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Diagnostic Hemoglobinopathies Laboratory Methods and Case Studies

Zia Uddin, PhD

St. John Macomb-Oakland Hospital Warren, Michigan

November 2013

Editorial Board

Diane M. Maennle, MD

Chairperson

Kenneth F. Tucker, MD

Member

Rita Ellerbrook, PhD

Member

Piero C. Giordano, PhD

Member

Kimberly R. Russell, MT (ASCP), MBA

Member

I

Contributors and Reviewers

Antonio Amato, MD Director Centro Studi Microcitemie Di Roma A.N.M.I. ONLUS Via Galla Placidia 28/30 00159 Rome, Rome Italy

Erol Omer Atalay, MD Professor, Medical Faculty Pamukkale University Kinikli, Denizli Turkey

Celeste Bento, PhD Laboratorio de Anemias Congenitas e Hematologia Molecular Servico de Hematologia, Hospital Pediatrico Centro Hospitalar e Universitario de Coimbra Portugal

Aigars Brants, PhD Scientific Affairs Manager Sebia, Inc 400-1705 Corporate Drive NorCross, GA 30093 USA

Thomas E. Burgess, PhD Technical Director, Quest Diagnostics Tucker, Georgia USA

Shahina Daar, MD, PhD Associate Professor Department of Hematology Sultan Qaboos University, Muscat Sultanate of Oman

II

Rita Ellerbrook, PhD Technical Director Emeritus Helena Laboratories, USA 1530 Lindberg Drive Beaumont, TX 77707 USA

Eitan Fibach, MD Professor, Department of Hematology Hadassah-Hebrew University Medical Center Ein-Kerem, Jerusalem Israel

Bernard G. Forget, MD Professor Emeritus of Internal Medicine Yale School of Medicine New Haven, CT 06520 USA

Piero C. Giordano, PhD Hemoglobinopathies Laboratory Human and Clinical Genetics Department Leiden University Medical Center The Netherlands

Dina N. Greene, PhD Scientific Director, Chemistry Regional Laboratories, Northern California The Permanente Medical Group Berkeley, CA 94710 USA

Rosline Hassan, PhD Professor of Hematology School of Medical Sciences University Sains Malaysia, Kelanran Malaysia

David Hockings, PhD Formerly with Isolab, USA and PerkinElmer Corporation, USA Raleigh-Durham, North Carolina USA

Prasad Rao Koduri, MD Division of Hematology-Oncology Hektoen Institute of Medical Research Chicago, Illinois 60612 USA

Elaine Lyon, PhD Associate Professor of Pathology University of Utah School of Medicine Medical Director, Molecular Genetics ARUP Laboratories, Salt Lake City, UT USA

Bushra Moiz, PhD Associate Professor Department of Pathology and Microbiology The Agha Khan University Hospital, Karachi Pakistan

Herbert L. Muncie, MD Professor, Department of Family Medicine School of Medicine, Louisiana State University 1542 Tulane Ave New Orleans, LA 70112 USA

Gul M. Mustafa, PhD Post-Doctorate Fellow Department of Pathology The University of Texas Medical Branch Galveston, TX 77555 USA

Diane M. Maennle, MD Associate Pathologist Department of Pathology St. John Macomb-Oakland Hospital

. Warren, MI 48093 USA

Jayson Miedema, MD Post-Doctorate Fellow Department of Pathology and Laboratory Medicine University of North Carolina Chapel Hill, North Carolina USA

Christopher R. McCudden, PhD Assistant Professor, Department of Pathology and Laboratory Medicine, University of Ottawa Ottawa, Ontario Canada

Michael A. Nardi, MS Associate Professor Department of Pediatrics and Pathology New York University School of Medicine New York, NY 100016 USA

John Petersen, PhD Professor, Department of Pathology The University of Texas Medical Branch Galveston, TX 77555 USA

Joseph M. Quashnock, PhD Laboratory Director PerkinElmer Genetics, Inc 90 Emerson Lane, Suite 1403 P.O. Box 219 Bridgeville, PA 15017 USA

