive NK cells in the graft ex-
erted a potent antileukemic effect in the con-
text of T-cell-depleted, HLA-haploidentical
HSCT.13 Significantly, there were no relapses in
a group of patients with high-risk AML when
NK cells exerted their effects due to donor-
patient KIR mismatching, compared to a 75%
relapse rate when no NK alloreactivity was
present.14
NK cells have also been administered in a
non-HSCT setting. Patients with poor-progno-
sis AML, Hodgkin disease, metastatic melano-
ma, or renal carcinoma were given haploiden-
tical NK cells.15 In-vivo expansion of these cells
occurred with assistance from the lymphope-
nia induced by a lympho-depleting prepara-
tive regimen, such as cyclophosphamide and
fludarabine. This regimen induced a marked
increase in endogenous IL-15 levels, which is
essential for donor NK cell expansion and sur-
vival in the patient. Significantly, these infused
and in-vivo-expanded NK cells did not induce
GVHD but did produce complete remissions
 CHAPTER 33 Blood/Marrow-Derived Non-HPC and Immune Cells
in 5 of 19 patients with AML. These results of-
fer another proof of the ability of NK cells to
separate GVL from GVHD with the aim of ex-
ploiting the former while minimizing the lat-
ter. As in the haploidentical transplant setting,
the effect was more marked when KIR-ligand
mismatched (indicating alloreactive) donors
were used.
In clinical protocols involving NK cells, ef-
fectors are enriched by magnetic CD3 deple-
tion followed by CD56 selection or through
combined CD3 (T-cell) and CD19 (B-cell) de-
pletion using the CliniMACS (Miltenyi Bio-
tech) to reduce the risk of T-cell-mediated
GVHD and EBV infections.16
Ex-Vivo Expansion
An alternative method to in-vivo expansion of
NK cells is expansion of NK cells in long-term,
GMP-compatible culture systems that gener-
ate large numbers of highly cytotoxic effectors.
This method has been established for clinical
“off-the-shelf” production of the highly cyto-
toxic NK cell line NK-92 [Conkwest (Del Mar,
CA) proprietary line].17,18 In contrast, GMP-
compatible expansion of primary NK cells has
remained challenging, although promising
protocols are under development.
New cell-culture media, including serum-
free media, are now available. Furthermore,
novel growth factors, such as IL-15, are being
used instead of the traditional IL-2 to further
optimize culture conditions with efficient bi-
asing of proliferation of the NK-cell popula-
tion within LAK cultures after long-term ex-
pansion.19
Highly purified NK cells are more chal-
lenging to expand. However, purified NK cells
can be grown on feeder cell sources of either
autologous or allogeneic origin. An elegant
way to obtain autologous feeder cells is to re-
tain the nonselected, NK-cell-negative frac-
tion after a magnetic isolation procedure. Fol-
lowing irradiation of this by-product to inhibit
unwanted cell proliferation during culture,
these cells can be seeded as a feeder layer to
support NK-cell stimulation and proliferation.
Other protocols have been established that in-
volve the use of allogeneic cell sources. Expo-
䡲 789
nential NK-cell expansion, for example, was
achieved with the EBV-transformed lympho-
blastoid cell line or the leukemic cell line K562,
engineered to present both IL-15 and the co-
stimulatory molecule CD137 (4-1BB) on the
surface (K562-CD137/IL-15). These protocols
are undergoing clinical testing.16,19
Automated cell processing within biore-
actors, such as Wave™ (GE Healthcare Life Sci-
ences, Freiburg, Germany) or G-Rex (Wilson
Wolf Manufacturing, New Brighton, MN), pro-
vides a developing platform for the mass pro-
duction of NK cells in less time with better re-
producibility.16 These methods are also used
for other immune effector therapies. Further-
more, shipment of fresh NK cells under IL-2
protection is feasible without the need for a
controlled incubator environment (37 C and
5% CO2) and facilitates international distribu-
tion of such products.20
NK-cell therapy products are generally
safe and well tolerated, especially when in-
vivo injections of IL-2 are not required. How-
ever, NK-cell products are not entirely risk free.
Two cases of significant hemolytic anemia af-
ter treatment with minor ABO-mismatched
NK-cell-enriched products have been report-
ed that were presumably due to the presence
of isoagglutinin-producing passenger B lym-
phocytes in the NK-cell product.21
Cytokine-Induced Killer Cells:
Polyclonal T Cells with NK-Cell Features
The traditional LAK cell culture protocol has
been further developed to generate a popula-
tion of rapidly expanding polyclonal T cells
known as “cytokine-induced killer” (CIK)
cells.22 Culture conditions for CIK expansion
are designed to obtain an increased propor-
tion of superior cytotoxic CD56+CD3+, double-
positive T cells or NK-like T cells. The genera-
tion of such CD56+ T cells is promoted in this
procedure through the initial addition of gam-
ma interferon (IFN; 1000 U/mL). The T-cell-
activating, anti-CD3 monoclonal antibody
OKT3 (50 ng/mL) and IL-2 (300 U/mL) are
added 24 hours later, and the cultures are
maintained under permanent IL-2-stimula-
tion for 21 to 28 days.23 The heterogeneous CIK
790 䡲 AABB TECHNICAL MANUAL
population at the end of culture comprises a
very small proportion (about 2%) of NK cells.
Most of the cells (>90%) in the culture are T
cells, of which about 35% express the double-
positive phenotype CD3+CD56+.
Characteristics of CIK Cells
CIK cells combine features of NK-cell
(CD56+CD3–) and T-cell (CD56–CD3+) effectors.
The different pathways involved in CIK cyto-
toxicity are under active investigation. Similar
to NK cells, CIK cells have cytolytic potential
that is immediate and independent of antigen
priming.24 However, studies have shown that
target cell recognition requires direct MHC
Class I engagement, a mechanism that is not
yet fully understood. As with NK cells, the criti-
cal role of the activating receptor NKG2D in tu-
mor lysis has been demonstrated for targets,
such as myeloma cells, that express its ligands
MIC A/B or ULBPs.
Clinical Trials
Results from clinical trials using autologous or
allogeneic CIK cells in a variety of hematologic
malignancies and solid tumors have been re-
ported. Overall, CIK cell therapy was well toler-
ated and associated with a low risk of GVHD. 25
Results for cancer clearance and increased
survival varied. Patients with a lower tumor
burden, such as those who had undergone
HSCT, are probably the most likely to benefit
from CIK effectors. In this sense, CIK cells may
be used as an alternative to DLI. Strategies to
increase the cytolytic potential of CIK cells, in-
cluding co-administration of IL-2 and engi-
neered CARs, are currently being investigated.
DCs in Immunotherapy
DCs play a critical role in the regulation of the
adaptive immune response. DCs have been
called “sentinels of the immune system,” and
they possess the ability to elicit a primary im-
mune response in resting naïve T lympho-
cytes. Naïve CD8+ T cells, for example, differ-
entiate into CTLs through interaction with
cognate antigens presented by mature DCs on
their MHC Class I receptors.26 DCs are capable
of eliciting full T-cell activation upon engage-
ment of costimulatory molecules (eg, B7:CD28
engagement) and mediate cytotoxic responses
against a broad range of malignancies. Partic-
ularly attractive is the intrinsic potential of
DCs to gain immunologic memory for com-
bating subsequent tumor invasions.27
Defining DCs and Their Subsets
Unlike T cells, no single cell surface marker is
exclusively expressed on DCs that can be used
to define these cells. Instead, a combination of
morphology and various surface markers is
used, including the expression of MHC Class II
antigens and absence of markers that define
other lineages, such as CD3 and CD19. DCs
also express a variety of adhesion molecules,
including CD11a (lymphocyte function-asso-
ciated antigen 1); the intercellular adhesion
molecule 1 family; and costimulatory mole-
cules, such as CD80 and CD86. Two additional
markers of mature DCs in humans are CD83
and CMRF-44.28
In the blood, DCs are present in different
subsets that are distinguished by CD303
(BDCA2, CLEC4C; plasmacytoid DC), CD1C
(or BDCA1; myeloid DC), and CD141 (BDCA3
or thrombomodulin; myeloid DC) markers.
Different functions could be attributed to
these subsets, with CD141-positive myeloid
DCs playing a role in eliciting CD8 T-cell re-
sponses.29
The rarity of DCs has made it difficult to
study them, although they were first described
more than 30 years ago.26 The ability to gener-
ate large numbers of DCs from CD34+ marrow
precursors or CD14+ monocytes in vitro has
expanded the field of DC-based cellular im-
munotherapy.
Expansion Protocols
Initial tissue-culture protocols for the genera-
tion of DCs (the first-generation DC vaccines)
used a combination of granulocyte macro-
phage colony-stimulating factor (GM-CSF)
with IL-4 for the stimulation of monocytes in
peripheral blood mononuclear cells (PBMCs)
to differentiate into DCs. However, the result-
ing DC vaccines usually contained a mixture of
 CHAPTER 33 Blood/Marrow-Derived Non-HPC and Immune Cells
immature or only partially mature DCs and of-
ten other additional cell types. Since then, re-
finements of protocols (known as “second-
generation” DC expansion protocols) have
been described using several new cytokine/
GM-CSF combinations that include IFN, tu-
mor necrosis factor (TNF), or IL-15.29 One such
cytokine cocktail consists of TNF, IL-1, IL-6,
and prostaglandin 2 for additional DC stimu-
lation. More recently, “third-generation” DC
vaccines have been developed that are polar-
ized toward a cytotoxic immune response (in-
nate and Type 1 immunity) and are currently
being tested in clinical trials.29,30
In-vitro manipulation of DCs includes
loading (pulsing) with tumor recognition mol-
ecules, such as tumor-antigen-derived pep-
tides, recombinant tumor antigens, or tumor-
derived ribonucleic acid or deoxyribonucleic
acid. Furthermore, DC tumor-hybrid vaccines
have been created with the potential to acti-
vate a combined CD4+ and CD8+ T-cell re-
sponse through simultaneous presentation of
several tumor antigens, including a direct fu-
sion of DCs to leukemic blasts, which are easi-
ly obtainable from patients with leukemia.31,32
However, release of suppressive factors, such
as TGF, by the tumor cell fused in the hybrid
may limit the vaccine’s efficacy.31
Cancer Vaccine Approved by Food and
Drug Administration
One of the successes of cellular therapy has
been in the treatment of prostate cancer. A
first-generation DC-based vaccine, sipuleucel-
T (Provenge; APC 8015; Dendreon Corpora-
tion, Seattle, WA), was approved by the US
Food and Drug Administration (FDA) based on
a 4-month improvement in survival compared
to placebo in the pivotal randomized clinical
trial of men with castration-resistant metastat-
ic prostate cancer.33 This is an autologous cel-
lular immunotherapy designed to stimulate a
patient’s own immune system to respond
against the prostate cancer. Sipuleucel-T con-
sists of autologous PBMCs that are enriched
for antigen-presenting DCs that have been ac-
tivated ex vivo with a recombinant fusion pro-
tein, prostatic acid phosphatase (PAP), fused
䡲 791
to GM-CSF. PAP is an immunogenic prostate
antigen whereas GM-CSF is an immune-cell
activator. Each dose is manufactured by ob-
taining a patient’s T cells via leukapheresis and
shipping them to the company to manufacture
the vaccine. After this process, the patient’s
own cells are reinfused into the patient to treat
the prostate cancer. Provenge is administered
intravenously in a three-dose schedule given
at about 2-week intervals.
The effectiveness of Provenge was studied
in 512 patients with metastatic prostate cancer
that was refractory to hormone treatment
in a randomized, double-blind, placebo-
controlled, multicenter trial.33 The median
survival duration for patients receiving
Provenge treatments was 25.8 months com-
pared to 21.7 months for those who did not re-
ceive the treatment.
MSCs
MSCs are a rare subset of cells of nonhemato-
poietic origin that were first discovered in 1968
by Friedenstein as an adherent fibroblast-like
population after isolation from the marrow,
where they represent 0.001% to 0.01% of total
cells.34 It was subsequently shown that MSCs
could be isolated from various tissues, includ-
ing amniotic fluid, adipose tissue, umbilical
cord blood, dental pulp, muscle connective
tissue, and placenta, and could be rapidly ex-
panded in vitro, offering the potential for use
in clinical cellular therapy.35 The rarity of MSCs
has meant that the majority of research to date
has relied on isolating them from the marrow
or adipose tissue before expansion in culture,
a prerequisite for clinical-scale therapies.
MSCs are often referred to as “multipotent”
due to their capacity for differentiation into
cells of mesodermal lineage, including osteo-
blasts, chondrocytes, adipocytes, and, under
specific conditions, several nonmesodermal
cell types, including neurons.36 It is this capac-
ity for multipotency that has generated a great
deal of interest in using MSCs for tissue engi-
neering and regenerative medicine.
Expanded MSC cultures do not all possess
the same level of multipotency. Although a low
frequency of self-renewing progenitors has
792 䡲 AABB TECHNICAL MANUAL
been identified among marrow-derived MSCs,
it remains unclear whether MSCs from other
tissues share these properties. This has result-
ed in the use of the term “multipotent MSCs”
as an alternative to “MSCs” because “multipo-
tent MSCs” does not imply that the cells have
“stem-cell-like” properties or self-renewal
ability without differentiation (Table 33-1).37
The defining characteristics of MSCs have
often differed among investigators, which
prompted the publication of the minimal cri-
teria for the definition of the phenotypic and
functional properties of MSCs by the Interna-
tional Society of Cellular Therapy (ISCT).38 The
ISCT guidelines for defining MSCs are listed in
Table 33-2.
MSCs have been identified as modulators
of several effector functions and immune re-
sponses through their interaction with both
the innate and adaptive immune systems.
They play a key role in the support of hemato-
poiesis, contributing to the maintenance of
the highly specialized microenvironment of
the marrow through the regulation of HSC
numbers and control of their maturation and
differentiation.39 MSCs have been shown to be
capable of exerting a profound immunosup-
pressive effect on both polyclonal and anti-
gen-specific T-cell responses through induc-
tion by cellular or humoral stimuli. They
largely have no immunologic restriction,
meaning that they can be used without HLA
matching.40 The immunoregulatory functions
of MSCs, including the suppression and inhi-
bition of T-, B-, and NK-cell functions and DC
activity, therefore offer a promising option for
the treatment of immune-mediated disorders,
TABLE 33-1. The Multipotentiality of MSCs
Self-Renewal Differentiation
Marrow cavity Mesodermal lineage
Connective stromal cells
Cartilage cells
Fat cells
Bone cells
MSCs = mesenchymal stem cells.
including GVHD, autoimmunity, and solid-or-
gan-transplant rejection. The suppressive
properties displayed by culture-expanded
MSCs are promoted through the expression of
indolamine 2,3-dioxygenase and other effector
molecules, many of which are augmented by
IFN- stimulation.41,42
Isolation and Expansion of MSCs
The isolation and initial expansion of MSCs is
performed in three distinct phases over a peri-
od of 3 to 4 weeks:
1. Isolation and seeding.
2. Cell passaging.
3. Final harvest.
The clinical production of MSCs for ther-
apeutic use is carried out in large tissue-cul-
ture flasks, although MSC production can be
industrialized in 25,000-cm2 culture chambers
or CellSTACKsTM (Sigma-Aldrich Corning, New
York, NY). Marrow aspirates/harvests remain
the most common starting material for the iso-
lation of MSCs. Following harvest, marrow is
collected into preservative-free heparin be-
fore transportation to the laboratory, where
the first step is the isolation of bone-marrow
mononuclear-cell (BMMNC) fraction by light-
density centrifugation. At this stage, MSCs are
isolated either by their ability to adhere to tis-
sue culture plastic, as described below, or are
directly purified using monoclonal antibodies
to CD105, CD146, or CD271 by immunomag-
netic or flow-cytometric sorting.43
Transdifferentiation
Ectodermal lineage Endodermal lineage
Epithelial cells Muscle cells
Neurons Gut epithelial cells
Lung cells
 CHAPTER 33 Blood/Marrow-Derived Non-HPC and Immune Cells
TABLE 33-2. Criteria for Identification of MSCs
䡲 Adherence to tissue culture plastic
䡲 Phenotype (levels of expression)
Positive (95% +) Negative (2% +)
䡲 CD73 䡲 CD45
䡲 CD90 䡲 CD34
䡲 CD105 䡲 CD14
䡲 CD11b
䡲 CD79
䡲 CD19
䡲 HLA-DR
䡲 Demonstration of in-vitro differentiation into
osteoblasts, adipocytes, chondrocytes
MSCs = mesenchymal stem cells.
For isolation by plastic adherence,
BMMNCs are seeded onto tissue-culture flasks
at a density ranging from 0.5 × 105 to 5 × 105
BMMNCs per cm.2 The BMMNCs are cultured
in alpha-modified minimum essential medi-
um supplemented with 10% fetal bovine se-
rum (FBS). Alternative MSC expansion proto-
cols have also been described using human
platelet lysate autologous serum as an alterna-
tive source of serum and growth-factor-sup-
plemented, serum-free media.35
Despite the prescreening of FBS for use in
clinical studies, its application in clinical-
grade cellular therapies still raises concerns
because, theoretically, it is a putative source of
prions and virus transmission. These concerns
make the future use of serum-free media in all
clinical products an attractive option.
The propagation of adherent cells is
maintained by the removal of nonadherent
cells on the third day and their subsequent
feeding every 3 to 5 days with fresh culture me-
dium. Cultures are reviewed daily for:
䡲 Adherence of cells to flasks.
䡲 Shape of adherent cells (round vs fibrocytic
appearance).
䡲 Confluence of cells.
䡲 Amount of debris in medium as an indica-
tion of medium change.
䡲 793
Adherent cells consistently achieve 80%
confluence after 9 to 14 days, at which point
the cells are passaged following enzymatic dis-
sociation from tissue culture flasks with tryp-
sin. Passaging of cell cultures once they have
achieved confluence is often a prerequisite
when an attempt is made to propagate cells in
sufficient quantity for therapeutic use. Howev-
er, continual passage of MSCs can lead to rep-
licative exhaustion and alterations in pheno-
type, which may play a role in modifying their
regenerative and immune-suppressive prop-
erties or potentially limiting their survival and
function in vivo.44 The malignant transforma-
tion potential of MSCs can be eliminated by
reducing cell passaging, although it has also
been reported that MSCs can be safely ex-
panded in vitro until the 25th passage.45 MSCs
are harvested and can be cryopreserved at the
recommended therapeutic dose or adminis-
tered “fresh.”
Immunophenotyping by flow cytometry
is an essential part of the quality assurance/
quality control process of manufacturing us-
ing the cell-surface markers CD73, CD90, and
CD105. Differentiation of MSCs into a specific
lineage often requires the addition of a com-
plex array of growth factors that are selected
based on the desired mature cell phenotype.
Standardization of MSC isolation and ex-
pansion is now gathering pace among the cell
therapy community as MSCs are increasingly
used in clinical trials and manufactured under
GMP conditions. The technology for culturing
large numbers of expanded MSCs for clinical
use has also undergone significant develop-
ment. Devices, such as The Quantum Cell Ex-
pansion System (Terumo BCT, Lakewood, CO),
a closed automated hollow fiber bioreactor
culture system with disposable sets, are now
commercially available and designed to repro-
ducibly grow adherent cells in GMP environ-
ments while minimizing both space and oper-
ator variability.
Clinical Applications of MSCs
The main indications for use of MSCs are in
the treatment of GVHD, Crohn disease, cardio-
vascular disorders, multiple sclerosis, spinal
794 䡲 AABB TECHNICAL MANUAL
cord injury, liver disease, diabetes mellitus,
and various bone and cartilage defects. MSCs
can be used without HLA matching because
they do not induce GVHD and are not rejected
by recipient T cells. These characteristics favor
banking of cryopreserved MSCs from third-
party donors. Such banked MSCs can be re-
leased and shipped as required for multiple
clinical applications as “off-the-shelf” prod-
ucts.
One of the earliest studies that illustrated
the regenerative properties of MSCs involved
their use in treating three children with osteo-
genesis imperfecta. 46 This disease is caused by
a deficiency in the production of Type I colla-
gen and results in defective connective tissue
formation, leading to multiple fractures, bony
deformities, and shortened stature. Following
the infusion of unmanipulated allogeneic
marrow, MSCs from within the graft migrated
to the bone and gave rise to osteoblasts, where
their presence correlated with an improve-
ment in bone structure and function. The
same group of researchers has since modified
their treatment protocol and demonstrated its
safety and efficacy after administering two
doses of marrow-derived, ex-vivo-expanded
MSCs following standard marrow transplanta-
tion in patients with this disease.47
The therapeutic role of MSCs in HSCT has
been largely targeted toward alleviation of
GVHD following transplantation. However, the
codelivery of MSCs at the time of transplanta-
tion has received attention because of their
potential role in graft tolerance and engraft-
ment. Marrow-derived MSCs have been used
successfully for the treatment of steroid-re-
fractory, severe, acute GVHD, for which there
is currently no established definitive therapy.
The clinical benefits of infusing MSCs to treat
GVHD were first demonstrated when haploi-
dentical MSCs were administered in two trans-
fusions to a 9-year-old boy with treatment-re-
sistant, Grade IV acute GVHD of the gut.48 This
has triggered great interest over the past de-
cade, and subsequent Phase I/II trials have
demonstrated both the safety and efficacy of
the use of MSCs in HSCT in the setting of ste-
roid-refractory GVHD.49
Optimal therapeutic doses have not been
defined, although clinical responses have been
documented with infusions of MSCs as low as
0.8 × 105/kg. However, a Phase III, industry-led
clinical trial examining the transfusion of high
doses of MSCs (2 × 106/kg) for the treatment of
steroid-refractory GVHD showed no increase
in overall complete response rate.44
The clinical use of MSCs for tissue regen-
eration has received the most interest for
cardiovascular repair.50 Many trials have had
encouraging results in the improvement of
cardiac function after injection of autologous-
marrow-derived MSCs. Adipose-tissue-derived
MSCs are also currently being investigated as
part of two industry-led clinical trials, APOLLO
and PRECISE, exploring their safety, feasibility,
and efficacy in patients with either acute or
chronic myocardial infarction.51
Although the therapeutic effects of MSCs
in cardiac repair have been acknowledged in
the scientific community, several issues re-
main unresolved, including the optimal dose
and the mechanism of improvement in cardi-
ac function. The differentiation of MSCs into
cardiomyocytes remains controversial, and
the literature in this field is still unclear and
contradictory. There have been a number of
recent publications providing evidence that in
vitro, MSCs may be induced to exhibit cardio-
myocyte-like features, but the exact culture
conditions are not clear. A number of com-
pounds are involved in their generation
through modulation of signaling pathways.
There has been no direct evidence of cardio-
myocyte differentiation in vivo following ad-
ministration of MSCs for cardiac repair, but
functional improvement has been observed.
These observations in preclinical and human
trials have led to the hypothesis that the im-
provements are related to the paracrine ef-
fects of transplanted cells, which induce myo-
cardial repair by releasing signals, including
cytokines and chemokines, into the surround-
ing tissue rather than generation of de-novo
cardiomyocytes.50-52
The multipotency of MSCs has also be-
come attractive to industry, and two commer-
cially driven clinical trials involve third-party
MSCs manufactured by Osiris Therapeutics
 CHAPTER 33 Blood/Marrow-Derived Non-HPC and Immune Cells
Inc. (Columbia, MD) for use in patients with
GVHD or Crohn’s disease and mesenchymal
precursor cells manufactured by Mesoblast
Ltd. (New York, NY) for the treatment of car-
diovascular, orthopedic, and neurologic dis-
ease. Although the clinical efficacy of such
MSC products has yet to be fully evaluated in
many of the proposed disease indications,
their application as cellular therapies contin-
ues to evolve rapidly.
MSCs and Tissue Engineering
The use of MSCs in tissue engineering has de-
veloped rapidly over the last few years with the
advent of scaffolds to improve the outcomes of
stem cell applications by offering more precise
targeting of MSCs combined with the possibil-
ity of making them biodegradable, depending
on the therapy. Scaffolds can be used in a
number of ways: 1) seeding of MSCs onto scaf-
folds in vitro and implantation after a short in-
cubation period, 2) MSC seeding and subse-
quent differentiation into lineage-specific cell
types in short-term culture (1-2 weeks) before
implantation, and 3) induction of differentia-
tion in culture before seeding onto scaffolds
and implantation shortly afterwards.
In 2008, the first fully tissue-engineered
bronchial transplant was reported. 53 In this
case, a nonimmunogenic, decellularized piece
of human donor trachea was reseeded with
autologous marrow-derived MSCs. The MSCs
were differentiated into chondrocytes in vitro
before surgical implantation to replace a bron-
chus that had been previously damaged by tu-
berculosis infection. The paucity of donor or-
gans has limited the use of this approach. As a
result, the use of biosynthetic nanocomposite
material seeded with MSCs has emerged as a
therapeutic option for tracheal replacement.54
IND UCED PLURIP OTE N T ST EM
CELLS
The archetypical pluripotent cell is the embry-
onic stem cell (ESC), which is derived from
embryos and is capable of long-term self-
renewal and differentiation into all cells and
tissues in the human body. The uniqueness of
䡲 795
these capabilities was called into question
when Gurdon55 demonstrated that the nuclei
of differentiated cells actually retain all of the
genetic information in pluripotent stem cells.
The cloning of Dolly the sheep, for example,
showed that the genome of even fully special-
ized cells remains genetically totipotent; that
is, the genome can support the development
of an entire organism.
The next key finding was that manipula-
tion of transcription factor expression can
change a cell’s fate.56 Experiments revealed
that lineage-associated transcription factors
can change cell fate when these factors are ec-
topically expressed in certain heterologous
cells. Lineage-associated transcription factors
help establish and maintain cellular identity
during development by driving the expression
of cell-type-specific genes while suppressing
lineage-inappropriate genes.
The third contributory result was ob-
tained from the Nobel Prize-winning work of
Yamanaka,57 who screened a pool of 24 pluri-
potency-associated candidate genes for fac-
tors. He determined that a core set of four
transcription factors comprising Klf4, Sox2,
c-Myc, and Oct4 was sufficient to produce in-
duced pluripotent stem cells (iPSCs). This re-
search was initially conducted in murine mod-
els, but the approach was also successful with
adult human skin fibroblasts. Many other in-
vestigators since then have demonstrated that
this cocktail of genes can be used to derive
iPSCs from other somatic cell populations, in-
cluding keratinocytes, neural cells, and stom-
ach and liver cells.
Now that iPSCs can be generated, the pri-
mary question is whether iPSCs and ESCs are
molecularly and functionally equivalent.
Comparisons of their genome-wide-expres-
sion patterns and global histone modifications
have demonstrated a high degree of similarity
between ESCs and iPSCs. However, unlike
ESCs, iPSCs are derived from somatic tissues
and therefore do not raise the ethical issues as-
sociated with ESCs.58 The ability to obtain
starting material from skin, blood, and umbili-
cal cord immediately opens up the horizon for
translational clinical applicability.59
796 䡲 AABB TECHNICAL MANUAL
Tumorigenicity
The use of integrating viruses in the genera-
tion of iPSCs raises concern about the poten-
tial for oncogenesis and teratoma formation.
iPSC-derived cells do indeed have a propensi-
ty to form tumors after transplantation, and
human iPSC-derived blood progenitor cells
also appear to undergo premature senes-
cence.60 Their translation to human applica-
tion will need to overcome this safety concern.
Recent approaches to generate iPSCs free of
potentially harmful mutagenic effects have
used either vectors that do not integrate into
the host cell genome or site-specific integrat-
ing vectors to direct the integration away from
known oncogenic sites. Finally, these tech-
niques have been refined so that iPSC genera-
tion no longer requires such integrating vec-
tors.60
Therapeutic Applications
The scope for iPSC-based clinical cell therapy
in the future is bewilderingly vast. The last few
years have seen continual refinements in tech-
niques to allow for large-scale GMP manufac-
turing. At the time of this writing, a new trial
has just received conditional approval in Japan
to use iPSCs to treat age-related macular de-
generation.
In-Vitro Generation of
Hematopoietic Cells
One of the most exciting prospects for using
iPSCs in cellular therapy is that of in-vitro gen-
eration of hematopoietic cells, including
erythrocytes. Current blood transfusion prac-
tice is very safe, but its past has been fraught
with infectious disease transmission. Blood
banks continue to contend with issues of red
cell compatibility and the need to have suffi-
cient donor pools and adequate inventory sys-
tems. These issues can potentially be resolved
with large-scale manufacturing processes us-
ing iPSCs.61
Disease Modeling
One important application of iPSCs is the
modeling of human diseases by generating
specific tissue types of interest followed by the
induction of necessary pathologic changes.
This process essentially replicates the disease
in a Petri dish. However, this approach may be
limited to single-gene disorders, such as sickle
cell disease. For now, it remains unclear
whether multigenic diseases, such as diabetes
or Alzheimer’s disease, are equally amenable
to in-vitro modeling using iPSCs.62
Drug Testing
If disease modeling using iPSCs is successful
and reproducible, these ex-vivo-derived
pathologic tissues can be subjected to phar-
maceutical testing to identify potential new
drugs that are more potent and targeted than
existing drugs.62
In conclusion, iPSCs hold great promise
for regenerative medicine and pharmaceuti-
cal evaluation. They represent a potentially vi-
able source of non-HSCs to treat a wide variety
of diseases. However, the path to final fruition
of their promise is laden with several challeng-
ing hurdles, and work is still necessary to over-
come these difficulties.
REGUL ATO RY AND OV ERSIGHT
ACT IV IT I E S
The cell therapy field has developed along the
same lines as blood banking. Both fields share
the strengths of multidisciplinary teams (phy-
sicians, nurses, laboratory officers, quality
managers, infectious disease experts, and im-
munologists). Both fields also share an em-
phasis on large-scale manufacture, automa-
tion, product release criteria, computer
systems for tracking and inventory, cold
chains for transportation, product traceability,
and monitoring of side effects.
Thus far, the majority of cell therapy
products have been manufactured from sourc-
es from a mixture of blood banks, academic
 CHAPTER 33 Blood/Marrow-Derived Non-HPC and Immune Cells 䡲 797