Semyon A. Risin, MD, PhD Professor of Pathology & Laboratory Medicine Director of Laboratory Medicine Restructuring & Strategic Planning Program University of Texas Health Science Center- Houston Medical School 6431 Fannin Street, MSB, 2.290 Houston, TX 77030 USA

V

Maria Cristina Rosatelli, PhD

Professor, Dipartimnto di Scienze Biomediche

e Biotecnologie Universit degli Studi di Cagliari

09121

Cagliari, Sardina

Italy

Donald L Rucknagel, MD, PhD Professor Emeritus Department of Human Genetics University of Michigan, School of Medicine Ann Arbor, Michigan

USA

Kimberly Russell, MT (ASCP), MBA Manager & Operations Coordinator Hematology and Blood Bank St. John Hospital & Medical Center

and affiliated hospitals of St. John Providence Health System, Michigan

USA

Luisella Saba, PhD

Professor, Dipartimnto di Scienze Biomediche

e Biotecnologie Universit degli Studi di Cagliari

09121

Cagliari, Sardina

Italy

Dror Sayar, MD, PhD

Department of Pediatrics,

Hematology-Oncology

Tel Hashmer Medical Center Ramat Gan

Israel

Upendra Srinivas, MD Department of Hematology

Kokilaben Dhirubhai Ambani Hospital

& Medical Research Institute

Mumbai, Maharashtra

India

Elizabeth Sykes, MD Clinical Pathologist William Beaumont Hospital Royal Oak, Michigan USA

Ali Taher, MD, PhD Professor Medicine, Hematology & Oncology American University of Beirut Medical Center Beirut Lebanon

Kenneth F. Tucker, MD Director, Hematology & Oncology Services Webber Cancer Center St. John Macomb-Oakland Hospital Warren, Michigan USA

Zia Uddin, PhD Consultant, Clinical Chemistry Department of Pathology St John Macomb-Oakland Hospital Warren, Michigan USA

Vip Viprakasit, MD, D. Phil Professor Department of Paediatrics & Thalassemia Center Faculty of Medicine Siriraj Hospital, Mahidol University 2 Prannok Road, Bangkoi Bangkok 10700 Thailand

Dr. Henri Wajcman Director of Research Emeritus Editor-in-Chief Hemoglobin INSERM U955 (Team 11) Hospital Henri Mondor 94010 Creteil France

VII

Winfred Wang, MD Professor of Pediatrics University of Tennessee College of Medicine Pediatric Hematologist & Oncologist St Jude Children’s Research Hospital Memphis, Tennessee USA

Andrew N Young, MD, PhD Department of Pathology & Laboratory Medicine Emory University School of Medicine Atlanta, GA 30303 USA

VIII

Financial Disclosure

I neither had nor will have financial relationship with any of the manufacturers or any other organization mentioned in the book.

Similarly all the contributors and reviewers of the book have worked with gratis to further the cause of education.

This book and its translations into several languages are provided at no charge.

November 2013

Zia Uddin, PhD

IX

Dedication

This book is dedicated with heartfelt thanks to my professors responsible for my PhD level education in Chemistry at the Illinois Institute of Technology, Chicago, Illinois, and post-doctoral education and training in Clinical Chemistry at the University of Illinois Medical Center, Chicago, Illinois.

Illinois Institute of Technology, Chicago, Illinois

Professor Kenneth D. Kopple, PhD Professor Paul E. Fanta, PhD Professor Robert Filler, PhD Professor Sidney I. Miller, PhD

University of Illinois Medical Center, Chicago, Illinois

Professor Newton Ressler, PhD

November 2013

X

Zia Uddin, PhD

Preface

Higher level education is one of the blessings of God. Unfortunately, primarily due to economic and logistic reasons a vast majority of the qualified candidates are denied this opportunity.

Internet has the potential of mass education at an infinitesimal cost. This is the 3rd book launched via Internet by me at no charge.

All the MD/PhD degree holders are most respectfully requested to utilize the Internet as a means of communication to launch books at no charge in their areas of expertise.

Love

God

Love

People

Serve

The World

November 2013

Zia Uddin, PhD

XI

Acknowledgement

During the past three years I contacted worldwide >200 family physicians, clinical chemists, pathologists, hematologists, public health officials and experts in diagnostic hemoglobinopathy for formatting this book. The contribution of all of these individuals is heartfelt and very much appreciated.

I am highly indebted to the following persons for their technical support:

Diane M. Maennle, MD Rita Ellerbrook, PhD Kimberly R. Russell, MT (ASCP), MBA Jennifer Randazzo, MS (Information Technology)

The following manufacturers and organizations provided technical support, and facilities for the collection of data for the book:

Helena Laboratories, USA Sebia, France PerkinElmer Corporation, USA Bio-Rad, USA ARUP Laboratories, USA Quest Diagnostics, USA College of American Pathologists, USA Seven Universities and four Newborn Screening Laboratories, USA (names are with held as per their request)

Mr. Mathew Garrin, Biomedical Communications and Graphic Arts Department, Wayne State University, School of Medicine, Detroit has worked on the figures, scans, and layout of the book. I am very grateful to him for his contribution.

Finally, I would like to thank the following persons for facilitating my work:

Adrian J. Christie, MD, Medical Director of Laboratories St. John Macomb-Oakland Hospital, Warren, Michigan, USA Anoop Patel, MD, Assistant Systems Medical Director St John Providence Health System Laboratories, Warren, Michigan, USA Mr. Tipton Golias, President & CEO Helena Laboratories, Beaumont, Texas, USA

November 2013

XII

Zia Uddin, PhD

Table of Contents

Chapter 1

Hemoglobin

1

Thomas E. Burgess, PhD

1.1 Hemoglobin Structure

1.2 Hemoglobin Function

1.3 Hemoglobin Synthesis

1.4 Hemoglobin Variants

Chapter 2

Diagnostic Laboratory Methods

2.1 Basic Concepts

10

Jayson Miedema, MD and Christopher R. McCudden, PhD

2.1.1 Unstable Hemoglobins

2.1.2 Altered Affinity Hemoglobins

2.1.3 Sickle Solubility Test

2.1.4 Serum Iron, TIBC, Transferrin, Ferritin

2.1.5 Soluble Transferrin Receptor

2.1.6 Hepcidin

2.2 Microcytosis

21

Diane Maennle, MD and Kimberly Russell, MT (ASCP), MBA

2.3 Hereditary Persistence of Fetal Hemoglobin

Bernard G. Forget, MD

28

2.3.1 Introduction

2.3.2 Deletions Associated with the HPFH Phenotype

2.3.3 Non-Deletion Forms of HPFH

2.3.4 HPFH Unlinked to the β-Globin Gene Cluster

2.3.5 Conclusion

2.3.6 Hemoglobin F Quantification

XIII

2.4

Flow Cytometry Measurements of Cellular Fetal Hemoglobin, Oxidative Stress and Free Iron in Hemoglobinopathies 41

Eitan Fibach, MD

2.4.1 Flow Cytometry of Blood Cells

2.4.2 Measurement of Fetal Hemoglobin-Containing Erythroid Cells

2.4.3 Staining Protocols for F-RBCs and F-Retics (15)

2.4.4 F-Cell Determination for Fetal-Maternal Hemorrhage (FMH) in Pregnant Patients wit β-Thalassemia- A single Case and General Conclusion (16)