GMP facilities, and commercial companies.
Harmonization in the terminology of cell-ther-
apy products is being undertaken by the Cellu-
lar Therapy Coding and Labeling Group, a
technical subgroup of ICCBBA that promotes
the use of ISBT 128 (the information technolo-
gy standard for transfusion medicine and
transplantation) labeling. This harmonization
will, of course, facilitate the transportation of
such products across national borders. In ad-
dition, the Alliance for Harmonization of Cell
Therapy Accreditation consists of representa-
tives from a variety of organizations involved
in HSCT and cell therapy. Its primary aim is to
create a single set of quality, safety, and profes-
sional requirements for cellular therapy.
Cell therapy products need to be gov-
erned under suitable regulatory frameworks,
including national ones that cover cells, tis-
sues, and organs. Such national regulatory
frameworks for cell-therapy products are not
in existence worldwide but will be important
as the field develops and the need grows for
cross-border transportation of cells.
Cellular products can be classified as ei-
ther substantially or minimally manipulated.
Substantially manipulated cell-therapy prod-
ucts are treated as biologics. In the United
States, these are known as “351” human cells,
tissues, and cellular and tissue-based products
and are regulated by the Center for Biologics
Evaluation and Research of the FDA. In Eu-
rope, substantially manipulated products are
known as advanced therapeutic medicinal
products (ATMPs). They are manufactured in
accordance with national legislation and over-
sight by the European Medicines Agency.
The question that arises next is how the
issuance and inventories of these biologics will
be managed in the local hospital environment.
The hospital pharmacy is the natural domain
for medicinal products oversight. However, the
unique qualities of cell therapy products ren-
der them more suitable for the sphere of activ-
ity and concern of the blood bank and GMP
processing facility.
Most importantly, the maturation of the
cell-therapy field and its clinical applicability
in many fields of medicine will mean that
more patients will be treated with this novel
therapeutic option. Because living cells are of-
ten administered to patients, short-term as
well as longer-term side effects and potential
toxicities should be systematically monitored.
Such monitoring systems have been in place
for pharmaceuticals (pharmacovigilance pro-
grams) and blood (hemovigilance programs),
and these programs could be extended to cel-
lular therapy products (cellulo-vigilance pro-
grams).
CO N CLUS IO N
The history of cell therapy has seen great ad-
vances of late with the entrance into the mar-
ket of the first FDA-approved therapeutic can-
cer vaccine, Provenge, as well as the dramatic
successes of antigen-specific T-cell therapy in
hematologic diseases. These successes will
certainly spur continuing interest in the devel-
opment of clinical trials. The attraction of an
“off-the shelf,” third-party product is that it
will increase the number of patients who can
be treated and heighten interest in the inter-
national transportation of such products.

KEY POINTS

1. Hematopoietic stem cell transplantation (HSCT) is well established for the treatment of he-
matologic diseases. A graft-vs-tumor (GVT) effect is largely mediated by cytotoxic immune
effector cells, such as T cells and natural killer (NK) cells.
2. Recent technological advances allow good manufacturing practice (GMP)-compliant isola-
tion, characterization, expansion, and modification of immune effector cells for application
as cellular therapeutics in clinics.
3. Donor lymphocyte infusion (DLI) has demonstrated considerable benefit as post-HSCT
treatment in relapsed patients, particularly in chronic myeloid leukemia (CML). These are
798 䡲 AABB TECHNICAL MANUAL

obtained from leukapheresis and constitute mainly T cells. A major side effect is graft-vs-
host disease (GVHD) as a consequence of a healthy host cell attack by alloreactive T cells.
4. NK cells are innate immune cells, do not require antigen priming, and do not induce GVHD.
Current promising developments to improve clinical outcome include treatment with allo-
reactive NK cells, mismatched for their inhibitory receptors, as well as in-vivo or ex-vivo ac-
tivation and expansion of NK cells.
5. Cytokine-induced killer cells (CIK) cells or NK-like T cells are expanded in long-term cul-
tures. Trials with CIK cell therapy have thus far demonstrated good tolerability with a low
risk of GVHD and some efficacy. They may be applied as an alternative to DLI.
6. Dendritic cells (DCs) play a key role in regulating the adaptive immune response to an anti-
gen-specific manner. A DC-based vaccine, sipuleucel-T (Provenge) is approved by the US
Food and Drug Administration as an autologous immunotherapy against prostate cancer.
7. Mesenchymal stem cells (MSCs) possess potent differentiation properties (multipotency)
and have sparked considerable clinical interest. They are generally isolated from marrow or
adipose tissue and expanded in vitro. They can give rise to structural components, such as
bone, cartilage, tendon, and fat, and appear to exert immunosuppressive functions. Indica-
tions for applications of MSCs include GVHD, Crohn disease, cardiovascular disorders, cord
injury, liver disease, diabetes mellitus and bone and cartilage defects.
8. Promising therapeutic effects of autologous MSCs in regenerative cardiac repair have been
reported although the potential for differentiation of MSCs into cardiomyocytes remains
controversial and more results are needed.
9. Third-party MSCs can be applied “off-the-shelf” without HLA matching and are not reject-
ed by recipient T cells.
10. The field of stem cell therapy has been transformed with the discovery that self-induced
pluripotent stem cells (iPSCs) can be generated from differentiated cells, including kerati-
nocytes, neural cells, and stomach and liver cells. The use of iPSCs involves considerably
fewer ethical issues compared to that of embryonic stem cells.
11. iPSCs can potentially be applied in a wide range of medical conditions and for in-vitro gen-
eration of hematopoietic cells. Techniques for large-scale GMP-compliant manufacturing
of iPSCs are being developed.
12. Cellular products are classified as either minimally or substantially manipulated, with a
more stringent regulatory framework for production of the latter in the US and Europe.

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 Index
Page numbers in italics
refer to figures or tables
A in transfusion reaction evaluation, 672
A antigen, 292-293, 295, 302, 457
A(B) phenotype, 296, 298
ABO compatibility
of apheresis platelets, 169
of Cryoprecipitated AHF, 375, 554, 583
of granulocytes, 173, 375, 525, 554, 584
in hemolytic transfusion reactions, 673
of HSC transplants, 632-633, 635, 716
of organ transplants, 491, 492, 783
of plasma, 375, 379, 554, 583, 635
of platelets, 375, 457, 513-514, 554
after HSCT transplantation, 635, 636
in hemolytic transfusion reactions, 675
in pediatric patients, 581, 589
of RBCs, 375, 378-379, 504, 554, 635
of tissue transplants, 777
of Whole Blood, 375
ABO discrepancies, 296, 298-301
ABO hemolytic disease of the fetus and
newborn, 566-567
ABO system, 291-301
acquired B phenotype, 296-297, 298
antibodies of
anti-A and anti-B, 293, 297
anti-A1, 297, 301
anti-A,B, 297
antigens of, 291, 292-293, 457
B(A) and A(B) phenotypes, 296, 298
biochemistry of, 292-293, 294, 302
in development and aging, 293
genetics of, 259, 266, 273, 293-295
O alleles, 295
phenotypes of, 292
subgroups of, 292-293, 295-296, 298, 300
ABO testing
of blood components, 225-226, 297-298, 378
with cold autoagglutinins, 300, 301, 438
comparison with previous records, 378
discrepancies in, 296, 298-301
hemolysis in, 297
interpretation of, 292, 296
for organ transplantation, 783
in pediatric patients, 385, 574, 587
in prenatal studies, 563
reagents for, 298
of recipients, 297-298, 375
of umbilical cord blood, 736
with warm autoagglutinins, 432
ABTI antigen, 361
Accidents, 45
Accreditation, 90, 91, 603-604, 698
ACE inhibitors. See Angiotensin-converting
enzyme inhibitors
Acid-elution stain (Kleihauer-Betke), 565-566
Acid elutions, 429
Acquired B phenotype, 296-297, 298
Activated partial thromboplastin time, 519
Activated protein C, 532
Acute disseminated encephalomyelitis, 653
Acute lung injury, 680. See also Transfusion-
related acute lung injury
Acute normovolemic hemodilution, 606
ADAMTS-13, 652-653
Additive solutions, 136-137, 138, 576-577
Additives, in serologic testing, 376-377
Adipose-derived stem cells, 755, 758-759, 762,
794
Adsorption, 407-408
allogeneic, 433-434
autologous cold, 438-439
autologous warm, 432-433
and elution, 409
Adverse reactions
to apheresis, 169, 170, 658-660, 720
in blood donors, 20, 22, 142-146
to donor lymphocyte infusions, 787
to G-CSF, 719-720
to HSC infusions, 724-725, 742
to IVIG, 529, 532
management of, 20-23
related to medical devices, 97
related to tissue allografts, 782
to transfusion (See Transfusion reactions)
transfusion-transmitted diseases (See
Transfusion-transmitted diseases)
AET. See 2-aminoethylisothiouronium
bromide
Age
of blood samples, 370-371, 410-411, 417
803
804 䡲 AABB TECHNICAL MANUAL

of donors, 119, 120 to platelet antigens, 515-516, 567-568, 637,

effect on antigens and antibodies, 293, 300,
410
of RBC units, 574, 577-578
Agglutination
in antiglobulin testing, 372
assays utilizing, 241-242, 279-280
interpreting and grading reactions, 372
mixed-field, 278, 298
principles of, 371
spontaneous
in ABO testing, 298, 300, 301
dispersing with sulfhydryl reagents, 432,
438
in Rh testing, 332
Agreements, 6-7, 9-10, 108
AHG serum, 372
AIDS, 124, 125, 182
Air embolism, 669, 685
Alarm systems, 36, 214
Albumin, bovine (reagent), 376, 417
Albumin solutions (colloid), 526, 647, 652
Aliquoting components, 224, 575-576, 583
Alleles, 262-264
defined, 256
frequencies of, 274, 275
polymorphic, 264
terminology for, 279, 280
Allergic reactions
to apheresis, 658
in HSCT patients, 639
to latex, 45
to transfusions, 547-548, 667-668, 678-680
Alloantibodies, 391-392. See also Antibodies
Allogeneic adsorption, 433-434
Allogeneic HSC transplantation, 715-718. See
also Hematopoietic stem cell
transplantation
Allografts, 774. See also Tissue
Alloimmunization. See also Antibodies
in delayed hemolytic transfusion reactions,
686
in hemolytic disease of the fetus and
newborn, 562
HLA
in HSC transplant recipients, 637, 639-
640, 716
management of, 670
in organ transplant recipients, 488-489
and plasma transfusions, 151
in platelet refractoriness, 458, 459-461,
488-489, 515-516
in TRALI, 489, 681, 682
in transfusion reactions, 489, 490, 677
689-690
prevention of, 329-330, 515
red cell, 391, 670
in sickle cell disease, 283, 329-330, 379, 588,
639
1-antitrypsin, 532
1-proteinase inhibitor, 532
American Rare Donor Program, 421
Aminocaproic acid, 608, 621-622
2-aminoethylisothiouronium bromide (AET),
301, 405, 407
Amniotic fluid-derived MSCs, 759-760
Amotosalen-UV treated plasma, 153, 205
Anaphylactic reactions, 658, 668, 678-679, 680
Anemia
acute, 500-502
and bleeding prevention, 508-509
chronic, 502, 610
defined, 604
in delayed transfusion reactions, 686
fetal (See Hemolytic disease of the fetus and
newborn)
hemolytic (See Hemolytic anemia)
in HSC transplantation, 634
iatrogenic, 609
in infants, 571-572, 578-579
iron deficiency, 605, 620
management of, 605
preoperative, 604-605
RBC transfusions in, 500-502
screening donors for, 121
signs and symptoms of, 604
tolerance, 609-610
Anesthesia, 607
Angioedema, 678
Angiotensin-converting enzyme inhibitors,
659, 669, 678, 679
Ankylosing spondylitis, 493-494
Antibiotics, in donors, 122, 129
Antibodies
autoreactive (See Autoantibodies)
detection of (See Antibody detection)
drug-induced, 392, 440-444, 465-466, 516
to Factor VIII, 527
granulocyte, 466-469
HLA (See HLA antibodies)
identification of (See Antibody
identification)
platelet (See Platelet antibodies)
to reagent components, 298, 300, 417
red cell (See Red cell antibodies)
structure of, 246-248
Antibody-dependent cellular cytotoxicity, 419