2.4.5 Oxidative Stress

2.4.6 Staining Protocols for ROS and GSH

2.4.7 Intracellular Free Iron

2.4.8 Staining Protocol for LIP

2.5 Solid Phase Electrophoretic Separation

61

Rita Ellerbrook, PhD, and Zia Uddin, PhD

2.5.1 Introduction

2.5.2 Cellulose Acetate Electrophoresis (alkaline pH)

2.5.3 Agarose Gel Electrophoresis (alkaline pH)

2.5.4 Agar Electrophoresis (acid pH)

2.5.5 Interpretation of Hemoglobin Agarose Gel (pH 8.6) and Agar Gel (pH 6.2) Electrophoresis

2.5.6 Requirements for the Identification of Complex Hemoglobinopathies

2.6 Capillary Zone Electrophoresis

Zia Uddin, PhD

73

2.6.1

Introduction

2.6.2

Basic Principle

2.6.3

Application of CZE in Diagnostic Hemoglobinopathies

2.6.4

Interpretation of CZE Results

XIV

2.7 Isoelectric Focusing

83

David Hockings, PhD

2.7.1 IEF of Normal Adult Hemoglobin: HbA (Adult), HbF ( Fetal), HbA 2

2.7.2 IEF of Normal Newborn Hemoglobins: HbF (Fetal) and HbA (Adult)

2.7.3 IEF of Beta-Chain Variant Hemoglobins

2.7.4 IEF of Alpha Chain Variant Hemoglobins

2.7.5 IEF of Thalassemias

Chapter 3

2.8 High Performance Liquid Chromatography

Zia Uddin, PhD

2.8.1 Introduction

2.8.2 Basic Principle

2.8.3 Illustrations

95

Globin Chain Analysis

3.1 Solid Phase Electrophoretic Separation

Zia Uddin, PhD

102

3.1.1 Cellulose Acetate Electrophoresis (Alkaline and Acid pH)

3.2 Reverse Phase High Performance Liquid Chromatography

106

Zia Uddin, PhD, and Rita Ellerbrook, PhD

3.3 Globin Chain Gene Mutations: DNA Studies

Joseph M. Quashnock, PhD

115

3.3.1 Introduction

3.3.2 Genotyping-PCR Methodology

3.3.3 Mutations

XV

3.4

Electrospray Ionization-Mass Spectrometry

132

Gul M. Mustafa, PhD and John R. Petersen, PhD

3.5 PCR and Sanger Sequencing

Elaine Lyon, PhD

147

3.5.1. Alpha Globin

3.5.2 Beta Globin

3.5.3 Sequencing

3.5.4 Reporting Sequence variants

3.5.5 DNA Sequence Traces

3.5.6 Conclusion

Chapter 4

Alpha and Beta Thalassemia

Herbert L. Muncie, MD.

4.1 Epidemiology

4.2 Pathophysiology

4.3 Alpha Thalassemia

4.4 Beta Thalassemia

4.5 Diagnosis

4.6 Treatment

4.7 Complications

157

4.8 Other Treatment Issues

4.8.1 Hypersplenism

4.8.2 Endocrinopathies

4.8.3 Pregnancy

4.8.4 Cardiac

4.8.5 Hypercoagulopathy

4.8.6 Psychosocial

4.8.7 Vitamin Deficiencies

4.8.8 Prognosis

XVI

Chapter 5

Neonatal Screening for

Zia Uddin, PhD

 

Hemoglobinopathies

178

5.1 Introduction

5.2 Methodologies

5.3 Laboratory Reports Format & Interpretation

5.4 Examples of Neonatal Screening

 
 

5.4.1.

Capillary Zone Electrophoresis

5.4.2

Isoelectric focusing

5.4.3

Isoelectric focusing and High Performance Liquid Chromatography

5.4.4

Isoelectric focusing, High Performance Liquid Chromatography and DNA studies

5.5 Genetic Counseling & Screening

 

Chapter 6

Prenatal Diagnosis of Beta-Thalassemia

and Hemoglobinopathies

202

Maria Cristina Rosatelli, PhD, and Luisella Saba, PhD

Chapter 7

Hemoglobin A 1 c

232

 

Zia Uddin, PhD

7.1 Introduction

7.2 HbA1 c Diagnostic Role in Diabetes Mellitus, and Glycemic Control in Adults

7.3 Measurement of HbA1 c

7.4 Factors Affecting the Accuracy of Hb A1 c Assay

XVII

Case Studies

244

Introduction

Case # 1

Normal Adult

247

Case # 2

Hemoglobin S trait

252

Case # 3

Hemoglobin S homozygous

258

Case # 4

Hemoglobin S with hereditary persistence

of fetal hemoglobin (HPFH)

264

Case # 5

Hemoglobin G-Philadelphia trait

272

 

Case # 6

Hemoglobin S-G Philadelphia

279

Case # 7

Hemoglobin G-Coushatta trait

287

Case # 8

Hemoglobin C trait

293

Case # 9

Hemoglobin C homozygous

299

Case # 10

Hemoglobin C with hereditary persistence

of fetal hemoglobin (HPFH)

306

Case # 11

Hemoglobin S-C disease

312

Case # 12

Hemoglobin D-Los Angeles (D-Punjab) trait

319

Case # 13

Hemoglobin S-D disease

326

XVIII

Case # 14

Hemoglobin E and Associated Disorders

333

Case # 14 a

Hemoglobin E trait

339

Case # 14 b

Hemoglobin E homozygous

344

Case # 14 c

Hemoglobin S-E disease

350

Case # 15

Hemoglobin S-Korle Bu (G-Accra)

356

Case # 16

Hemoglobin O-Arab trait

362

Case # 17

β-Thalassemia trait

368

Case # 18

Hemoglobin S-β + - thalassemia

374

Case # 19

Hemoglobin C-β o thalassemia

381

Case # 20

Hemoglobin Hasharon trait

387

Case # 21

Hemoglobin Zurich trait

394

Case # 22

Hemoglobin Lepore trait

400

 

Case # 23

Hemoglobin J-Oxford trait

408

Case # 24

Hemoglobin J-Baltimore trait

415

Case # 25

Hemoglobin Malmo trait

421

 

Case # 26

Hemoglobin Koln trait

432

Case # 27

Hemoglobin Q-India trait

441

 

Case # 28

Hemoglobin Dhofar trait

454

XIX

Chapter 1

Thomas E. Burgess, PhD

Hemoglobin

To attempt a full treatise on hemoglobin in this textbook would be an effort in

futility as the purpose is not to duplicate knowledge already present in the literature.

Rather, this chapter is to provide basic information to the reader which will allow him/her

to properly identify hemoglobin variants in their laboratory. A basic knowledge of the

hemoglobin molecule is absolutely critical to that effort and the sections printed below

are written expressly for that purpose. For a complete treatise on hemoglobin, textbooks

such as Disorders of Hemoglobin 1 edited by Steinberg, Forget, Higgs and Nagel should

be consulted.

1.1 Hemoglobin Structure

Composed of 2 distinct globin chains, the complex protein molecule known as

hemoglobin (“heme” + “globin”) is arguably THE primary component of the red blood cell

in human beings. In “normal” adults, the globin chains are either alpha (α), beta (β),

gamma (ϒ) or delta (δ). In addition, during embryonic life in utero, zeta (ζ) and epsilon

(ε) chains are present in the first several weeks of life, being rapidly converted to alpha,

beta and gamma chains as development occurs.

1

Figure 1. stages of life (Huehns ER, Dance N, Hecht S, Motulsky AG. Human embryonic

Figure 1.

stages of life (Huehns ER, Dance N, Hecht S, Motulsky AG. Human embryonic hemoglobins. Cold Spring Harbor Symp Quant Biol 1969; 29: 327-331). Adopted with permission from Blackwell Publishing (Barbara J. Bain, Haemoglobinopathy Diagnosis, 2 nd Edition, 2006).