Antibody detection, 375-377
with autoantibodies, 432-435, 438-439
by ELISA, 243-244
frequency of testing, 419
of granulocyte antibodies, 469
of HLA antibodies, 460, 487
interpreting results of, 381, 382
methods for, 376-378
in pediatric patients, 385, 574-575, 587
of platelet antibodies, 243, 456, 463-464,
465-466
in prenatal evaluations, 563
reagents for, 376-377, 393, 396
with rouleaux present, 416
solid-phase assays for, 243
specimen requirements for, 370-371
Antibody identification
anomalous reactions in, 416-417
of antibodies to high-prevalence antigens,
394-395, 415-416
of antibodies to low-prevalence antigens,
416
with antibodies to reagent components,
417
with autoantibodies, 432-435
autologous control in, 400, 403-404, 417-
419
complex problems, 402, 403-404
exclusion (“rule-out”) in, 398
factors affecting, 410-411
frequency of testing, 419
immunohematology reference labs for, 419
interpreting results of, 397
with multiple antibodies, 408, 410, 412, 414
with no discernible specificity, 411-412
positive and negative reactions in, 397-398
with positive DAT, 401, 404, 415-416, 417-
419
preanalytical considerations for, 392-393
in prenatal evaluations, 563
probability values in, 400
reagents for, 393, 396
and selection of blood, 274, 379, 419-421
specimen requirements for, 393
test methods for, 396
adsorption, 407-408
alteration in pH, 406
combined adsorption-elution, 409
dispersing autoagglutination, 438
elution, 408-409
enzymes, 402, 405
flowcharts for, 403-404
identification panels, 393, 396-400
inactivation of antigens, 405, 406-407
increased incubation time, 405
INDEX 䡲 805
increased serum-to-cell ratio, 405
inhibition techniques, 406
LISS and PEG, 402
phenotyping autologous red cells, 401-
402
selected cells, 397, 398
temperature reduction, 405
titration studies, 409-410, 563
variations in antigen expression in, 410,
411-412
Antibody screen. See Antibody detection
Antibody specificity prediction method, 460
Anticoagulant medications
in blood donors, 129
reversal of, 517-518, 519, 622-623
Anticoagulant-preservative solutions, 136, 137
Anticoagulation, in apheresis, 658
Antifibrinolytic agents, 522, 608, 621-622
Antigen capture assays, 464-465
Antigen-matching, 281, 329-330, 379, 588, 654
Antigens. See also specific blood groups
acquired, 296-297, 298
antithetical, 263-264
of blood group collections, 361-362
of blood group systems, 278-279
chromosomal location of, 259-261
granulocyte, 466-468
high-prevalence, 361-362, 415-416, 421
HLA, 475-476, 480-483
inactivation of, 405, 406-407
low-prevalence, 362, 416
methods for detecting, 241-243, 244-246,
279-285
platelet, 243, 453-458
prevalence of, 274
terminology for, 278-279, 280
variations in expression of, 410
Antiglobulin test, 371-372
in crossmatching, 380
direct, 372, 425-430
indirect, 372
reagents for, 372, 396
sources of error in, 373-374
use of IgG-coated cells, 376
Antihistamines, 548, 679-680
Antiplatelet agents, 122, 129, 169, 509
Antipyretics, 547
Antithrombin, 531
Apheresis
complications of, 169, 658-660, 720
component collection by, 167-176
adverse donor reactions in, 169, 170
donation requirements for, 121, 122,
123, 168-169, 171
granulocytes, 168, 172-173, 175-176
806 䡲 AABB TECHNICAL MANUAL

instrumentation for, 168, 173-176 warm

multicomponent donations, 171-172, 174 ABO testing with, 432
plasma, 168, 170, 173-174 adsorption of, 432-435
platelets, 168-170, 174-175 with alloantibodies, 418-419, 432-435,
quality control of, 171 438-439
RBCs, 168, 171-172, 175 antibody identification with, 418-419
HSCs collected by (See Peripheral blood disease associations with, 392
HSCs) mimicking alloantibodies, 434-435
records of, 170, 172 in mixed-type AIHA, 439
therapeutic (See Therapeutic apheresis) in phenotyping problems, 401
Aplastic anemia, 640 Rh testing with, 332, 432
Arterial puncture, in donors, 145 specificity of, 435
Arthritis, 763 transfusion with, 436-437, 506
Aspirin, 122, 169, 509 in warm AIHA, 431-435
Assessments Autoclaving biohazardous waste, 55
of blood utilization, 24-25, 611, 697-707 Autografts, 774, 782-783. See also Tissue
competency, 7-8 Autoimmune hemolytic anemia
external, 25, 86-87 classification of, 430
internal, 24 cold agglutinin disease, 437-439
management of, 13, 23-26 DAT-negative, 437
proficiency testing, 25-26, 91 mixed-type, 439-440
quality indicators, 24 paroxysmal cold hemoglobinuria, 440
risk, 98-99, 102 serologic findings in, 431
Audits, transfusion, 25 transfusion in, 436-437, 439-440, 506
concurrent, 699, 700, 703, 706 warm, 430-437
prospective, 611, 699-700, 701-702, 706 Autoimmune neutropenia, 468
retrospective, 699, 701-702, 703-704, 707 Autoimmune thrombocytopenic purpura, 463,
selecting a process for, 705 465, 517, 568
Auto control. See Autologous control Autologous adsorption, 432-433, 438-439
Autoadsorption. See Adsorption Autologous blood
Autoagglutination collection of
in ABO typing, 298, 299, 300, 301 by acute normovolemic hemodilution,
dispersing with sulfhydryl reagents, 432, 606
438 by intraoperative blood recovery, 606-
in Rh testing, 332 607
Autoantibodies low-volume units, 141
cold by preoperative blood donation, 605-606
in ABO testing, 300, 301, 438 donor selection for, 120, 131
adsorption of, 438-439 infectious disease testing of, 190-191
antibody identification with, 418 for rare phenotypes, 421
in cold agglutinin disease, 308-309, 437- separation from transfused cells, 401
439 Autologous control
in mixed AIHA, 439 in antibody identification, 400, 403-404
in paroxysmal cold hemoglobinuria, 440 positive, 404, 417-419
in Rh testing, 332, 438 in pretransfusion testing, 377, 382
use of sulfhydryl reagents with, 438 Autologous HSC transplantation, 638, 714-
in warm autoimmune hemolytic 715, 718-719. See also Hematopoietic stem
anemia, 431 cell transplantation
defined, 391 Automated testing systems, 378
disease associations with, 392 Autosomal inheritance, 265-267
distinguishing from alloantibodies, 283
drug-induced, 443 B
low-affinity, 437 B antigen, 292-293, 302
in phenotyping problems, 401 acquired, 296-297, 298
platelet, 461, 465, 568 on platelets, 457

B(A) phenotype, 296, 298
Babesiosis, 126, 201
Bacterial contamination, 198-199
detecting, 198-199
inspecting components for, 149, 224
during phlebotomy, 141, 198
of platelets, 150, 198-199, 221
of RBCs, 198
reactive testing results for, 188
of tissue allografts, 777, 782
in transfusion-associated sepsis, 669,
676
Band 3 glycoprotein, 352-353
Bernard Soulier syndrome, 456
Bg (Bennett-Goodspeed) antigens, 362, 480,
490
Bilirubin, 562, 564, 579
Bioassays, for radiation monitoring, 61
Biocontamination control system, 41
Biohazardous waste, 53-55, 108
Biological product deviations, 22, 89, 90
Biological products, regulations for, 84, 85
Biological response modifiers, 681
Biological safety cabinets, 49, 50-51
Biologics license applications, 735, 744, 745,
746
Biosafety, 47-55
biological safety cabinets for, 49, 50-51
biosafety levels, 48, 73
Blood-borne Pathogens Standard, 47
decontamination for, 49, 52
in donor room, 53
emergency response plan for, 53
engineering controls for, 49-52
hazard identification and communication
for, 48-49
in laboratory, 53
personal protective equipment for, 52
safe work practices for, 52-53
standard precautions, 48
storage of biohazards, 52, 54-55
training for, 48
waste management, 53-55
Biovigilance. See Hemovigilance
Bleeding
acute, transfusion for, 501-502
and anemia, 508-509
assessing risk of, 518-521, 605
microvascular, 684
Bleeding disorders. See Coagulopathy
Blood administration
baseline assessment of recipient, 546
blood warmers for, 548, 572, 584, 685-686
delays in starting transfusion, 550
delivering blood to patient area, 549-550
INDEX 䡲 807
emergency equipment for, 549
emergency release, 158, 227-228, 385-386,
506, 555-556
errors in, 551, 675
filters for, 551-552, 554, 585
identification procedures in, 550-551
infusion pumps for, 548-549, 584
infusion rates for, 553, 554, 585
infusion sets for, 551-552, 585
IV solutions for, 552
medical history in, 546
medical order for, 546-547, 551, 699-700,
703-704
monitoring patient in, 553, 555
in neonates, 584-585
out-of-hospital, 556
patient consent in, 545-546, 601
patient education in, 546
post-administration events, 555
premedications for, 547-548, 677, 679-680
pressure devices for, 549
starting the transfusion, 552-553
for surgery and trauma, 555-556
syringe infusion pumps for, 549
transfusion reactions in, 553, 555, 666
venous access for, 547, 580, 584
Blood-borne Pathogens Standard, 47, 53
Blood clots, in RBCs, 149
Blood collection
additive solutions for, 136-137, 138, 576-
577
adverse donor reactions in, 20, 22, 142-
146
anticoagulant-preservative solutions for,
136, 137
autologous, 605-606
blood containers for, 136-139, 140
of blood samples, 141, 368, 370, 384
of components by apheresis, 167-176
in disaster planning, 100-102
donation intervals for, 120, 122
donor and component identification in,
139-140
donor care after, 142
equipment quality control, 38
mobile sites for, 41
phlebotomy in, 140-141
process of, 141
safety of, 41, 53
of umbilical cord blood, 733-734
volume collected, 120, 141-142, 143
Blood component selection
ABO/Rh compatibility in, 375, 378-379, 504,
513-515, 554, 635
after non-group-specific transfusions, 396
808 䡲 AABB TECHNICAL MANUAL
with clinically significant antibodies, 274,
379, 419-421
for exchange transfusions, 574, 579-580
for HSC transplantation, 633-634
for intrauterine transfusions, 564
for massive transfusions, 386
of platelets, 375, 459-461, 513-514, 581-582
rare types, 421
of red cell components, 375, 378-379, 504
for red cell exchange, 654
in urgent situations, 228, 385-386
in warm autoimmune hemolytic anemia,
435-436
Blood components. See also specific
components
administration of (See Blood
administration)
aliquoting, 224, 575-576, 583
bacterial contamination of, 198-199, 224
CMV-reduced-risk, 190, 525, 564, 639
costs of, 602
cryopreservation of, 155
density of, 143
expiration of, 213, 215-220, 551
identification of, 139-140, 226, 384-385,
549, 550, 551
inspection of, 148, 149, 224, 226, 385, 549,
550-551
irradiated, 155-156, 222, 590, 688, 689
issuing, 226, 384-385, 549-550
labeling, 139, 158-159, 226, 384-385
leukocyte reduction of, 154-155, 222-223,
504-505, 590
monitoring use of, 697-707
ordering, 226, 367-368, 546, 699-700, 703-
704
pooling, 156-157, 223-224
preparation and processing of, 146-148,
221-224
quality control of, 159-160
quarantine of, 157-158, 189, 224
rare, 421
receiving into inventory, 225
records of, 384-385
regulations regarding, 83
retrieval of prior donations, 187-188, 189-
190
return and reissue of, 226-227, 550
selection of (See Blood component
selection)
shortages of, 101-102
storage of, 213-214, 215-220, 221, 503-504
testing
ABO and Rh, 225-226, 297-298, 378
infectious disease, 182-191, 192-204
phenotyping, 281, 329-330, 379, 420,
588, 654
traceability of, 225
transporting, 100-101, 146-147, 213-214,
215-220, 225
volume reduction of, 157, 223, 513-514,
581-582, 590
washed, 223, 590-591, 639
Blood containers
additive solutions in, 136-137, 138
for aliquots, 576
anticoagulant-preservative solutions in,
136, 137
configurations of, 136-137, 139
diversion pouch on, 139, 141
modification by sterile connecting device,
139, 140
properties of, 136
Blood donation. See Blood collection; Donors
Blood exposure, 44-45, 47-48, 49, 53, 124
Blood group genomics, 278-287
for antigen-negative blood donors, 285
to confirm D type of donors, 285
discrepancies between phenotype and
genotype, 240, 285-287, 402
to distinguish alloantibody from
autoantibody, 283
to predict phenotype of recently transfused
patients, 240, 281
to predict phenotype when red cells are
coated with IgG, 283, 401-402, 436
in prenatal practice, 283-285
Blood group systems. See also specific blood
groups
clinical significance of antibodies in, 338-
340, 413-414
defined, 279
genetics of, 255-279
chimerism, 278
gene mapping, 259-261, 277-278
inheritance patterns, 265-273
population genetics, 274-275
principles of, 256-258, 261-264
relationship testing, 276-277
terminology for, 278-279, 280
Blood loss
phlebotomy-related, 609
signs and symptoms of, 501
transfusion for, 501-502
Blood management. See Patient blood
management
Blood order schedules, 227
Blood pressure
of donors, 120
hypotension
 INDEX 䡲 809

during apheresis, 659 donor lymphocyte infusions for, 786-

associated with ACE inhibitors, 659, 669, 787, 788
678 natural killer cells in, 788
deliberate, 607 platelet transfusions in, 507
in recipients, 674, 679 prostate, 791
Blood pressure cuffs, 38 Carboprost, 626
Blood recovery, 606-607, 608-609 Cardiac patients
Blood samples platelet transfusions in, 509
age of, 370-371, 410-411, 417 red cell transfusions in, 500-501
from arterial or central lines, 609 treatment with MSCs, 763, 794
collection of, 141, 368, 370, 384 Carriers, of traits, 261
confirming linkage with blood requests, Cash reserves, 104
370 Catheters
for hemoglobin/hematocrit screening, 121 for apheresis, 660
hemolyzed, 370 drawing specimens from, 609
incompletely clotted, 370 for neonates, 580, 584
labeling, 370, 547 for transfusions, 547
lipemic, 370 Cause-and-effect diagrams, 27, 28
for PCR, 235, 236 CCI. See Corrected platelet count increment
for pretransfusion testing, 368, 370-371, CD11a/CD11b, 468
393, 547 CD34 cells, 723, 736
retention and storage of, 371 CD36, 458
transportation and shipment of, 63 CD59, 361
Blood spills, 53, 54 CD109, 456-457
Blood transfusions. See Transfusions CD177, 466-467, 469
Blood utilization review, 25, 697-707 Cefotetan, 442
interventions to change transfusion Ceftriaxone, 442
practice, 610-611, 699, 704-705 Cell counters, 38
rationale for, 698-699 Cell counts, of HSCs, 723, 736
selecting audit process, 705 Cell division, 258, 262, 263
types of audits Cell washers, 36
concurrent, 699, 700, 703, 706 Cellular immunotherapy
prospective, 611, 699-700, 701-702, 706 with cytokine-induced killer cells, 789-790
retrospective, 699, 701-702, 703-704, 707 with dendritic cells, 790-791
Blood volume, of pediatric patients, 572 donor lymphocyte infusions, 786-788
Blood warmers, 37, 548, 572, 584, 685-686 with induced pluripotent stem cells, 795-796
Bombay phenotype, 292, 293, 303 with natural killer cells, 788-789
Bone grafts, 123, 775 regulation and oversight of, 796-797
Bone marrow. See Marrow Centrifugation, in component preparation,
Bovine serum, 764-765, 793 147-148
Brain natriuretic peptide, 682 Centrifuges, 36, 38
Bruising, in donors, 145 Centromere, 257
Buerger disease, 763 Cephalosporins, 442, 443
Buffy-coat concentration of marrow, 721 Ceppellini effect, 321, 323
Buffy-coat method of platelet preparation, Cerebral diseases, 763
146, 147, 150, 156-157 cGMP. See Good manufacturing practice
cGTP. See Good tissue practice
C Chagas’ disease, 126, 200-201
C1-esterase inhibitor deficiency, 522 Change control, 33
Calcium supplementation, 658, 670, 683 Charts
Calibration, equipment, 10, 11, 33 Pareto, 27, 29
Cancer pedigree, 265, 266
in blood donors, 126, 128 process flow, 27
leukemia run and control, 24, 33
cytapheresis in, 654 Check cells, 376
810 䡲 AABB TECHNICAL MANUAL
Chemical hygiene plan, 55
Chemical/organic solvent elutions, 429
Chemical safety, 55-60
chemical categories, 57, 76-77
chemicals found in blood banks, 74-75
emergency response plan for, 59, 78-82
engineering controls for, 58
hazard identification and communication,
56-58
personal protective equipment for, 58
safe work practices for, 58-59
training for, 56
waste disposal, 60
Chemiluminescence assays, 419
Chemotherapy, 720
Chido/Rodgers system, 260, 338, 356-357, 406
Chido substance, 406
Chikungunya virus, 202
Children. See Neonates; Pediatric patients
Chimeric antigen receptors, 788
Chimerism, 278, 298, 489-490
Chlamydia trachomatis, 191
Chloroquine diphosphate, 407
Choline transporter-like protein 2, 467-468
Chromatids, 257, 258
Chromosomes, 256-258, 259-261, 277-278. See
also Genes
Chronic granulomatous disease, 348-349, 524,
525
Circular of Information for the Use of Human
Blood and Blood Components, 158-159
Circulatory overload, transfusion-associated,
669, 682-683
Cirrhosis, 518-519, 764
Cis position, 272, 329
Citrate toxicity, 658, 670, 683
CJD. See Creutzfeldt-Jakob disease
Clean rooms, 40-41
Cleaning and decontamination, 49, 52
Clinical Laboratory Improvement
Amendments (CLIA), 89-90
Clocks, 37
Clonogenic assays, 723, 736
Clotting factors. See Coagulation factors
CMV. See Cytomegalovirus
Coagulation factors
concentrates, 124, 126, 526-527, 637-638
in Cryoprecipitated AHF, 153-154, 522-523
half-lives of, 521
and INR, 520
in neonates, 582
replacement of, 524
in Thawed Plasma, 221
Coagulopathy
in apheresis, 659
in blood donors, 126, 128-129
in hemolytic transfusion reactions, 674
in massive transfusion, 591, 684-685
in neonates, 582-583
plasma transfusions for, 517-522
Codominant inheritance, 265
Cold agglutinin disease, 437-439
antibody detection in, 438-439
serologic findings in, 431, 437-438
specificity of autoantibodies in, 308-309,
392, 439
Cold autoadsorption, 438-439
Cold autoantibodies
ABO typing with, 300, 301, 438
adsorption of, 438-439
antibody identification with, 418
in cold agglutinin syndrome, 308-309, 437-
439
in mixed AIHA, 439
in paroxysmal cold hemoglobinuria, 440
phenotyping with, 401
in Rh testing, 332, 438
use of sulfhydryl reagents with, 438
in warm autoimmune hemolytic anemia,
431
Cold-reactive alloantibodies, 300, 301
Cold stress. See Hypothermia
Collections, blood group, 361-362
Colloid solutions, 526
Colony-forming cell assays, 723, 736
Colton system, 260, 339, 355
Column agglutination technology, 377, 396
Committee, for transfusion oversight, 24-25
Communications, emergency plans for, 105-
107
Compatibility testing. See Pretransfusion
testing
Competency assessments, 7-8
Complement
in acute transfusion reactions, 673-674
in autoimmune hemolytic anemia, 431,
437
role in immunity, 248-250
Complications. See Adverse reactions
Complotype, 478
Computer crossmatch, 380-381
Computer systems
alternative systems for, 20
backup of, 18, 19-20
management of, 19-20
security of, 19
validation of, 15-16
Computerized provider order entry, 611, 699,
700
Concurrent audits, 699, 700, 703, 706
 INDEX 䡲 811

Confidentiality of information, 19 Cordocentesis, 563-564, 567, 568

Consanguineous mating, 266 Corrected platelet count increment, 458, 512,
Consent 516
for apheresis, 170 Corrective action, 26, 27
for blood donation, 119 Corticosteroids, 172, 548
for cord blood donation, 731 Cost collection, 361
for donation of HSCs, 718 Costs, health-care, 602
for transfusion, 545-546, 601 Counter-flow centrifugal elutriation, 721-722
Contact lists, in disaster planning, 105 CPOE. See Computerized provider order entry
Containers Creutzfeldt-Jakob disease, 202-203
for blood collection, 136-139, 140 and plasma derivatives, 526
for MSC storage, 761 screening donors for, 125, 126
for shipping transmission through transplantation, 777
dry shippers, 724, 738, 739 variant, 125, 202-203
HSCs, 724-725, 738 Crohn’s disease, 764
quality control intervals for, 38 Cromer system, 260, 339, 357-358
temperature of, 724-725, 734, 738, 739, Cross-reactive groups, 482
779-780 Crossing-over, gene, 270-271, 479
validation of, 147, 225 Crossmatch-to-transfusion ratios, 227, 381
Contamination Crossmatching, 379-381
bacterial, 198-199 with alloantibodies, 420-421
detecting, 198-199 antiglobulin test in, 380
inspecting components for, 149, 224 computer, 380-381
during phlebotomy, 141, 198 HLA (lymphocyte), 487, 491-492
of platelets, 150, 198-199, 221 immediate-spin, 380
of RBCs, 198 “in-vivo,” 419, 506-507
reactive testing results for, 188 interpretation of results, 381, 382
of tissue allografts, 777, 782 in pediatric recipients, 385, 574-575
in transfusion-associated sepsis, 669, 676 platelet components, 460-461, 489, 516
fungal, 777, 782 Cryoprecipitate Reduced Plasma, 152, 219-
in PCR, 236 220, 652
Continuity of Operations Plan, 102-109 Cryoprecipitated AHF, 153-154
alternate facilities in, 103-104 ABO compatibility of, 375, 554, 583
cash reserves in, 104 coagulation factors in, 153-154, 522-523
decision trees in, 103 dose of, 523
elements of, 103 expiration of, 157, 218, 222
emergency communications plan in, 105- in HSC transplantation, 637
107 indications for, 522-523
essential functions in, 103 infusion of, 554
insurance in, 104 for pediatric patients, 578, 583-584
logistics in, 107-108 pooled, 157, 218, 222, 224, 523
security and safety in, 104 preparation of, 153
staffing in, 108-109 storage of, 153, 218, 222
utilities in, 108 thawing, 222
vital records in, 104 topical application of, 523
Contracts, 9-10, 108 transportation and shipping of, 218
Control charts, 24, 33 Cryopreservation
Control process, 3 agents for, 155, 722
Controls of HSCs, 722-723
autologous, 377, 382, 400, 403-404, 417-419 of platelets, 155
IgG-coated cells, 376 of RBCs, 155
for Rh typing reagents, 331-332 of tissue allografts, 775
Copper sulfate, 38, 64, 121 Crystalloids, 501-502, 526, 652
Cord blood. See Umbilical cord blood CTL2, 467-468
Cord blood banks, 729-730, 742-746 Cytapheresis, 653-654, 655
812 䡲 AABB TECHNICAL MANUAL
Cytokine-induced killer cells, 789-790
Cytomegalovirus, 190
and CTL infusions, 788
in neonates, 589-590
preventing with leukocyte reduction, 190,
590, 639
screening donors for, 191
Cytomegalovirus-reduced-risk products, 190,
525, 564, 590, 639
Cytotoxic T lymphocytes, 787-788
D for cryopreservation of products, 155, 722,
D antigen, 323-328
antibody to (anti-D)
clinical significance of, 338
in HDFN, 338, 562
and partial D, 328
passively acquired, 566
and platelet transfusions, 515
in tissue transplantation, 777
titrations of, 563
and weak D, 328
clinical considerations for, 328
D epitopes on RHCE, 325, 326
D-negative, 321, 327
D-positive, 323-325, 327
elevated D, 325, 327
in fetus, 283-284
nonfunctional RHD, 325
partial D, 324, 326, 327, 328, 333
in recipients, 327-328, 375
testing for (See Rh testing)
weak D, 323-324
in donors, 285, 327, 328
in fetus, 565
in prenatal patients, 565
in recipients, 327-328, 375
DARC glycoprotein, 349-351
DAT. See Direct antiglobulin test
DAT-negative autoimmune hemolytic
anemia, 437
DDAVP. See Desmopressin
Decontamination procedures, 49, 52,
55
Deep vein thrombosis, in donors, 145
Deferasirox, 690
Deferiprone, 690
Deferoxamine, 690
Deferrals. See Donor deferrals
Deglycerolization, 155, 222
Delayed transfusion reactions
hemolytic, 250, 588, 670, 686-687
management of, 670-671
serologic, 686, 687
Dendritic cells, 785, 790-791
Dengue virus, 202
Density, of blood cells and components, 143
Derivatives. See Plasma derivatives
Design output, 3, 33
Designated donations, 130
Desmopressin, 624
Dexamethasone, 525
DIC. See Disseminated intravascular
coagulation
Diego system, 259, 338, 352-354
Dimethyl sulfoxide (DMSO)
761
functional effect on HSCs, 741
toxicity to, 724, 764-765
2,3-Diphosphoglycerate (2,3-DPG), 574
Diploid, defined, 258
Direct antiglobulin test, 425-430
false positive/negative results in, 373-374
method for, 426-427
positive test
after HSC transplantation, 428
in antibody identification, 401, 404, 415-
416, 417-419
causes of, 426
in cold agglutinin syndrome, 431
drug-induced, 428, 440-444, 448-451
elution with, 428-430
evaluation of, 427-430
medical history in, 427-428
in mixed-type AIHA, 431, 439
in paroxysmal cold hemoglobinuria,
431, 440
predicting phenotype with, 283
in warm autoimmune hemolytic
anemia, 431
in pretransfusion testing, 372, 377
principles of, 426-427
reagents for, 372, 426-427
specimens for, 427
in transfusion reaction evaluation, 426, 672
Direct thrombin inhibitors, 518
Directed donations, 131
Disaster management
for blood and transfusion services, 100-102
business operations planning in, 102-109
cycle of, 98
emergency management agencies in, 99-100
lessons learned from recent disasters, 112
overview of, 97
planning team in, 102
regulatory issues in, 109-111
risk assessment in, 98-99
testing disaster plans, 111-112
Disease modeling, 796

Disinfectants
to clean work surfaces, 49, 52
for venipuncture, 140-141, 198
Disposal, waste, 54-55, 60, 63, 108
Disseminated intravascular coagulation, 674-
675
Dithiothreitol (DTT)
to disperse autoagglutination, 301, 432, 438
for inactivating blood group antigens, 405,
407, 413-414
Diversion pouches, 139, 141, 198
DMSO. See Dimethyl sulfoxide
DNA, 232-233, 237
DNA-based assays. See Nucleic acid analysis
Documents, 13, 16-18. See also Records
Dolichos biflorus lectin, 295, 301
Dombrock system, 260, 339, 354-355
Donath-Landsteiner test, 312, 440
Donation identification number, 135, 139-140,
159, 551
Donor deferrals
for allogeneic donors, 122-126
for bleeding conditions or blood diseases,
126, 128-129
blood donation intervals for, 122, 123, 169, 170
for blood exposure, 124
for blood transfusions, 123, 125
for bone, skin or dura mater grafts, 123, 126
for cancer, 126, 128
for clotting factor concentrates, 126
developing criteria for, 127-130
during disasters, 101, 110
for drugs taken by donor, 122, 125, 129-130,
169
for heart and lung conditions, 126, 129
for hemoglobin/hematocrit, 121
for incarceration, 125
for infectious diseases, 124-126
for needlesticks, 124
for piercings, 124
for pregnancy, 122
for reactive infectious screening tests, 187-
188, 189
records of, 118, 119
regarding sexual contacts, 124, 125
for tattoos, 124
for transplants, 123
for travel outside the US or Canada, 125,
126
for vaccinations or shots, 123
Donor History Questionnaire, 121-127, 130
Donor lymphocyte infusion, 786-788
Donor room, biosafety in, 53
Donor selection
for autologous donations, 118, 131
INDEX 䡲 813
blood-center-defined criteria for, 127-130
consent of donor in, 119-120
for directed donations, 131
Donor History Questionnaire in, 121-127
education of donor in, 119-120
for exceptional medical need, 130
for frequent donors, 130
hemoglobin or hematocrit in, 120, 121
for HSC transplantation, 714-717
identification of donor, 118-119
overview of, 117-118
physical examination in, 120
registration process in, 118-119
Donor-specific antibodies, 716
Donor testing
ABO, 297-298
for blood group antigens, 285, 379, 420
for cord blood donation, 732-733
for HSC transplantation, 714-716
for infectious diseases, 182-191, 192-204
Rh, 285, 327, 328
silenced or nonexpressed genes in, 286
weak D, 327
Donors
adverse reactions in, 20, 22, 142-146
age of, 119, 120
of apheresis RBCs, 123, 171
autologous, 131, 190-191, 605-606
with bleeding conditions or blood diseases,
126, 128-129
with cancer, 126, 128
consent of, 119, 170, 718, 731
deferral of (See Donor deferrals)
designated or directed, 130-131
donation intervals for, 120, 122, 123, 169,
170
educational materials for, 119-120, 122
effect of disasters on, 101, 110
family members as, 421
frequent or repeat, 130
general health of, 122
hearing- or vision-impaired, 120
with heart and lung conditions, 126, 129
hemoglobin/hematocrit in, 120, 121
for HLA-matched platelets, 488-489
for HSC transplantation, 714-717
identification of, 118-119, 139
legally incompetent, 119
leukapheresis, 122
medications taken by, 122, 129-130, 169
non-English-speaking, 120
notification of abnormal test results, 189
phlebotomy of, 140-146
physical examination of, 120
plasmapheresis, 122, 170
814 䡲 AABB TECHNICAL MANUAL
plateletpheresis, 122, 168-169
postphlebotomy care of, 142
pregnancy in, 122
qualification requirements for, 121-127
with reactive screening tests, 186-190
records of, 119
registration of, 118-119
sexual contacts of, 124, 125, 126
temperature of, 120
of tissue allografts, 773-774
and TRALI, 682
of umbilical cord blood, 730-733
Dosage effect, 264, 393, 410
Dosimeters, 61, 156
2,3-DPG, 574
Drug-induced immune hemolytic anemia,
440-444
antibodies in, 441-443
classification of, 441-443
drugs associated with, 448-451
laboratory investigation of, 443-444
mechanisms of, 441
Drug-induced thrombocytopenia, 462, 465-
466
Drug testing, with induced pluripotent stem
cells, 796
Drugs
administered for leukapheresis, 172
antiplatelet agents, 122, 129, 169, 509
causing positive DAT, 428, 440-444, 448-
451
hemostatic agents, 608, 621-622
intravenous use of, 125
platelet antibodies induced by, 465-466, 516
pretransfusion medications, 547-548, 677,
679-680
removal of, in apheresis, 659-660
taken by donors, 122, 129-130, 169
Dry ice, 225
Dry shippers, 724, 738, 739
DTT. See Dithiothreitol
Duffy system, 349-351
antibodies of, 338, 350
antigens, 259, 349-350
Duffy glycoprotein, 350-351
and malaria, 351
phenotypes and genotypes, 349
silenced alleles in, 287
Dura mater transplants, 126
Dysfibrinogenemias, 523
E quality control of, 159
ECMO. See Extracorporeal membrane
oxygenation
Education
for donors, 119-120, 122
for patients, 546
for physicians, 611-612
for umbilical cord blood donation, 730-731
Electrical safety, 46-47
ELISA. See Enzyme-linked immunosorbent assay
Elutions, 408-409, 428-430
Elutriation, 721-722
Embolism, air, 669, 685
Emergency Communications Plan, 105-107
Emergency equipment, 549
Emergency management. See Disaster
management
Emergency Management Agencies, 99, 100,
106-107, 111-112
Emergency release of blood, 158, 227-228, 385-
386, 506, 555-556
Emergency response plans, 44
for biohazards, 53
for chemical spills, 59, 78-82
for electrical emergency, 47
for fires, 46
for radiation safety, 62
Emergency showers, 58
Employees. See Personnel
End-product test and inspection, 33
Endothelial barrier dysfunction, 763-764
Engineering controls
for biosafety, 49-52
for chemical safety, 58
for electrical safety, 47
for fire prevention, 46
general guidelines for, 44, 72
for radiation safety, 62
Engraftment, in HSC transplantation, 717-718
Enhancement media, 376-377, 396, 417
Enzyme-linked immunosorbent assay, 243-
245, 464, 465-466
Enzymes, 377, 402, 405, 413-414
Epinephrine, 679
EPO. See Erythropoietic stimulating agents
Epsilon-aminocaproic acid (EACA), 608, 621-
622
Equipment
apheresis, 168, 173-176, 645-646
critical, 10-11
decontamination of, 49, 52
emergency, 549
management of, 10-11, 13
manufacturers directions for, 11
personal protective, 44, 52-53, 70-71
alarm systems, 36
performance intervals for, 36-38
thermometers, 37

regulations for, 83-84, 87
for transfusions, 548-549
validation of, 15
Er antigens, 361
Ergonomics, 41-42
Errors
in ABO and Rh typing, 300, 332
fatalities due to, 551
identification, 551, 675
mislabeled specimens, 370
quarantine release, 192, 195-196
sources of, in antiglobulin test, 373-374
wrong blood in tube, 378, 384
Erythroblastosis fetalis, 561
Erythrocytapheresis, 646, 655
Erythroid phenotypes, 345, 362-363
Erythropoiesis, in neonates, 572
Erythropoietic stimulating agents, 572, 605,
620-621, 634
Estrogen, conjugated, 625
Ethnic groups, differences in
in antibody identification, 392-393, 394-
395, 415
in Duffy system, 349
in Kell system, 347
in Kidd system, 351-352
in MNS system, 341
in Rh system, 318-319, 320, 321, 323, 324,
325, 327
Exchange transfusions
blood warmer for, 572
component choice for, 574, 579-580
for hemolytic disease of the fetus and
newborn, 564
for hyperbilirubinemia, 579
techniques for, 580
vascular access for, 580
volume of, 580
Executive management, 5
Expiration, of components, 215-220, 551
Exposure control plan, 47, 48
Extracorporeal membrane oxygenation, 585-
586
Extracorporeal photopheresis, 646, 654, 656
Eyewashes, 72
F weak D in, 565
Face shields, 71
Facilities
alternate, during disasters, 103-104
clean rooms in, 40-41
design and workflow of, 40
ergonomic design of, 41-42
housekeeping in, 40
licensure and registration of, 84, 86
INDEX 䡲 815
mobile sites, 41
quality management of, 5-6, 7, 12
regulatory agencies for, 39-40, 68-69
restricted areas in, 41
safety program of, 7, 12, 42-64
Factor VIIa, recombinant, 528, 623, 637-638,
685
Factor VIII
antibodies to, 527
concentrates, 524, 526-527
in Cryoprecipitated AHF, 153, 154, 522
Factor IX complex concentrate, 518, 527-528,
623
Factor IX concentrates, 524, 527
Factor Xa inhibitors, 518
Factor XIII, 638
Failure modes and effects analysis, 28
False positive/negative test results, 332, 373-
374
Fatalities
during apheresis, 660
of blood donors, 20, 145-146
due to identification errors, 551
due to TRALI, 681
due to transfusions, 20, 691
of employees, 45
related to medical devices, 87
reporting, 20, 45, 87, 145, 691
Fatigue, in donors, 145
Fc receptors, 248, 249, 250, 466
FDA. See Food and Drug Administration
Febrile nonhemolytic transfusion reactions,
489, 548, 667, 677
Fenwal apheresis systems, 168, 173, 174, 175
Fetal and neonatal alloimmune
thrombocytopenia, 461, 567-568
Fetal bovine serum, 764-765, 793
Fetal hemoglobin, hereditary persistence of,
362
Fetomaternal hemorrhage, 565-566
Fetus
genotyping, 283-284, 330, 563, 567
hemolytic disease in, 561-564, 566-567
pretransfusion testing of, 563
thrombocytopenia in, 461, 567-568
transfusions in, 563-564
Fever, 489, 667, 676, 677, 686
FFP. See Fresh Frozen Plasma
Fibrin clots, 300
Fibrinogen
abnormalities of, 522, 523
in Cryoprecipitated AHF, 153-154, 522
Fibrinogen concentrate, 529, 624
Ficin, 402
816 䡲 AABB TECHNICAL MANUAL
Filters
Leukocyte reduction, 154, 223, 552, 554
microaggregate, 551-552, 585
standard in-line, 551, 554, 585
Fire prevention, 45-46
First aid, 44-45
Fish-bone diagrams, 27, 28
Flow cytometry, 246
in HLA antibody testing, 487, 491
to measure fetomaternal hemorrhage, 565
to measure residual leukocytes, 154-155
in platelet antibody detection, 460, 464, 465
in red cell survival studies, 419
Fludarabine, 443
Fluids, replacement, in apheresis, 522, 647,
652
Fluorescent methods for nucleic acid analysis,
238-240
Focal segmental glomerulosclerosis, 653
Food and Drug Administration
inspections by, 86-87
regulations and guidance
for biological products, 83, 84, 85
cGMP, 7, 8, 20, 213, 742, 760, 761
cGTP, 8, 20, 87, 743-744, 775, 777
for HPCs, 87-89, 743-746
for infectious disease testing, 182, 184,
187-188
for licensure and registration, 84, 86
for medical devices, 83-84, 87
for recalls and withdrawals, 89
for tissues, 777
reporting adverse events to, 20, 22, 87
reporting fatalities to, 20, 87, 145, 691
variances to requirements, 11
Forensic testing, 493
Forms, 17, 18. See also Documents; Records
Forssman system, 261, 340, 360
Freeze-thaw elutions, 429
Freezers, 36, 214
Freezing
cryoprotective agents for, 155, 722, 761
Fresh Frozen Plasma, 151
hematopoietic progenitor cells, 722-723,
736-737
platelets, 155
RBCs, 155
Frequency, allele (gene), 274, 275-276
Fresh Frozen Plasma, 151
ABO compatibility of, 375, 554, 583, 635
aliquoting, 583
expiration of, 151, 218-219, 221
leukocyte content of, 505
in massive transfusion, 502, 685
for pediatric patients, 578, 582-583, 589
preparation of, 147, 151
quarantine, 152
as replacement fluid in apheresis, 522, 647,
652
storage of, 151, 218-219
thawing, 151, 221
transfusion of, 522, 554
transportation and shipping of, 218-219
Fume hoods, 58
Fungal contamination, 777, 782
G
G-CSF. See Granulocyte colony-stimulating
factor
Gastrointestinal diseases, 764
Gel-column agglutination technology, 377,
396
Gene locus, 256
Genes, 256-258
of blood group systems, 259-261, 277-278
frequencies of, 275-276
of major histocompatibility complex, 476-
480
mapping, 259-261, 277-278
mutation of, 264
nucleic acid structure in, 232-233
position effect, 272-273
silent or amorphic, 264, 267, 286-287
suppressor or modifier, 273
syntenic, 271
Genetic principles
alleles, 262, 264, 275, 276
blood group gene mapping, 259-261, 277-
278
cell division, 258, 262, 263
chimerism, 278
genes and chromosomes, 256-258
genotype and phenotype, 261-262
inheritance patterns, 265-273
autosomal, 265-267
gene interaction and position effect,
272-273
independent segregation and
independent assortment, 270
linkage and crossing-over, 270-271, 272,
479
linkage disequilibrium, 271, 479-480
pedigrees, 265, 266
sex-linked, 267-269
of major histocompatibility complex, 476-
480
polymorphism, 264
population genetics, 274-276
relationship testing, 276-277
X chromosome inactivation, 258, 261