Globin chains concentration changes in embryonic, fetal and post-natal

Each of these globin chains has associated with it a porphyrin molecule

known as heme whose primary function in the red blood cell is the facilitation of

transport of oxygen to the tissues of the human body. The globin portion of the

molecule serves several functions, not the least of which is protection. The internal

pocket of the molecule formed from the convergence of the four globin chains,

2

provides a hydrophobic environment in which the heme molecules reside. This

pocket protects the heme from oxidation and facilitates oxygen transfer to the

tissues of the body. The previously mentioned ζ and ε chain-containing

hemoglobins have very high oxygen affinities, a factor very important in the early

embryonic life of the fetus.

The hemoglobin molecule can be looked at in four different ways; primary,

secondary, tertiary and quaternary structural views. While outside of the scope of

this volume, each of these structures contributes definitive unique properties to the

various hemoglobin molecules from normal hemoglobins to the very rare and

functionally diverse molecules. The primary structure of all hemoglobins is the order

of amino acids found in the globin chains of the molecule. It is this unique sequence

that is the major differentiator of hemoglobin from each other. The secondary

structure of hemoglobin is the arrangement of these amino acid chains into alpha

helices separated by non-helical turns 2 . The tertiary structure is the 3-dimensional

arrangement of these globin chains forming the “pocket” of hemoglobin that cradles

the iron molecule in its grasp. The quaternary structure is the moving structure of the

molecule that facilitates the oxygenation of the heme molecules in response to the

physiological needs of the human body.

3

Figure 2. hemoglobin molecule (Adopted with permission from Blackwell Publishing, Barbara J. Bain, Haemoglobinopathy

Figure 2.

hemoglobin molecule (Adopted with permission from Blackwell Publishing, Barbara

J. Bain, Haemoglobinopathy Diagnosis, 2 nd Edition, 2006).

Tertiary structure of a β globin chain and the quaternary structure of

The forthcoming sections will elucidate the effects that these structural

considerations have on the hemoglobin molecule and, more specifically, the

abnormal and atypical hemoglobin variants.

1.2 Hemoglobin Function

As mentioned above, the primary function of hemoglobin is to reversibly

transport oxygen to the tissues of the body. In addition, however, this flexible

molecule can also transport carbon dioxide (CO 2 ) and nitrous oxide (NO).

The

transport of CO 2 is facilitated by reversible carbamoylation (formation of carbamoyl

moiety, i.e., H 2 NCO-) of the N-terminal amino acids of the α globin chains and can,

4

via proton scavenging, keep CO 2 in the soluble bicarbonate form 3 .

Nitrous oxide is

handled in two different ways by hemoglobin: one as a transporter and the other as

a scavenger. Blood levels of NO are therefore, by definition, a balance between NO

production and NO removal by binding to oxyhemoglobin. Since NO is an extremely

potent vasodilator, hypoxic patients will have lower oxyhemoglobin and therefore

higher amounts of free NO. This free NO can cause significant vasodilation, a

physiological effect that is very desirable in hypoxia.

All hemoglobin molecules, either normal or variant, share the same

functionality in the human body. Therefore, the primary structural differences

mentioned above and in more complete treatises (i.e., amino acid

substitutions/deletions) will be the prime reason for functional differences. It is these

amino acid variances that, along with the secondary, tertiary and quaternary

structural differences, will determine if the variant hemoglobin is either benign or

clinically important.

The bottom line is this whether the hemoglobin is normal or variant in

nature, the prime reason for determining the hemoglobin phenotype of the patient is

to assess the functionality of the hemoglobin. If the variant is normally functioning in

both the heterozygous and homozygous states, the clinical picture is benign. If,

however, the variant has normal properties in the heterozygous state (i.e., “trait”) but

clinical issues in the homozygous state (i.e., “disease”), the phenotypic analysis and

subsequent interpretation becomes ultimately important to the patient.