Genotype
defined, 261-262
DNA-based assays for, 282
for antigen-negative blood donors, 282,
285
to distinguish alloantibody from
autoantibody, 282, 283
of fetus, 283-284, 330, 563, 567
platelet genotyping, 465
in prenatal practice, 283-285, 330
in recently transfused patients, 240, 281,
282, 330
of Rh system, 285, 330
in sickle cell disease patients, 330
when red cells are coated with IgG, 282,
283, 401-402, 436
frequencies of, 275-276
nomenclature for, 280
and phenotypes, 240, 261-262, 285-287, 402
Gerbich system, 260, 339, 357
Gill system, 261, 340, 359-360
Glanzmann thrombasthenia, 455
Globoside collection, 260, 309, 310, 311, 340
Gloves, 70-71
and latex allergy, 45
use in donor room, 53, 140
Glycerolization of red cells, 155
Glycine-HCl/EDTA, 407
Glycoproteins, platelet, 453, 454-455, 455-457,
458
GMP. See Good manufacturing practice
Goggles, safety, 71
Gonorrhea, in donors, 124
Good manufacturing practice
for component manufacturing, 213
for HCT/Ps, 742
for MSCs, 760, 761
for nonconforming events, 20
for training personnel, 8
for work environment, 7
Good tissue practice
for infectious disease testing, 87
for nonconforming events, 20
for tissue processing, 775, 777
for training personnel, 8
for umbilical cord blood, 743-744
Grading test results, 372
Graft-vs-host disease
and donor lymphocyte infusions, 787
in HSC transplantation, 638-639, 717
transfusion-associated, 671, 687-688
HLA system in, 489-490, 688
in neonates, 573
treatment with MSCs, 762, 763, 794
Graft-vs-tumor effect, 715, 717, 785
INDEX 䡲 817
Granulocyte agglutination test, 469
Granulocyte colony-stimulating factor (G-
CSF)
for HSC mobilization, 719-720
in leukapheresis, 172-173, 524, 525
side effects of, 719-720
Granulocyte immunofluorescence test, 469
Granulocytes
ABO compatibility of, 173, 375, 525, 554,
584
antigens and antibodies, 466-469, 681, 682
apheresis collection of, 168, 172-173
CMV-reduced-risk, 525
dose of, 584
expiration of, 217
HLA-matched, 525
in HSC transplantation patients, 638
increasing yields of, 172-172, 525
indications for, 525, 584, 589
infusion of, 173, 523-526, 554
irradiation of, 173, 217, 525
laboratory testing of, 173
in pediatric patients, 584
storage of, 173, 217, 221, 525
transportation and shipping of, 217
Growth factors, recombinant, 172-173, 719-
720
Growth hormone, in donors, 129
GTP. See Good tissue practice
GVHD. See Graft-vs-host disease
H
H system, 301-304
alloanti-H, 303, 339
autoanti-H, 303
autoanti-HI, 303, 339
biochemistry and genetics of, 260, 292-293,
301-303
Bombay phenotype, 292, 293, 303
H antigen, 292, 293, 301
Para-Bombay phenotype, 303
transfusion practice for, 304
Hand washing, 72
Haploid, defined, 258
Haplotype
ancestral, 480, 493
defined, 271
of HLA system, 478, 479, 480
of Rh system, 319, 320, 321-322
Hardy-Weinberg equilibrium, 275-276
Hazardous areas of facilities, 41
Hazardous materials
biohazards, 47-55
chemicals, 55-60
classification of, 56
818 䡲 AABB TECHNICAL MANUAL
identification and communication of, 43-44
for biosafety, 48-49
for chemical safety, 56-58
for electrical safety, 47
for fire safety, 46
radioactive, 60-63
safety plan for, 42
shipping, 63
waste management of, 53-55, 63-64
Hazards, risk assessment of, 98-99, 102
HBsAg. See Hepatitis B surface antigen
HBV. See Hepatitis B virus
HCT/Ps (human cells, tissue, and cellular and
tissue-based products). See also Stem cells;
Tissue
biological product deviations of, 22
infectious disease testing on donors of, 191-
192, 714-715, 774
regulation of, 87-89, 743-746, 777-778, 796-
797
HCV. See Hepatitis C virus
HDFN. See Hemolytic disease of the fetus and
newborn
Health history questionnaires. See Donor
history questionnaire
Heart disease, in blood donors, 126, 129
Heart transplants, 493-494
Heat elutions, 429
Heating blocks, 37
Hemagglutination, 241-242, 279-280, 396
Hematocrit
in blood donors, 121
of blood in exchange transfusions, 580
in neonates, 585
of red cell components, 149
as transfusion threshold, 692
of Whole Blood, 148
Hematologic disorders, in blood donors, 128-
129
Hematoma, in donors, 145
Hematopoietic growth factors, 172-173, 719-720
Hematopoietic progenitor cells. See
Hematopoietic stem cells
Hematopoietic stem cell transplantation
ABO typing discrepancies in, 298, 300
adverse reactions to, 724-725, 742
allogeneic, 632-638, 715
autologous, 638, 714-715
and CMV, 639
diseases treated with, 713-714
donor lymphocyte infusion after, 786-788
donor requirements for, 714-717
engraftment kinetics in, 717-718
graft-vs-host disease after, 638-639, 717, 794
graft-vs-neoplasm effect in, 717
histocompatibility in, 491, 715-716
history of, in blood donors, 123
incompatible
bidirectional ABO, 633, 635
major ABO, 632-633, 635, 716
minor ABO, 633, 635, 716
related to non-ABO antigens, 633
patient care in, 724-725
patient survival after, 718
positive DAT after, 428
products for (See Hematopoietic stem cells)
records of, 640
regulation of, 87, 88, 89, 725, 743-746
standards for, 746
transfusion support for
autologous recipients, 638
component processing, 638-639
Cryoprecipitated AHF, 637
factor concentrates, 637-638
incompatible transplants, 632-633
information portability, 640
in patients with HLA/HPA antibodies,
637, 639-640
in patients with neutropenia and
infection, 638
in pediatric patients, 639-640
plasma, 637
platelets, 634, 635, 636-637
RBCs, 634
selection of components, 633-634, 635
transfusion reactions after, 639
Hematopoietic stem cells
collection of, 718-720, 733-734
cryopreservation of, 722-723, 736-737
donors of, 714-717
infusion of, 724-725
irradiation of, 638
processing, 720-722
quality control of, 723, 736
regulation of, 87, 88, 89, 725
shipping and transport of, 723-724, 737-738
sources of, 717-720
storage of, 736-737
thawing, 721
washing, 721, 740-741
Hemizygous, defined, 263, 267
Hemoglobin
in blood donors, 120, 121
decreased, tolerance for, 609-610
in infants, 571-572, 578-579, 585
in Red Blood Cells, 149
in red cell apheresis donors, 171
testing methods for, 121
as transfusion threshold, 499-501, 505-506,
578-579, 610

Hemoglobin F, 362
Hemoglobin S, 564, 587, 588
Hemoglobin substitutes, 507
Hemoglobinometers, 38
Hemoglobinopathies, 587-589. See also Sickle
cell disease
Hemolysis
in ABO testing, 297
in antiglobulin tests, 372
extravascular, 250, 430, 673-674
in hemolytic disease of the fetus and
newborn, 562
intravascular, 251, 430, 672-673
nonimmune, 660, 669, 675-676
passenger lymphocyte syndrome, 633
in patient samples, 370, 672
Hemolytic anemia
autoimmune
classification of, 430
cold agglutinin disease, 437-439
DAT-negative, 437
mixed-type, 439-440
paroxysmal cold hemoglobinuria, 440
serologic findings in, 431
transfusion in, 436-437, 439-440, 506
warm, 430-437
drug-induced immune, 440-444
neonatal, 561-564, 566-567
positive DAT in, 426, 428
Hemolytic disease of the fetus and newborn,
561-564
ABO, 566-567
antibodies associated with, 338-340, 347-
348, 413-414, 562
blood selection and administration in, 564
diagnosis of, 562-563
maternal alloimmunization in, 562
neonatal management in, 564
pathophysiology of, 562
pregnancy monitoring in, 563
preventing, 565-566
testing in, 563
antibody titers, 409, 563
elutions, 429
Rh testing, 283-285, 332, 563, 565
treatment of, 563-564
Hemolytic transfusion reactions
acute, 667, 672-675
antibodies associated with, 338-340,
413-414
clinical evaluation and management of,
666, 667, 672
due to misidentification errors, 551
elutions in, 429
HLA antibodies in, 490
INDEX 䡲 819
delayed, 250, 588, 670, 686-687
intravascular hemolysis in, 251
nonimmune, 669, 675-676
Hemolytic uremic syndrome, 653
Hemophilia, 524, 526-527, 528
Hemorrhage
and anemia, 508-509
assessing risk of, 518-521, 605
fetomaternal, 565-566
intraventricular, 581
transfusion for, 501-502
Hemostasis
during massive transfusion, 591, 684-685
in neonates, 582-583
tests for, 518-521, 582
Hemostatic agents, 608, 621-627
Hemovigilance, 22, 33, 665-666
Heparin, 462, 465-466, 658, 675
Heparin-induced thrombocytopenia, 462,
465-466, 517, 528
Hepatitis, non-A,non-B, 179, 182, 196
Hepatitis B immune globulin, 45
Hepatitis B surface antigen, 179, 196
Hepatitis B virus, 196
employee exposure to, 44-45
nucleic acid testing for, 186, 196
prophylaxis for, 44
reactive testing results for, 187, 188, 189
residual transfusion risk of, 192, 194, 196
screening donors for, 124, 126, 179, 184,
191, 196
transmission through transplantation, 774,
777, 782
Hepatitis C virus, 196-197
employee exposure to, 44-45
nucleic acid testing for, 185-186, 197
reactive testing results for, 187, 188, 189
residual transfusion risk of, 192-193, 194,
197
screening donors for, 124, 126, 179, 184,
191, 197
transmission through transplantation, 774,
777, 782
Hepatitis E virus, 203-204
Herbal supplements, 605
Hereditary hemochromatosis, 476
Hereditary persistence of fetal hemoglobin
syndrome, 363
Heredity, genetics of. See Genetic principles
HES. See Hydroxyethyl starch
Heterozygous, defined, 263-264
High-prevalence antigens
901 series, 361-362
antibodies to, 415-416, 564
blood selection for, 421
820 䡲 AABB TECHNICAL MANUAL
High-titer, low-avidity antibodies, 409-410
Histocompatibility. See HLA system
HIV. See Human immunodeficiency virus
Hives, 667, 678, 679-680
HLA alleles, 483-484
HLA antibodies
detection of, 460, 487
donor specific, 716
in HSC transplant recipients, 637, 639-640,
716
management of, 670
and plasma transfusions, 151
in platelet refractoriness, 458, 459-461, 488-
489, 515-516
in TRALI, 489, 681, 682
in transfusion reactions, 489, 490, 677
HLA antigens
Bg, 362, 480, 490
Class I and Class II, 475, 480-482, 484
configuration of, 481
cross-reactive groups, 482
identification of, 484-487
nomenclature for, 481-482
on platelets, 458
“public,” 482-483
“splits,” 482
HLA-matched platelets, 459-460
HLA Matchmaker program, 460, 488-489
HLA system
biochemistry, tissue distribution and
structure of, 480-484
biologic function of, 484
and chimerism, 489-490
disease associations with, 493-494
genetics of, 476-480
absence of antigens, 478-479
crossovers, 479
finding HLA-identical siblings, 478
linkage disequilibrium, 479-480
organization of, 476-478
patterns of inheritance in, 478-480
in graft-vs-host disease, 489-490, 688
importance of, 475-476
HLA typing
cellular assays for, 486-487
crossmatching, 487, 491-492
DNA-based assays for, 485-486, 493
in forensic testing, 493
of Granulocytes, 525
lymphotoxicity assays for, 486
of platelets, 459-460, 488, 516
in relationship testing, 493
in transplantation, 491-493, 715-716
HNA. See Human neutrophil antigens
Homolog, defined, 267
Homozygous, defined, 263-264
Hook effect, 245
Hospitals, 91, 602-603
Housekeeping, 40
HPA. See Human platelet alloantigens
HSC. See Hematopoietic stem cells
HSCT. See Hematopoietic stem cell
transplantation
HTLV. See Human T-cell lymphotropic virus
Human immunodeficiency virus, 194-196
educating donors about, 119
employee exposure to, 44-45
nucleic acid testing for, 185-186, 195
reactive testing results for, 187, 188, 189
reporting new cases of, 782
residual transfusion risks for, 192-193, 194,
195
screening donors for, 124, 125, 126, 182,
184, 191, 194-196
transmission through transplantation, 774,
777, 782
Human neutrophil antigens, 466-468
Human platelet alloantigens, 453-457
Human resources, 7-9, 12, 109
Human T-cell lymphotropic virus, 197
reactive testing results for, 187, 188, 189
screening donors for, 184, 191, 197
transmission through transplantation, 774,
782
Human urea transporter 11, 352
Hydatid cyst fluid, 312, 406
Hydrops fetalis, 561
Hydroxyethyl starch, 172, 722
Hyperbilirubinemia, 579
Hyperhemolytic syndrome, 588
Hyperkalemia, 555, 683-684
Hyperviscosity, 652
Hypocalcemia, 555, 658, 670, 683
Hypofibrinogenemia, 522-523
Hypogammaglobulinemia, 300, 529, 659
Hypoglycemia, 580
Hypokalemia, 683-684
Hypotension
in apheresis, 659
associated with ACE inhibitors, 659, 669,
678
deliberate, 607
in transfusion recipients, 674, 679
Hypothermia, 548, 572-573, 607, 670, 685-686
Hypovolemia, 526, 659
I
I and i antigens, 306-309
anti-I, 308, 340
anti-i, 308

in cold agglutinin disease, 308-309, 392, 439
disease associations with, 307, 392
genetics of, 260, 307-308
phenotypes, 306-307
transfusion practice with, 309
Iatrogenic anemia, 609
ICAM-4, 355-356
Identification
of blood components
before administration, 226, 551
before issue, 226, 549-550
donation identification number, 135,
139-140, 159
labeling, 158-159, 384-385
of donors, 118-119, 139
of equipment, 11
errors in, 551, 675
of personnel, 19
of persons issuing blood, 385
of phlebotomists, 370, 547
of problems and solutions, 26-29
of recipients, 226, 368-370, 384-385, 547,
549, 550, 551
of umbilical cord blood, 739
Idiopathic thrombocytopenia, 463, 465, 517,
568
IgA, 247, 248, 678-679, 680
IgE, 247, 248
IgG, 247, 248, 249, 573
IgG-coated cells (check cells), 376
IgM, 246, 248
cold-reactive autoagglutinins, 437-439
in complement activation, 249
dispersing autoagglutination caused by,
301, 438
in infants, 573
in intravascular hemolysis, 251
structure of, 247
Immediate-spin crossmatch, 380
Immune complexes, 652
Immune thrombocytopenic purpura, 463, 465,
517, 568
Immunity
antibodies in, 246-248
complement in, 248-250
extravascular hemolysis in, 250
Fc receptors in, 248, 249, 250
in infants, 573
intravascular hemolysis in, 251
Immunoglobulin products, 526, 529-531, 532
Immunoglobulins, 246-248. See also specific
immunoglobulins
Immunohematology reference laboratories,
419
Immunomagnetic cell separation, 722
INDEX 䡲 821
Immunomodulation, 504, 758, 762-763, 764
In-vivo testing, 419, 506-507
Incarceration, donor history of, 125
Incidence, defined, 274
Incinerators, hospital/medical/infectious
waste, 55
Incubation times, 405
Incubators, platelet, 36, 214
Independent assortment, 270
Independent segregation, 270
Indian system, 260, 339, 358-359
Indirect antiglobulin test, 372, 373-374
Induced pluripotent stem cells, 755, 795-796
Infants. See Neonates; Pediatric patients
Infection, in transplant patients, 638, 782
Infectious disease screening
approaches to, 183
of autologous donations, 190-191
for Babesia, 201
for bacterial contamination, 198-199
for chikungunya virus, 202
for CMV, 190
for dengue virus, 202
for HBV, 196
for HCV, 196-197
for HEV, 203-204
historical overview of, 179-182
for HIV, 194-196
in HSC transplantation donors, 191-192,
714-715
for HTLV, 197
international variations in, 192
logistics of, 183
for malaria, 199-200, 201-202
nucleic acid testing, 185-186
for parvovirus B19, 203
for prions, 202-203
reactive test results in, 186-190
residual infectious risks of transfusion, 192-
194
serologic testing, 185
for syphilis, 198
in tissue transplantation, 774
for Trypanosoma cruzi, 200-201
for umbilical cord blood, 732
in the United States, 184
for West Nile virus, 200
Infectious diseases
emerging agents, 203, 204
safety precautions for, 47-55
screening for (See Infectious disease
screening)
transmitted by plasma derivatives, 203
transmitted by tissue transplantation, 777,
782
822 䡲 AABB TECHNICAL MANUAL
Infectious waste, 53-55
Inflammation modulation, 763-764
Information management, 13, 19-20
Infusion pumps, 548-549, 584
Infusion rates, 553, 554, 555, 585
Infusion sets, 551-552, 585
Inheritance patterns
autosomal, 265-267
crossing-over, 270-271, 479
gene interaction and position effect, 272-
273
independent segregation and independent
assortment, 270
linkage, 270-271, 272
linkage disequilibrium, 271, 479-480
of major histocompatibility complex, 478-
480
pedigrees, 265, 266
sex-linked, 267-269
Inhibition tests, 406
Injuries, 45, 49, 87
INR. See International normalized ratio
Inspections
of components
before administration, 550-551
before release, 224, 226, 385, 549
after preparation, 148, 149
documentation of, 224
of incoming supplies, 10
of tissue grafts, 779-780
Insulin, bovine, 129
Insurance, 104
Integrins, 455-456
Internal event reports, 20, 21-22
International normalized ratio (INR), 518, 519,
520
Intraoperative blood recovery, 606-607
Intrauterine transfusions, 563-564
Intravenous immune globulin, 529-531
ABO discrepancies with, 300
adverse effects of, 529, 532
in antibody identification problems, 392
applications of, 530-531
for fetal and neonatal immune
thrombocytopenia, 568
in HDFN, 564
in platelet refractoriness, 516
positive DAT with, 428
Intravenous solutions, 552
Intraventricular hemorrhage, 581
Inventory, blood
during disasters, 101-102, 110
management of, 227-228
and patient blood management, 603
receiving components into, 225
Investigational new drug application, 731, 744,
745
Iron, supplemental, 605, 620
Iron chelation therapy, 690
Iron deficiency anemia, 605, 620
Iron overload, 588, 671, 690
Irradiated products, 155-156, 222
expiration of, 215, 217, 222
granulocytes, 173, 217, 525
in HSC transplantation, 638
indications for, 688, 689
for intrauterine transfusions, 564
for pediatric patients, 590
platelets, 156, 217, 459-460
potassium leak in, 573
quality control of, 156
RBCs, 156, 215
storage of, 215, 217
transportation of, 215, 217
Whole Blood, 215
Irradiators, blood, 37, 62, 155-156
Ishikawa diagrams, 27, 28
Isografts, 774
Isohemagglutinins, 292
Issuing components, 226
delivering blood to patient area, 549-550
identification of recipient and component
before, 384-385, 549-550
inspections prior to, 226, 385, 549
reissue, 226-227, 550
in urgent situations, 158, 227-228, 385-386,
506, 555-556
ITP. See Immune thrombocytopenic purpura
IVIG. See Intravenous immune globulin
J-K
John Milton Hagen system, 260, 339, 359
JR system, 261, 340, 360
Juran’s Quality Trilogy, 2-3
Karyotype, 256
Kell system, 345-349
allele frequencies in, 275-276
antibodies of, 338, 347-348
antigens of, 259, 346-347
biochemistry and genes of, 259, 346
functional aspects of, 348
in HDFN, 284, 347-348, 562, 563
Kmod, 348
Ko (null) phenotype, 348
phenotypes of, 346
position effect in, 272-273
Kernicterus, 562, 564, 579
Kidd system, 351-352
antibodies of, 338, 352

antigens of, 259, 351-352
genetics of, 259
Kidd glycoprotein, 351, 352
phenotypes of, 351
in transfusion reactions, 352
Kidney transplantation, 491-492
Kleihauer-Betke acid-elution test, 565-566
Knops system, 260, 339, 358
Kx system, 348-349
antibodies of, 339
genetics of, 258, 260, 261, 269
McLeod phenotype, 258, 261, 269, 348
L phenotypes of, 304-305
Labels
for biohazardous materials, 49
for blood components, 139, 158-159, 226,
384-385
for blood samples, 370, 547
control of, 17, 18
for hazardous chemicals, 57-58
ISBT 128 system for, 159
for shipments, 738
for umbilical cord blood, 739
Laboratories
biosafety precautions for, 53
regulations for, 89-91
Laboratory coats, 70
Lan system, 261, 340, 360-361
Landsteiner-Wiener system, 260, 339, 355-356
Latex allergies, 45, 136
LDL apheresis, 656
Leadership, organizational, 5-6, 12, 105
LEAN, process improvement, 28-29
Lectins, 295, 301
Leukapheresis
collection of granulocytes by, 175-176
donation intervals for, 120, 122
indications for, 646, 654, 655
instrumentation for, 168, 175-176
Leukemia
in blood donors, 126, 128
cytapheresis in, 654
donor lymphocyte infusions for, 786-787, 788
natural killer cells in, 788
platelet transfusions in, 507
Leukocyte-reduced components, 504-505
expiration, transportation and storage of,
216, 217
leukocyte content in, 154-155, 505
for pediatric patients, 590
platelets, 154, 156, 169-170, 217, 505
poststorage filtration, 222-223
prestorage filtration, 154-155, 223, 552
to prevent CMV infection, 190, 590, 639
INDEX 䡲 823
RBCs, 149, 154, 216, 504-505
to reduce alloimmunization, 515
to reduce incidence of PTP, 690
Leukocyte reduction filters, 154, 223, 552, 554
Leukocytes, in components, 154-155, 505
Lewis substance, 406
Lewis system, 304-306
antibodies of, 305-306, 338
antigens of, 259, 302, 304
biochemistry and synthesis of, 302, 304
disease associations with, 306
expression in children, 305
genetics of, 259, 304-305
saliva testing for, 406
transfusion practice with, 306
Licensure, of facilities, 84, 86
Likelihood ratio, 277
Linkage, 270-271, 272
Linkage disequilibrium, 271, 479-480
Lipemic samples, 370
Liquid nitrogen
for shipping, 724, 738
for storage, 723, 737, 761
Liquid Plasma, 152, 220
LISS, 376, 402, 417
Liver, transplantation of, 493-494
Liver disease, 764
LKE antigen, 309, 310, 313
Look-back investigations, 187-188, 189-190, 782
Low-prevalence antigens, 362, 416
Low-volume units, 141, 142, 149
Lui freeze thaw elution, 429
Luke antigen, 309, 310, 313
Lung conditions, in blood donors, 126, 129
Lung transplants, 493-494
Lutheran system, 344-345
antibodies of, 338, 345
antigens of, 259, 344-345
genetics of, 259, 267, 272, 273
Lymphocyte crossmatching, 487
Lymphocytes, T cytotoxic, 787-788
Lymphocytotoxicity assays, 485, 486, 487
Lymphoproliferative disease, 788
Lyonization, 258, 261
M
MACE (modified antigen capture ELISA), 464
MAIGA (monoclonal antibody immobilization
of granulocyte antigens), 469
MAIPA (monoclonal antibody immobilization
of platelet antigens assay), 464
Major histocompatibility complex, 475. See
also HLA system
Class I and Class II antigens in, 480-482
824 䡲 AABB TECHNICAL MANUAL
genetics of, 476-480
public antigens, 482-483
split and cross-reactive groups in, 482
Major histocompatibility complex multimer
method, 787
Malaria, 201-202
and Duffy glycoprotein, 351
screening donors for, 125, 126, 202
Markers, defined, 255
Market withdrawals, 782
Marrow
autologous, 714-715
collection of, 718-719
cryopreservation of, 722-723
donor requirements for, 714-716
engraftment kinetics of, 717-718
histocompatibility of, 715-716, 717
infectious disease testing of, 191, 715
infusion of, 724-725
MSCs derived from, 755, 762, 763
processing, 720-722
quality control of, 723
red cell reduction in, 720-721
regulations for, 87, 88, 89, 725
shipping and transport of, 723-724
transplantation outcomes using, 718
Marrow aplasia, 787
Masks, 71
Massive transfusions
complications of, 591, 683-685
component replacement in, 501-502, 685
defined, 228, 386
Factor VIIa, recombinant in, 685
in pediatric patients, 591
pretransfusion testing in, 228, 330, 386
Material safety data sheets, 57, 58
Materials management, 9-10, 12
Maximum surgical blood order schedules, 227
McLeod phenotype, 258, 261, 269, 348-349
2-ME. See 2-mercaptoethanol
Mechanical hemolysis, 660, 676
Media, working with, 107
Medical devices, regulations for, 83-84, 87
Medical history
in antibody identification, 392
in blood donor selection, 121-127
for cord blood donation, 731-732
in evaluation of positive DAT, 427-428
of recipients, 546
Medical waste, 53-55
Medication Deferral List, 129-130
Medications. See Drugs
Meiosis, 258, 263
Membrane attack complex, 249-250
2-mercaptoethanol (2-ME), 407, 432, 438
Mesenchymal stem cells
autologous vs allogeneic use, 754
future directions for, 765-766
identification criteria for, 793
isolation and expansion of, 758-760, 792-
793
multipotentiality of, 754-755, 791-792
properties of, 756-757, 758, 786, 791-792
research and development on, 764-765
shipping, 761-762
sources of, 754-758
standardization of methods for, 760
storage and banking, 761
therapeutic applications of, 762-764, 793-795
and tissue engineering, 795
tumor-forming potential of, 765
Message mapping, 107
Messenger RNA, 233
Metabolic abnormalities, in infants, 573-574
Methergine, 625
Methyldopa, 443
Methylene blue-treated plasma, 153, 205
Microaggregate filters, 551-552, 585
Microarray assays, 245
Microlymphocytotoxicity tests, 485, 486, 487
Microsatellites, 277
Microvascular bleeding, 684
Minisatellites, 277
Misoprostol, 626
Mitosis, 258, 262
Mixed-field agglutination, 278, 298, 362
Mixed leukocyte culture, 486-487
Mixed-type autoimmune hemolytic anemia,
431, 439-440
MNS system, 337, 341-344
antibodies of, 338, 343-344, 406
antigens of, 259, 341, 344
effect of enzymes on, 314
genetics of, 259, 341, 343
glycoproteins of, 341, 342, 343
linkage disequilibrium in, 271
phenotypes of, 341
S–s–U– phenotype, 343
Mobilization regimens
for granulocytes, 172-173
for HSCs, 719-720
Molecular immunohematology. See Blood
group genomics
Monoclonal antibody immobilization of
granulocyte antigens, 469
Monoclonal antibody-specific immobilization
of platelet antigen, 464
Monocyte monolayer assay, 419
Mortality. See Fatalities
MSC. See Mesenchymal stem cells

Multiple sclerosis, 653
Multiplex nucleic acid amplification, 238
Multipotent stromal cells. See Mesenchymal
stem cells
Mutations, genetic, 264, 362-363
Myeloma, IgM, 652
N 589
Nageotte hemocytometry, 154-155
Narcolepsy, 494
NAT (nucleic acid testing), 185-186
in HBV testing, 196
in HCV testing, 197
in HIV testing, 195
Natural killer cells, 788-790
Near-miss events, 26, 33
Needlesticks, 49, 124, 141
Neisseria gonorrhea, 191
Neonatal alloimmune neutropenia, 468
Neonatal alloimmune thrombocytopenia, 461,
567-568
Neonates (younger than 4 months)
ABO and Rh typing in, 300, 385, 574
ABO antigens and antibodies in, 293, 300
anemia in, 571-572
antibody screening in, 385
antigenic variations in, 305, 410, 411
blood volume in, 572
compatibility testing in, 385, 574-575
ECMO in, 585-586
erythropoietic response in, 572
hemoglobin in, 571-572, 578-579
hemolytic disease in, 561-564, 566-567
hemostasis in, 582-583
hypothermia in, 572-573
immunologic status of, 573
Lewis antigens in, 305
metabolic problems in, 573-574
neutropenia in, 468, 525
polycythemia in, 585
thrombocytopenia in, 461, 567-568, 580-581
transfusion-associated GVHD in, 573
transfusions in
administration of, 584-585
age of units for, 577-578
aliquots for, 224, 575-576, 583
of Cryoprecipitated AHF, 578, 583-584
dosing for, 578
exchange, 564, 574, 579-580
of FFP, 578, 582-583
of granulocytes, 584
indications for, 574, 575, 578-579
of platelets, 578, 580-582
of RBCs, 574-580
safety of additive solutions in, 576-577
INDEX 䡲 825
transfusion thresholds in, 578-579
vascular access for, 547, 580, 584
Nerve injury, in donors, 145
Neutralization techniques, 406
Neutropenia
autoimmune, 468
granulocyte transfusions for, 523-525, 584,
in HSC transplant patients, 638
of infancy, 468
neonatal alloimmune, 468
NMDP registry, 725
Nomenclature
for blood group systems, 259-261, 278-279,
280
for granulocyte antigens, 467
for HLA system, 481-482, 483-484
for human platelet alloantigens, 453-455
of Rh system, 318-319, 319, 320
Nonconformances
biological product deviations, 22, 89, 90
classification of, 22-23
internal event reports, 20, 21-22
management of, 13, 20-23
Nonimmune-mediated hemolysis, 660, 669,
675-676
NOR phenotype, 311
Normovolemic hemodilution, 606
Notifications, of reactive screening tests, 189-
190
Nucleic acid analysis
detection of amplification products in, 238-
240
for genotyping, 282
for antigen-negative blood donors, 282,
285
to distinguish alloantibody from
autoantibody, 282, 283
of fetus, 283-284, 330, 563, 567
platelets, 465
in prenatal practice, 283-285, 330
in recently transfused patients, 240, 281,
282, 330
of Rh system, 284-285, 287, 330
in sickle cell disease patients, 330
when red cells are coated with IgG, 282,
283, 401-402, 436
for granulocyte antigens, 469
in HLA typing, 485-486, 493
hybridization-based methods of, 233
for infectious diseases, 185-186, 195, 196, 197
isolation of nucleic acids in, 233
nucleic acid sequence-based amplification
in, 237-238
overview of, 231-232
826 䡲 AABB TECHNICAL MANUAL
for platelet antigens, 465
polymerase chain reaction in, 233-237, 280-
281
in pretransfusion testing, 378
in relationship testing, 277
reverse-transcriptase PCR in, 236-237
of single nucleotide polymorphisms, 240
transcription-mediated amplification in,
237-238
in West Nile virus testing, 200
Nucleic acid sequence-based amplification,
237-238
Nucleic acids, structure of, 232-233
O 431, 440
Obstetrics, controlling bleeding in, 625-626
Octreotide, 626-627
Oh (Bombay) phenotype, 292, 293, 303
Ok system, 260, 339, 359
Oligonucleotide probes, 485
Orders, physician
auditing, 697-707
computerized provider order entry, 611,
699, 700
pretransfusion, 381, 383, 384, 546-547
sample form for, 711
surgical blood orders, 227
verifying prior to transfusion, 551
Organ transplantation
ABO compatibility in, 491, 492, 783
history of, in blood donors, 123
HLA testing in, 491-492, 493
kidney, 491-492
paired donations, 492
positive DAT after, 428
regenerative therapy in, 754
rejection of, 653, 654, 656
transfusion support for, 783
Organizations
for emergency management, 99-100, 109-
110
for quality system regulation, 1-2
for safety, 39-40, 68-69
structure of, 5-6, 12
Orientation programs, 7-8
Osteoarthritis, 763
Osteogenesis imperfecta, 794
Outpatients, 368, 556
Oxytocin, 625
P surgical blood orders in, 227
P blood groups, 309-313
antibodies of, 312, 338
antigens of, 259, 260, 309-312
biochemistry of, 310-311
disease associations with, 312-313, 392, 440
genetics of, 259, 260
molecular biology of, 311-312
phenotypes of, 309-310
transfusion practice with, 312
P1 substance, 312, 406
P1PK system, 259, 309, 338
Panel-reactive antibody, 487, 492
Panels, red cell, 393, 396-400
Papain, 402
Para-Bombay phenotype, 303
Pareto analysis, 27, 29
Paroxysmal cold hemoglobinuria, 312, 392,
Partial D, 324, 326, 327, 328, 333
Partial thromboplastin time, 519
Parvovirus B19, 187, 203, 313
Passenger lymphocyte syndrome, 633
Paternal samples, testing
DNA-based testing, 284-285, 563
in relationship testing, 276-277
Pathogen reduction technology, 204, 205
and emerging infectious agents, 194
for plasma, 153
for plasma derivatives, 203, 526
to reduce risk of septic reactions, 199
Patient blood management
activity levels of, 612, 629
acute normovolemic hemodilution in, 606
anemia assessment in, 604-605
anesthesia in, 607
auditing in, 24-25, 697-707
bleeding risk assessment in, 605
blood recovery in, 606-607, 608-609
blood utilization review in, 604, 611
changing physician behavior in, 610-611
coordinators for, 611
definition of, 599
increased tolerance of anemia in, 609-610
limiting phlebotomy in, 609
medical education in, 604, 611-612
pharmacologic agents in, 608, 620-627
point-of-care testing in, 607-608
preoperative autologous blood donation in,
605-606
program development, 612
rationale for, 600-603
responsibilities for, 629
scope of, 600, 604
surgical techniques in, 607
transfusion algorithms in, 608
transfusion thresholds in, 610
 INDEX 䡲 827

PBSC (peripheral blood stem cells). See
Peripheral blood HSCs
PCC. See Prothrombin complex concentrates
PCR. See Polymerase chain reaction
PEDI-PAK system, 576, 577
Pediatric patients (older than 4 months). See
also Neonates
ABO antigens and antibodies in, 293, 300
CMV prevention in, 589-590
Lewis antigens in, 305
pretransfusion testing in, 300, 587
thrombocytopenia in, 581
transfusions in
aliquoting for small volumes, 224, 575-
576
of CMV-reduced-risk components, 590
of Cryoprecipitated AHF, 578, 583
of FFP, 578, 583, 589
of granulocytes, 584, 589
in HSC transplantation patients, 639-
640
of irradiated components, 590
of leukocyte-reduced components, 590
massive, 591
of platelets, 578, 581, 582, 589, 590
of RBCs, 578, 586-589
with sickle cell disease, 587-588, 639
syringe infusion pumps for, 549
with thalassemia, 588-589, 640
vascular access for, 547
of volume-reduced components, 590
of washed components, 590-591
Pedigrees, 265, 266
Peer review, 24-25, 697-707
PEG. See Polyethylene glycol
Penicillin, 442
Performance improvement standards, 26
Periadventitial cells, 758
Pericytes, 758
Peripheral blood HSCs
allogeneic, 715-717
autologous, 714-715
collection of, 718, 719-720
cryopreservation of, 722-723
donor eligibility of, 714-717
engraftment kinetics of, 717-718
infectious disease testing on donors of, 191,
714-715
infusion of, 724-725
mobilization of, 719-720
patient survival with, 718
processing, 720-722
quality control of, 723
regulations for, 87, 88, 89, 725
shipping and transport of, 723-724
Personal protective equipment, 44, 70-71
for biosafety, 52-53
for chemical safety, 58
gloves, 45, 53, 70-71
Personnel
accidents and injuries in, 45, 49
blood exposure in, 44-45, 47-48, 49
competency assessment of, 7-8
contact list of, 105
disaster plans for, 108-109
essential, 105, 109
hepatitis prophylaxis for, 44
identification of, 19
latex allergies in, 45
orientation program for, 7-8
protective equipment for, 44, 52-53, 70-71
records, 19
safety monitoring programs for, 44
selection of, 7
staffing plan, 9, 108-109, 110-111
training (See Training)
PF4 ELISA, 465-466
pH, 406, 411
pH meters, 37
Phenotype. See also specific blood groups
calculations for, 274, 421
defined, 261-262
and genetic mutations, 264
and genotypes, 240, 261-262, 285-287, 402
nomenclature for, 280
prevalence of, 274
rare, 394-395, 421
Phenotyping
antigen-matching, 281, 329-330, 379, 588,
654
autologous red cells, 401-402
with DNA-based assays, 280-281, 282
for antigen-negative donors, 282, 285
to confirm D type of donors, 285, 330
to distinguish alloantibody from
autoantibody, 282, 283
in prenatal practice, 283-285, 330
in recently transfused patients, 240, 281,
282, 330
when red cells are coated with IgG, 282,
283, 401-402, 436
donor units, 420
solid-phase assays for, 242-243
Phlebotomy. See also Blood collection
adverse reactions to, 142-146
blood loss due to, 609
for collection of blood samples, 368, 370
disinfection methods for, 140-141
of donors, 140-146
vein selection for, 140
828 䡲 AABB TECHNICAL MANUAL
Photopheresis, 646, 654, 656
Physical assessment
of apheresis patients, 660-661
of donors, 120
of recipients, 546
Physicians
changing behavior of, 610-611, 704-705
educating, 611-612
perspective on patient blood management,
601-602
Physiologic anemia of infancy, 571-572
Piercings, 124
Piperacillin, 442, 443
Pipettes, recalibration of, 37
PlA1 (HPA-1) antigen, 455, 567
Plasma
ABO compatibility of, 375, 554, 583, 635
aliquoting, 583
coagulation factors in, 221
collection by apheresis, 168, 170
cryoprecipitate reduced, 152, 219-220, 652
donors of, 682
expiration of, 151, 218-220, 221
fresh frozen (See Fresh Frozen Plasma)
frozen within 24 hours after Phlebotomy,
152
liquid, 152, 220
pathogen-reduced, 153, 205
preparation of, 147-148, 151
for pretransfusion testing, 392, 393
recovered (for manufacture), 152-153, 220,
526
as replacement fluid in apheresis, 522, 647,
652
solvent/detergent-treated, 153, 205
source, 170, 526
storage of, 151, 218-220
thawed, 151, 152, 219, 221, 522
thawing, 221
transfusion of (See Plasma transfusions)
transportation and shipping of, 218-220,
225
Plasma derivatives
1-antitrypsin, 532
activated protein C, 532
albumin, 526
antithrombin, 531
clotting factor concentrates, 526-527
fibrinogen concentrate, 529, 624
infectious disease screening for, 203
intravenous immune globulin, 529-531, 532
pathogen reduction for, 203, 526
prothrombin complex concentrates, 527-
528, 623
recombinant Factor VIIa, 528, 623
Plasma exchange. See Therapeutic plasma
exchange
Plasma transfusions
to correct PT/INR, 518, 519-520
dose and timing of, 521
and HLA antibodies, 151
indications for, 517-518, 522
in cirrhosis patients, 518-519
in correction of anticoagulation, 518, 519
in HSC transplantation, 635, 637
in massive transfusion, 502, 685
in pediatric patients, 578, 582-583, 589
infusion of, 554
types of plasma for, 522
Plasmapheresis, 170
consent for, 170
donation intervals for, 120, 122, 170
instrumentation for, 168, 173-174
plasma transfusion in, 522
red cell losses in, 170
therapeutic plasma exchange, 647-653
adverse effects of, 658-660
in hemolytic disease of the fetus and
newborn, 564
indications for, 647, 648-651, 651-653
replacement fluids for, 646, 647, 652
Platelet antagonists, 122, 129, 169, 509
Platelet antibodies
anti-HPA, 455-457
autoantibodies, 461, 465, 568
in autoimmune thrombocytopenic
purpura, 463, 568
detecting, 243, 456, 463-464, 465-466, 639-
640
drug-induced, 462, 465-466
in fetal and neonatal alloimmune
thrombocytopenia, 455, 456, 461, 567
in HSC transplant patients, 637
in platelet refractoriness, 515-516
in posttransfusion purpura, 455, 456, 461-
462, 689-690
Platelet antigens
ABO antigens, 457
GPIV/CD36, 457, 458
GPVI, 457, 458
HLA antigens, 458, 488
HPA, 453-457
Platelet counts
corrected platelet count increment, 458,
512, 516
in infants, 568
in plateletpheresis donors, 169
posttransfusion platelet recovery, 458, 512
as transfusion threshold, 507-509, 581, 589,
636-637

Platelet disorders
drug-induced thrombocytopenia, 462, 465-
466, 516
fetal and neonatal alloimmune
thrombocytopenia, 461, 567-568
immune thrombocytopenia purpura, 463,
465, 517, 568
in massive transfusion, 684
platelet transfusion refractoriness, 458-461
posttransfusion purpura, 461-462, 671, 688-
690
Platelet factor 4 ELISA, 465-466
Platelet gel, 222, 517
Platelet incubators, 36, 214
Platelet-rich plasma, 146, 148, 150, 517
Platelet transfusions
ABO compatibility of, 375, 457, 513-514, 554
after HSCT transplantation, 635, 636
in hemolytic transfusion reactions, 675
in pediatric patients, 581, 589
contraindications to, 517
to correct thrombocytopathy, 509-510
dosage of, 510-511, 581, 637
in fetus, 567-568
in HSC transplantation, 634, 635, 636-637
indications for, 507, 509-510, 580-581
infusion of, 554
in massive transfusion, 502, 685
in pediatric patients, 578, 580-582, 589
prophylactic vs therapeutic, 507, 510-511,
636
refractoriness to, 458-461
ABO compatibility in, 457
causes of, 459
HLA antibodies in, 458, 459-461, 488-
489, 515-516, 670
HLA-matched platelets for, 459-460,
488-489
HPA antibodies in, 453-457
managing, 514, 516-517
platelet crossmatching for, 460-461, 489,
516
preventing alloimmunization in, 515-
516
selection of platelets for, 459-461, 488-
489
response to, 512
Rh matching, 515
thresholds for, 507-509, 581, 589, 636-637
unit type and age, 511, 512, 513
Plateletpheresis, 168-170
adverse reactions to, 169
donor selection and monitoring in, 120,
122, 168-169
instrumentation for, 168, 174-175
INDEX 䡲 829
records of, 170
therapeutic, 654
volume collected in, 169
yields from, 510
Platelets, Apheresis
additive solution in, 217
agitation of, 214, 221
bacterial contamination of, 198-199,
221
biochemical changes in storage of, 221, 511,
513
collection of, 168-170
compared to Whole-Blood-derived
Platelets, 511, 512
crossmatching, 460-461, 489, 516
donors of, 168-169
expiration of, 217
HLA matched, 459-460, 488-489, 516
irradiated, 156, 217, 459-460
laboratory testing of, 169-170
leukocytes reduced, 169-170, 217, 505
pathogen reduction for, 205
storage of, 214, 217
transfusion of (See Platelet transfusions)
transportation and shipping of, 217
volume-reduced, 157, 223, 513-514
washed, 223
Platelets (Whole-Blood-derived), 150-151
agitation of, 150, 214, 221
bacterial contamination of, 150, 198-199,
221
biochemical changes in storage of, 221, 511,
513
clumping in, 148
compared to Apheresis Platelets, 511, 512
cryopreservation of, 155
expiration of, 150, 156, 216-217, 224
irradiated, 156, 217
leukocytes-reduced, 154, 156, 218, 505
pathogen reduction for, 205
pooled, 156-157, 217, 223-224, 512
preparation of, 146, 147-148, 150
storage of, 150-151, 214, 216-217, 221
transfusion of (See Platelet transfusions)
transportation and shipping, 150-151, 216-
217
visual inspection of, 148, 199
volume-reduced, 157, 223, 581-582, 590
washed, 223, 590-591
Plerixafor, 720
Point-of-care testing, 607-608
Policies, 11, 17, 18
Polyagglutination, 300, 332
Polycythemia, 585
Polyethylene glycol (PEG), 376-377, 402
830 䡲 AABB TECHNICAL MANUAL
Polymerase chain reaction, 233-237
in genotyping, 280-281
in HLA typing, 485
oligonucleotide probes, 485
problems with, 235-236
real-time, 238-240
reverse transcriptase, 236-237
sequence-based typing, 486
sequence-specific primers, 485-486
Polymorphism, 264, 276-277
Pooled components, 156-157, 223-224
Cryoprecipitated AHF, 157, 218, 224, 523
platelets, 156-157, 217, 223-224, 512
Reconstituted Whole Blood, 224
storage, transportation, and expiration of,
217, 218, 223-224
Population genetics, 274-276
Position effect, 272-273
Postoperative blood recovery, 608-609
Posttransfusion platelet recovery, 458, 512
Posttransfusion purpura, 461-462, 671, 688-
690
Postzone effect, 242
Potassium, 573-574, 683-684
PPR. See Posttransfusion platelet recovery
Preadmission testing, 368
Pregnancy
in blood donors, 122
in patient history, 370-371, 392
testing during (See Prenatal studies)
umbilical cord blood recruitment in, 730-
731
Premedication, 547-548, 677, 679-680
Prenatal studies
ABO/Rh testing, 563
antibody detection, 563
antibody identification, 416
antibody titration, 409, 563
DNA-based testing, 283-285
on fetus, 283-284, 330, 563
of paternal samples, 284-285, 563
in pregnant women, 284, 330
in fetal and neonatal immune
thrombocytopenia, 567
Preoperative autologous donation, 131, 605-
606
Preservatives, antibodies to, 417
Pressure devices, 549
Pretransfusion testing
ABO and Rh typing, 375
after non-group-specific transfusions, 386
antibody detection and identification, 375-
378, 381
antiglobulin test, 371-372, 373-374
with autoantibodies, 432, 438-439
autologous control, 377
and blood availability, 384
blood samples for, 368, 370-371, 384, 547
in cold agglutinin disease, 438-439
comparison with previous records, 378
component selection, 375, 378-379
crossmatching, 379-381
donor unit testing, 378
identification of recipients, 368-370
in massive transfusions, 228, 386
orders for, 381, 383, 384, 546-547
in pediatric recipients, 385, 574-575, 587
reading and interpreting reactions, 372,
381, 382
requests for transfusion, 367-368, 383, 546,
699-700, 703-704
serologic testing, principles of, 371-372
tubeless methods, 377-378
turnaround times, 547
in urgent situations, 227-228, 385-386
Prevalence, of phenotypes, 274
Preventive action, 26, 27
Primed lymphocyte typing, 486, 487
Primers, PCR, 235-236
Prions, 202-203
Probability values, in antibody identification,
398, 400
Proband, 265
Problem identification and resolution, 26-29
Procedures, 11, 17, 18, 23
Process
capability, 33
control, 3, 33
flow charts, 27
improvement, 13, 26-29
management, 3-4, 11, 13, 14-16
validation, 14
Processes, 11, 17, 18
Production, principles of, 4
Proficiency testing, 25-26, 91
Propositus, 265
Prospective audits, 699-700, 701-702, 706
Prostate cancer, 791
Protamine, 622
Protein analysis, 240-247
agglutination-based methods for, 241-242
ELISA for, 243-245
flow cytometry for, 246
protein microarrays for, 245
SPRCA for, 242-243
Western blotting for, 245-246
Protein C, 532
Protein microarrays, 245
Prothrombin complex concentrates, 518, 527-
528, 623
 INDEX 䡲 831

Prothrombin time, 519-520, 521 Quality oversight, 5-6

Proton pump inhibitors, 626
Provenge, 791
Prozone effect, 241-242
PRP. See Platelet-rich plasma
Pseudogenes, 476, 478
Psoralen-treated plasma, 153, 205
PT. See Prothrombin time
PTT. See Partial thromboplastin time
Public antigens, 482-483
Pulmonary disease, in blood donors, 126, 129
Pulmonary edema, 659, 680, 681, 682
Pulse, of donor, 120
Pumps, infusion, 548-549, 584
Q for ABO testing, 298
Quad packs, 576
Qualification
defined, 33
of equipment, 15
of personnel, 7
of suppliers, 9, 779, 780
Quality assurance, 1-2, 34
Quality control, 16
of blood components, 159-160, 171
of copper sulfate solution, 38
defined, 2, 34
of equipment, 36, 37
of HSCs, 723, 736, 739, 741
performance intervals for, 36-38
records of, 16
unacceptable results for, 16
Quality improvement, 3
Quality indicators, 24, 34
Quality management systems
Code of Federal Regulations references, 35
components of, 4-5, 12-13
customer focus, 6-7, 12
documents and records, 13, 16-19
equipment management, 10-11, 13
facilities, work environment and safety, 7,
12
general concepts of, 1-5
human resources, 7-9, 12
information management, 13, 19-20
management of nonconforming events, 13,
20-23
monitoring and assessment, 13, 23-26
organization and leadership, 5-6, 12
process improvement, 13, 26-29
process management, 11, 13, 14-16
quality control performance intervals, 36-38
suppliers and materials management, 9-10,
12
terminology of, 33-34
Quality planning, 2-3
Quality System Essentials, 2
Quarantine
of collected blood, 157-158
of nonconforming products, 224, 225
of repeatedly reactive units, 187-188, 189
Quarantine FFP, 152
Quarantine release errors, 192, 195-196
R
Radiation safety, 60-63
Raph system, 260, 339, 359
RBCs. See Red Blood Cells
Reagents
antibodies to components of, 298, 300, 417
antiglobulin, 372, 396, 426-427
bovine albumin, 376
chloroquine diphosphate, 407
contamination of, 332
DTT, 405
for elutions, 429
enhancement media, 396, 417
enzymes, 377, 402, 405
glycine-HCl/EDTA, 407
LISS, 376, 402
manufacturers directions for, 11
PEG, 376-377, 402
for phenotyping, 420
quality control intervals for, 38
red cells, 376, 393, 396
for Rh testing, 326, 327, 331-332
sulfhydryl, 407
ZZAP, 407
Real-time PCR, 238-240
Recalls, 89, 90, 782
Recipients
ABO and Rh testing, 297-298, 327-328, 375
antibody detection in, 357-377
baseline assessment of, 546
consent of, 545-546, 601
crossmatching in, 379-381
education of, 546
identification of, 226, 368-370, 384-385,
549, 550, 551
immunocompromised, 190
medical history of, 392, 546
monitoring during and after transfusions,
553, 555
pediatric patients (See Neonates; Pediatric
patients)
phenotyping, 329-330, 401-402
records of, 378, 672
tracing (look-back), 187-188, 189-190, 782
832 䡲 AABB TECHNICAL MANUAL

of unknown identity, 368, 370 Red Blood Cells, Deglycerolized

weak D in, 327-328, 375 expiration, storage, and transportation of,
Recombination, 271 215, 222
Reconstituted Whole Blood, 224 leukocyte content of, 505
Records preparation of, 155, 222
altering or correcting, 18-19 quality control of, 222
apheresis, 170, 172 rejuvenated, 216
blood component, 384-385 Red Blood Cells, Frozen
checking before blood issue, 226, 384-385, expiration, storage, and transportation of,
550 215
comparing testing results to, 378 preparation of, 155
confidentiality of, 19 rejuvenated, 216
donor, 119 thawing and deglycerolizing, 155, 222
electronic, 18, 19 Red cells, in-vitro generation of, 796
HSC transplantation, 640 Red Blood Cells, Leukocytes Reduced, 504-505
management of, 13, 16, 18-19 expiration, storage, and transportation of,
personnel, 19 216
protection of, during disasters, 104, 111 leukocyte content of, 149, 505
quality control, 16 prestorage filtration, 154
storage of, 19 Red Blood Cells (RBCs)
of tissue allografts, 781-782 additive solutions for, 136-137, 138, 576-
transfusion, 226, 555, 640, 672 577
Recovered Plasma, 152-153, 220 age of, 574, 577-578
Red Blood Cell transfusion, 499-507 aliquoting, 224, 575-576
ABO/Rh compatibility of, 375, 378-379, 504, anticoagulant-preservative solutions for,
554, 635 136, 137
in autoimmune hemolytic anemias, 436- bacterial contamination of, 198
437, 439-440, 506 biochemical changes of storage in, 221,
in chronic anemia, 502 503-504
dose of, 505-506, 610 collected by apheresis, 171-172
in emergency release, 227-228, 385-386, cryopreservation of, 155
506, 555-556 deglycerolized, 155, 215, 222, 505
exchange transfusion, 579-580 expiration of, 149, 215-216
hemoglobin targets in, 499-501, 505-506 frozen, 155, 215, 222
in hemorrhagic shock, 501-502 hemoglobin/hematocrit of, 149
in HSC transplantation, 634 irradiated, 156, 215
of incompatible units, 506-507 leukocyte content of, 505
indications for, 499-502, 574, 575, 578-579 leukocytes reduced, 216, 504-505
infusion of, 554, 585 low-volume units, 141, 142, 149
intrauterine, 563-564 pathogen reduction of, 205
leukocyte reduced, 504-505 phenotyping, 420, 588
in massive transfusion, 228, 386, 501-502, preparation of, 147-148
683-685 rare, 421
in pediatric patients, 574-580, 586-589 in red cell exchange, 654
restrictive vs liberal strategies for, 499-501 rejuvenated, 216
Red Blood Cells, Apheresis, 171-172 storage of, 214, 215-216, 221, 410-411
collection of, 168, 175 substitutes for, 507
donation requirements for, 120, 122, 123, survival studies of, 419, 506-507
171 transfusion of (See Red Blood Cell
expiration, storage, and transportation of, transfusion)
216 transportation and shipping of, 215-216,
leukocytes reduced, 216 225
quality control of, 171 visual inspection of, 149
records of, 172 washed, 216, 223, 505, 590-591

Red cell antibodies. See also specific blood
groups
with autoantibodies, 432-435
and with HDFN, 338-340, 347-348, 413-414,
562
and with hemolytic transfusion reactions,
338-340, 413-414, 686-687
clinical significance of, 338-340, 347-348,
376, 392, 413-414, 419
defined, 391
detection of (See Antibody detection)
disease associations with, 392
distinguishing alloantibodies from
autoantibodies, 283
dosage effect of, 264, 393, 410
effect of DTT on, 413-414
effect of enzymes on, 413-414
to high-prevalence antigens, 392-393, 394-
395, 415-416, 564
high-titer, low-avidity, 409-410
identification of (See Antibody
identification)
low-affinity, 437
to low-prevalence antigens, 416
multiple, 412, 414
naturally occurring, 391, 392
nonhemolytic, 251-252
in selection of units, 379, 419-421
serologic reactivity of, 413-414
in sickle cell disease, 329-330, 379, 588
in tissue transplant patients, 777
Red cell exchange, 646, 653-654, 655, 675
Red cell losses, in apheresis, 170, 171
Red cell reduction, 720-721
Red cell substitutes, 507
Reference laboratories, 419
Refrigerators, 36, 214
Regenerative medicine, 753-754. See also Stem
cells
Registration
of donors, 118-119
of facilities, 84, 86
Regulatory issues, 83-91
for biological products, 84, 85
for blood-related devices, 83-84, 87
for cellular therapy products, 760, 796-
797
for emergencies, 109-111
FDA inspections, 86-87
hospital regulations and accreditation, 91,
603-604
for HSCs, 87-89, 725, 743-746
licensure and registration, 84, 86
medical laboratory laws and regulations,
89-91
INDEX 䡲 833
for quality systems, 1-2
for radioactive materials, 60-61
recalls and withdrawals, 89, 90
safety, 39-40, 60-61, 68-69
for tissue, 777-778
Reissuing blood products, 227-228, 550
Rejuvenated RBCs, 216
Relationship testing, 276-277, 493
Relative risk, 494
Remedial action, 26, 27
Renal failure, 652, 674
Reports
of adverse events related to tissue grafts,
782
facility quality, 27
of fatalities, 20, 45, 87, 145, 691
of injuries, 45, 87
internal event, 20, 21-22
Requests for transfusion, 367-368, 383, 546,
699-704
Requirement, defined, 34
Respiratory distress, 658-659, 680-681
Restriction fragment length polymorphism
analysis, 238
Retrospective audits, 699, 701-702, 703-704,
707
Reverse transcriptase PCR, 236-237
RFLP. See Restriction fragment length
polymorphism analysis
Rh Immune Globulin, 565-566
antepartum administration of, 564, 565
in antibody identification problems, 392
dosage for, 565-566
positive DAT after, 428
postpartum administration of, 565-566
serology and mechanism of, 566
use in platelet transfusions, 515
Rh system, 317-333
antibodies of, 331, 338
antigens of, 259, 318-319, 321-330
C/ c and E/ e, 328-330
D, 323-328
G, 328
characterization of, 317, 319
clinical considerations for, 328
ethnic differences in, 318-319, 320, 321,
325, 326, 327
genes and proteins of, 259, 272, 273, 319,
320, 321, 325
genotypes, 321, 322-323
haplotypes in, 319, 320, 321-322
phenotypes, 321-323
RhAG, 261, 331, 340, 360
Rhnull, 273, 331
terminology for, 318-319, 319, 320
834 䡲 AABB TECHNICAL MANUAL
Rh testing
with autoagglutinins, 332, 432, 438
of blood components, 225-226, 285, 327,
328, 378
for C, c, E, e antigens, 322-323
comparison with previous records, 378
in component selection, 379, 515
for D antigen, 327-328, 330
discrepancies in, 328, 333
DNA-based assays, 284-285, 287, 330
false-positive/negative results in, 332
of fetus, 283-284, 330, 563
in hemolytic disease of the fetus and
newborn, 332, 563, 565
in HSC transplantation, 634
in multitransfused patients, 330
in pediatric patients, 332, 385, 574, 587
phenotyping, 322-323
in prenatal evaluation, 284, 330, 563
reagents for, 326, 327, 331-332
of recipients, 327-328, 375
for sickle cell disease patients, 283, 329-330
for weak D, 327, 328, 375, 565
RhAG system, 261, 319, 331, 340, 360
Rheopheresis, 646, 647
Rheumatoid arthritis, 763
Riboflavin-treated plasma, 153, 205
Risks
assessment of, 98-99, 102
of bleeding, 605
relative, 494
of transfusion, 192-194, 600-601
RNA, 233
Rodgers blood group. See Chido/Rodgers
Rodgers substance, 406
Root cause analysis, 27, 28, 29
Rosette test, 565
Rotational thromboelastometry, 520
Rouleaux
in ABO testing, 298, 300
in antibody detection/identification, 416
in Rh testing, 332
saline replacement technique for, 301,
417
Run charts, 24
S Sequence-specific oligonucleotide probes, 485
Safe work practices
for biosafety, 52-53
for chemical safety, 58-59
for electrical safety, 47
for fire prevention, 47
general guidelines for, 44, 72
for radiation safety, 62
Safety goggles, 71
Safety program
accidents and injuries, 45, 49
biosafety, 47-55, 73
chemical safety, 55-60, 74-82
in disaster management, 104
electrical safety, 46-47
emergency response plan, 44
employee health services, 44-45
engineering controls, 44, 72
fire prevention, 45-46
first aid and follow-up, 44-45
hazard identification and communication,
43-44
hepatitis prophylaxis, 44
latex allergies, 45
management controls, 42-43, 44
personal protective equipment, 44, 70-71
quality management of, 7, 12
radiation safety, 60-63
regulations and recommendations for, 39-
40, 68-69
safe work practices, 44, 72
safety officers, 42, 56, 61
safety plan, 42
shipping hazardous materials, 63
training, 43
waste management, 63-64
Saline replacement technique, 301, 417
Samples, blood. See Blood samples
Sandwich ELISA, 244
Scianna system, 259, 339, 354
Scoring reactions, 372
SD (solvent/detergent-treated) plasma, 153,
205
Sda antigen, 361-362, 372, 406
Sda substance, 406
Secretors
genetics of, 301, 302, 303, 304-305
linkage with Lutheran group, 271, 272
Security, 104
Sedimenting agents, 172
Segments, of RBCs, 149
Selective absorption, 646
Sepsis, transfusion-associated, 198, 199, 669,
676
Sequence-based typing, 486
Sequence-specific primers, 485-486
Serotonin release assay, 466
Serum proteins, in typing discrepancies, 298,
301, 332
Serum-to-cell ratio, 405
Services, critical, 4, 9-10
Sex-linked inheritance, 267-269
 INDEX 䡲 835

Sexual contacts, of blood donors, 124, 125, 126 Source Plasma, 170
Sharps injuries, 49, 141 Specific gravity, of blood cells and
Shipping components, 143
blood components, 146-147, 150-151, 213- Specification, defined, 34
214, 215-220, 225 Spills
containers for, 38, 225, 724-725, 738 blood, 53, 54
in disaster planning, 107-108 chemical, 59, 78-82
frozen products, 225, 724, 738 radioactive, 62
hazardous materials, 63 “Splits,” 482
HSCs, 723-724, 734, 737-738 SPRCA. See Solid-phase red cell adherence
labeling, 738 testing
monitoring temperature during, 213-214, SSOP. See Sequence-specific oligonucleotide
724-725, 738, 739, 779-780 probes
MSCs, 761-762 SSP. See Sequence-specific primers
plasma derivatives, 220 Staffing, 9, 108-109, 110-111
samples, 63 Standard Operating Procedures. See
tissue, 220, 779-780 Procedures
Shock, 501-502, 674 Standard precautions, 48
Short tandem repeat analysis, 277 Standards
Showers, emergency, 58 for biosafety, 47
Siblings, 266, 478 for performance improvement, 26
Sickle cell disease for quality management, 1- 2
alloimmunization in, 329-330, 379, 588, for tissue transplantation, 778-779
639 Staphylococcal protein A absorption, 657
blood selection in, 379, 587-588, 654 Stem Cell Therapeutic and Research Act, 743
delayed transfusion reactions in, 588, 687 Stem cells
genotyping for, 283, 330 adipose-derived, 755, 758-759, 762, 794
in HSCT patients, 639 embryonic, 795
in pediatric patients, 587-588, 639 hematopoietic
red cell exchange in, 587, 654 collection of, 718-720, 733-734
separation of transfused from autologous cryopreservation of, 722-723, 736-737
cells in, 401 donors of, 714-717
transfusion in, 379, 587-588 infusion of, 724-725
Side effects. See Adverse reactions irradiation of, 638
Signs, safety, 46, 48-49, 57, 58 processing, 720-722
Silent mutations, 264, 267, 286-287 quality control of, 723, 736
Single nucleotide polymorphisms, 240, 264 regulation of, 87, 88, 89, 725
Sipuleucel-T, 791 shipping and transport of, 723-724, 737-
Six Sigma, 28-29 738
Skeletal repair, with MSCs, 763 sources of, 717-720
Skin appearance, in donors, 120 storage of, 736-737
Skin grafts, 123, 775, 777 thawing, 721
Solid-phase red cell adherence testing washing, 721, 740-741
for detection of HLA antibodies, 487 induced pluripotent, 755, 795-796
for detection of platelet antibodies, 463-464 mesenchymal
for phenotyping red cells, 242-243 autologous vs allogeneic use, 754
for platelet crossmatching, 460 future directions for, 765-766
for pretransfusion testing, 377, 396 identification criteria for, 793
Soluble substances, 406 isolation and expansion of, 758-760,
Solutions 792-793
additive, 136-137, 138, 576-577 multipotentiality of, 754-755, 791-792
anticoagulant-preservative, 136, 137 properties of, 756-757, 758, 786, 791-792
intravenous, 552 research and development on, 764-765
Solvent/detergent-treated plasma, 153, 203, shipping, 761-762
204, 205 sources of, 754-758
836 䡲 AABB TECHNICAL MANUAL

standardization of methods for, 760 techniques in, 607

storage and banking of, 761
therapeutic applications of, 762-764,
793-795
and tissue engineering, 795
tumor-forming potential of, 765
Sterile connection devices, 37, 139, 140, 576
Sterility testing, of HSCs, 723, 736
Storage
of biohazardous material, 52, 54-55
of blood components, 213-214, 215-220, 221
biochemical changes in, 221, 503-504,
511, 513
Cryoprecipitated AHF, 153, 218, 222
granulocytes, 173, 217, 221, 525
plasma, 151, 218-220
platelets, 150-151, 216-217, 511, 513
RBCs, 214, 215-216, 221, 503-504
red cell antigen deterioration with, 410-
411
Whole Blood, 147, 215
of blood samples, 371
in disaster plan, 108
equipment for, 214
of hazardous chemicals, 58-59
of HSCs, 736-737
liquid nitrogen, 723, 737, 761
of MSCs, 761-762
of plasma derivatives, 220
of records, 19
temperature for, 213-214, 215-220, 221
of tissue grafts, 220, 775, 780-781, 783
Storage lesion, 221, 503-504, 511, 513
STR. See Short tandem repeat analysis
Stroke, 763
Stromal-vascular fraction, 759, 762, 763
Sulfhydryl reagents, 407, 438
Suppliers, 9-10, 12, 779, 780
Supplies, critical, 9-10, 11, 107, 108
Surgery
acute normovolemic hemodilution in, 606
anemia assessment before, 604-605
anesthesia in, 607
assessing bleeding risk in, 605
blood administration in, 555-556
blood ordering practices for, 227
blood recovery in, 606-607, 608-609
deliberate hypotension in, 607
fluid management in, 607
maintaining normothermia in, 607
pharmacologic agents in, 608, 620-627
point-of-care testing in, 607-608
positioning in, 607
preoperative autologous donation for, 605-
606
temperature regulation in, 607
transfusion algorithms in, 608
Survival studies of red cells, 419, 506-507
Syncope, 144-145
Syntenic genes, 271
Syphilis, 198
reactive testing results for, 187, 188, 189
screening blood donors for, 124, 184, 198
screening tissue donors for, 774
Syringe aliquoting devices, 576
Syringe infusion pumps, 549, 584
T
T-activation, 300
TA-GVHD. See Transfusion-associated graft-
vs-host disease
TACO. See Transfusion-associated circulatory
overload
Tattoos, 124
Temperature
of antibody reactivity, 405, 413-414
of collected whole blood, 147
for component storage, 213-214, 215-220,
221
of donors, 120
monitoring systems for, 214
of recipients, 546, 676, 677
regulation of, during surgery, 607
for shipping containers, 213-214, 225, 734,
738, 739, 779-780
Teratogens, 122, 129
TerumoBCT apheresis systems, 168, 174, 175
Testing. See also specific testing methods;
Pretransfusion testing
grading results of, 372
of incoming supplies, 10
method validation, 14
point-of-care, 607-608
regulations for, 90
Thalassemia, 588-589, 640
Thawed Plasma, 151, 152, 219, 221, 522
Thawing
Cryoprecipitated AHF, 222
devices for, 37
frozen RBCs, 222
HSCs, 721
plasma, 151, 221
Therapeutic apheresis
adverse effects of, 658-660
anticoagulation in, 658
cytapheresis, 653-654, 655
extracorporeal photopheresis, 646, 654, 656
indications for, 646, 647, 648-651, 655, 656,
657

modalities of, 645, 646, 647
patient evaluation in, 660-661
principles of, 645, 646, 647
replacement fluids in, 522, 646, 652
selective absorption, 656-658
therapeutic plasma exchange, 646, 647-653
vascular access in, 660
Therapeutic plasma exchange, 647-653
adverse effects of, 658-660
in hemolytic disease of the fetus and
newborn, 564
indications for, 646, 647, 648-651, 651-653
replacement fluids for, 646, 647, 652
Thermal amplitude studies, 419, 438
Thermometers, 37
Thrombocytapheresis, 646, 654, 655
Thrombocytopathy, 509-510
Thrombocytopenia
drug-induced, 462, 465-466, 516
fetal and neonatal alloimmune, 461, 567-568
heparin-induced, 462, 465-466, 517, 528
immune, 463, 465, 517, 568
management of, 514
in pediatric patients, 580-581
in plasmapheresis, 659
platelet transfusions in, 507-517
posttransfusion purpura, 461-462, 671, 688-
690
thrombotic thrombocytopenic purpura,
517, 522, 652-653, 713
Thrombocytosis, 654
Thromboelastography, 520-521, 608
Thrombosis, in blood donors, 145
Thrombotic thrombocytopenic purpura, 517,
522, 652-653, 713
Time, incubation, 405
Timers, 37
Tissue
autografts, 782-783
collecting, 773, 782-783
expiration of, 220
hospital-based services for, 778-783
infectious disease testing on donors of, 191-
192, 774
oversight responsibility for, 778-779
processing, 775
recall of, 782
receipt and inspection of, 779-780
regulations and standards for, 87, 88, 89,
777-778
standard operating procedures for, 779
storage of, 220, 775, 780-781, 783
suppliers of, 779, 780
traceability and records of, 781-782
transplantation of, 773-777
INDEX 䡲 837
ABO compatibility in, 777
adverse events in, 782
antibody development after, 777
background of, 774-775
clinical uses for, 775-777
disease transmission through, 777
donor eligibility for, 773-774
history of, in blood donors, 123, 126
look-back investigations in, 782
and MSCs, 795
organ transplantation, 783
transfusion support for, 783
types of grafts for, 774
transporting, 220, 779-780
Tissue banks and distributors, 777
Tissue engineering, 795
Titration of antibodies, 409-410, 563
Topical hemostatic agents, 608, 625
TPE. See Therapeutic plasma exchange
Traceability, 225, 781-782
Training
biosafety, 48
cGMP and cGTP, 8
chemical safety, 56
disaster, 109, 111-112
electrical safety, 47
fire safety, 46
general safety, 43
new employees, 7-8
radiation safety, 61-62
Traits, 256, 265
TRALI. See Transfusion-related acute lung
injury
Tranexamic acid, 608, 621-622
Trans position, 272
Transcription-mediated amplification, 237-
238
Transfusion-associated circulatory overload,
669, 682-683
Transfusion-associated graft-vs-host disease,
671, 687-688
HLA system in, 489-490, 688
in neonates, 573
Transfusion-associated sepsis, 198, 199, 669,
676
Transfusion committee, 24-25
Transfusion reactions
acute hemolytic, 667, 672-675
air embolus, 669, 685
allergic, 547-548, 667, 678-680
alloimmunization, 670 (See also
Alloimmunization)
anaphylactic, 658, 668, 678-679, 680
biovigilance programs in monitoring, 665-
666
838 䡲 AABB TECHNICAL MANUAL
clinical evaluation and management of,
666, 667-671
coagulopathy, 591, 684-685
delayed hemolytic, 250, 588, 670, 686-687
febrile nonhemolytic, 489, 548, 667, 677
graft-vs-host disease, 671, 687-688
in HSCT patients, 639
hyperkalemia and hypokalemia, 555, 683-
684
hypocalcemia, 555, 658, 670, 683
hypotension, 659, 669, 678
hypothermia, 548, 572-573, 670, 685-686
identification of, 553, 555, 666
iron overload, 588, 671, 690
laboratory investigation of, 672
nonimmune hemolysis, 669, 675-676
platelet refractoriness, 458-461
posttransfusion purpura, 461-462, 671, 688-
690
records of, 672
reporting, 22
signs and symptoms of, 666, 667-671
TACO, 669, 682-683
TRALI, 668, 680-682
transfusion-related sepsis, 198, 199, 669, 676
Transfusion-related acute lung injury, 468,
489, 668, 680-682
Transfusion-related immunomodulation, 504
Transfusion safety officers, 611
Transfusion thresholds
in pediatric patients, 578-579
for platelet transfusions, 507-509, 581, 589,
636-637
for red cell transfusions, 499-501, 578-579,
610, 634
Transfusion-transmitted diseases
Babesiosis, 201
chikungunya virus, 202
dengue virus, 202
hepatitis B virus, 192, 194, 196
hepatitis C virus, 192-193, 194, 196-197
human immunodeficiency virus, 194-196
human T-cell lymphotropic virus, 197
malaria, 201-202
parvovirus B19, 203
prions, 202-203
safety precautions for, 47-55
syphilis, 198
Trypanosoma cruzi, 200-201
West Nile virus, 200
Transfusions
administration procedures for (See Blood
administration)
algorithms for, 608
auditing, 25, 697-707
in blood donors, 123, 125
chimerism after, 489-490
consent for, 545-546, 601
costs of, 602
of Cryoprecipitated AHF, 522-523, 583-584
during disasters, 101
documentation of, 226, 555
exchange, 564, 574, 579-580
fatalities due to, 20, 551, 681, 691
of granulocytes, 173, 523-526, 584
guidelines for, 611, 704
and HLA system, 488-490
in HSC transplantation, 633-640
intrauterine, 563-564
massive, 228, 386, 501-502, 591, 683-685
in medical history, 123, 370-371, 392, 417-
418, 428, 546
monitoring appropriateness of, 697-707
non-group-specific, 396
in operating room and trauma, 555-556
in organ transplantation, 783
out-of-hospital, 556
in pediatric patients, 574-591
of plasma, 517-522, 582-583
of plasma derivatives, 526-532
of platelets, 507-517, 580-581
of RBCs, 499-507, 574-580
requests for, 367-368, 383, 546, 603-604,
699-700
risks of, 192-194, 600-601
selection of components for, 375, 378-379
in urgent situations, 227-228, 385-386, 506,
555-556
of Whole Blood, 502-503
Transmissible spongiform encephalopathy,
202-203
Transplantation. See specific types of
transplantation
Transportation
of blood components, 146-147, 150-151,
213-214, 215-220, 225
containers for, 38, 225, 724-725
in disaster plan, 107-108
of frozen products, 225, 724, 738
of hazardous materials, 63
of HSCs, 723-724, 734, 737-738
labeling requirements, 738
monitoring temperatures during, 213-214,
738, 739, 779-780
of MSCs, 761-762
of plasma derivatives, 220
of samples, 63
of tissue, 220, 779-780
Trauma, blood administration in, 555-556
Travel, by blood donors, 125, 126, 202
 INDEX 䡲 839

Treponema pallidum, 191, 198 V

TRIM. See Transfusion-related
Vaccines, 44, 123, 791
immunomodulation
Validation
Trypanosoma cruzi, 184, 187, 190, 192, 200-201
of computer systems, 15-16
TTP. See Thrombotic thrombocytopenic
definition of, 34
purpura
of equipment, 15
Tumorigenicity, of stem cells, 765, 796
of processes, 14
Two-unit red cell collection, 120, 122, 123, 168,
of shipping containers, 147, 225
171-172
of test methods, 14
Type and crossmatch, 383, 384
validation plans, 14-15
Type and hold, 381, 383
Vapors, hazardous, 59
Type and screen, 382, 383, 384
Variable number of tandem repeats (VNTR),
U 277
Vascular access
UCB. See Umbilical cord blood for apheresis, 660
Ulex europaeus lectin, 295 in pediatric patients, 547, 580, 584
Umbilical cord blood for transfusions, 547
antigen expression on, 410, 411 Vasovagal reactions, 143-145, 169, 659, 678
DAT testing on, 427 Vector-borne diseases, 199-202
for transplantation Vel system, 261, 340, 361
advantages of, 729 Venipuncture, 140-141
cell expansion, 722 Verification, defined, 34
collection, 733-734 Viability assays for HSCs, 723, 736
consent for collection, 731 View boxes, 37
cryopreservation, 736-737 Viruses
donor recruitment for, 730-731 donor screening for, 194-197, 200
donor testing, 715, 716, 717, 732-733 transmitted by tissue transplants, 774, 782
economic issues regarding, 742-743 Viscoelastic coagulation testing, 520-521, 608
engraftment kinetics, 717-718 Viscosity, plasma, 652
health history and medical evaluation, Vital signs, 546, 553, 555, 724, 742
731-732 Vitamin K, 518, 622
HLA matching, 716, 736 Volume of blood
infusion, 741-742 in exchange transfusions, 580
labeling, 738, 739 in intrauterine transfusions, 564
legislation regarding, 743 in neonatal transfusions, 578
patient survival, 718 in pediatric patients, 572
post-thaw testing, 737 in whole blood collections, 120, 141-142, 143
processing, 734-736 Volume overload. See Transfusion-associated
quality control testing, 736, 739, 741 circulatory overload
receipt of, 738-740 Volume-reduction
regulations and standards, 87, 88, 89, of HSCs, 720
743-746 of platelets, 157, 223, 513-514, 581-582, 590
shipping, 734, 737-738 von Willebrand disease, 129, 523, 524, 584
storing, 736-737 von Willebrand factor, 154, 523
thawing and washing, 721, 740-741
Umbilical cord blood banks, 729-730, 742-743 W
Uniforms, 70
Warfarin, 518, 519, 622
United Network for Organ Sharing, 783
Warm autoantibodies
Urgent release of blood, 227-228, 385-386, 506,
ABO testing with, 432
555-556
adsorption of, 432-435
Urine neutralization, 362, 406
with alloantibodies, 418-419, 432-435, 438-
Urticaria (hives), 667, 678, 679-680
439
Utilities, in disaster plan, 108
antibody identification with, 418-419
Utilization of blood. See Blood utilization
disease associations with, 392
review
840 䡲 AABB TECHNICAL MANUAL
mimicking alloantibodies, 434-435
in mixed-type AIHA, 439
in phenotyping problems, 401
Rh testing with, 332, 432
specificity of, 435
transfusion with, 436-437, 506
in warm AIHA, 431-435
Warm autoimmune hemolytic anemia, 430-
437
adsorption testing in, 432-435
autoantibody specificity in, 435
blood selection in, 435-436
serologic characteristics of, 431-432
serologic problems of, 432
transfusions in, 436-437
Warmers, blood, 37, 548, 572, 584, 685-686
Washed components, 223
HSCs, 721, 740-741
for pediatric patients, 573-574, 590-591
platelets, 223, 590-591, 639
RBCs, 216, 223, 505, 590-591, 639
Waste management, 63-64
biohazardous, 53-55
chemical, 60
disposal, 54-55
radioactive, 63
treating, 55
Waterbaths, 37
WB. See Whole Blood
Weak D, 323-324
in donors, 285, 327, 328
in fetus, 565
in prenatal patients, 565
in recipients, 327-328, 375
testing for, 327, 328, 375, 565
West Nile virus, 200
reactive testing results for, 187, 188, 190
screening donors for, 184, 186, 192, 200
Western blotting, 245-246
Whole Blood, 148-149
ABO compatibility of, 375
collection of, 135-146
adverse donor reactions in, 142-146
containers for, 136-139, 140
donation intervals for, 120, 122
donor and blood identification in, 139-
140
donor care after, 142
handling after, 146-147
phlebotomy, 140-141
process of, 141
volume collected, 120, 141-142, 143
expiration of, 148-149, 215
hematocrit of, 148
indications for, 502-503
irradiated, 215
leukocyte content of, 505
processing, 146, 147-148
reconstituted, 149, 224
storage of, 215
temperature for, 147
transportation of, 146-147, 215, 225
Wipe tests, 61
Withdrawals, 89, 90
WNV. See West Nile virus
Work environment, 7, 12, 39-42
Work instructions, 17
Wristbands, patient, 368
Wrong blood in tube, 378, 384
X-Z
X-borne genes, 267-269
X chromosome inactivation, 258, 261
Xenografts, 775
Xg system, 354
antibodies of, 339, 354
genes and antigens of, 259, 354
inheritance pattern of, 258, 268
XK gene, 258, 260, 261, 269, 348
Yt system, 259, 338, 354
Zygosity, 264, 393, 410
ZZAP, 407, 438