5

1.3

Hemoglobin Synthesis

The synthesis of hemoglobin, as mentioned before, is under the control of

gene loci on two chromosomes: chromosome 11 (the beta globin or “non-alpha

gene) and chromosome 16 (the alpha globin gene). Hemoglobin variants (alpha,

beta, gamma, delta and fusion) are the result of alterations in the nucleotide

sequences of the globin genes and can occur for more than one reason. Mutations

such as point mutations, insertions and deletions can have major, minor or no

influences on hemoglobin function or structure. That being said, the site of the

synthetic variance can in some cases alter the ability of the hemoglobin molecule to

function in a normal manner, i.e., stability, oxygen affinity, solubility or other critical

functions. These alterations truly determine whether the variant hemoglobin is

classified as benign (i.e., no abnormal or pathological effect) or pathological (a

significant physiological effect).

The actual nature of the alteration is not of initial

importance to the hemoglobinopathy interpreter.

However, once assigned, the

identity of the variant hemoglobin may become of importance when looking at

second generation offspring from the variant carrier, i.e., the pregnant female. For

most hemoglobin variants, the synthetic pathway is of no clinical interest in that the

resulting hemoglobin is benign. It may, however, be of academic interest in that the

identification of the synthetic anomaly can, indeed point to the genetic locus or loci

involved in the alteration, thus giving information to the genetic counselor as to

possible genetic details of the hemoglobinopathy.

6

As mentioned before, the true reason for identifying the abnormal hemoglobin

or hemoglobins in patients is to identify any associated functional anomalies

associated with these hemoglobins. The actual hemoglobin identification in and of

itself is merely of academic interest.

1.4 Hemoglobin Variants

All hemoglobin variants have one thing in common they all involve the

hemoglobin molecule and its functionality.

Whether alpha, beta, gamma, delta,

fusion variant, etc., the variant and its effect are judged not on its migration or

concentration but rather on its functionality.

The amino acid variation (e.g., glutamic

acid → valine at position 6 on the beta chain for hemoglobin S) is the prime effector

of the variant’s functional alteration(s) and will in most cases be the causative factor

in any abnormal migration that the variant may have versus the “normal”

hemoglobins (A, F, A 2 ).

Most variants therefore will have altered electrophoretic or

chromatographic migrations when compared to the normal variants. Some, such as

hemoglobin Chicago, are not separable by normal electrophoretic techniques and

rely on high performance liquid chromatographic (HPLC) separations to identify its

presence in the blood. As previously mentioned, the presence of variant “traits” (i.e.,

AS, sickle trait) may or may not be of clinical consequence. Where these traits

really are of importance is in the homozygous state (i.e., SS for hemoglobin S). The

clinical picture dramatically changes with significant physiological changes being

directly associated with the homozygous state. This therefore requires the

interpreter to have several pieces of information specific to the patient at hand

7

during the interpretation of the hemoglobinopathy. This data includes, but is not

limited to, pregnancy, transfusion history and ethnicity. All of these pieces of

information can be critical to the proper identification/interpretation of the

hemoglobin variant in the patient’s specimen. For example, an elevation of

hemoglobin F in a female patient with a normal hemogram may be evidence of

hereditary persistence of fetal hemoglobin; whereas, if this female is pregnant, the

elevation may be a normal physiological response to the fetal presence in her body.

These data may not be readily available and may require contact with the ordering

healthcare professional to obtain these facts. However obtained, they are

necessary for the proper identification of the hemoglobin variant or variants in the

patient’s bloodstream and therefore are important in the assignment of a benign or

pathological assessment of the variant hemoglobin.

The variants described in the following chapters all obey the aforementioned

differences, i.e., amino acid substitutions, genetic deletions, sequence modifications,

etc. While not critical, the exact identification of the variant in and of itself is not

normally life-threatening, especially in the heterozygous state, i.e., “trait”. It is

essential that the variant be properly identified as a mis-identification can lead to

other issues. For example, a mis-interpretation of a hemoglobin G trait (AG) as a

sickle trait (AS), while not in and of itself is clinically an issue, presents real

difficulties for a couple expecting a child. If both partners are AS, there is a 1 in 4

chance that a child born to this couple could be homozygous SS or sickle cell

disease. In the case of an AS mother and an AG father (or vice versa), there is a 1

in 4 chance of a child being born with a phenotype of SG. While on the surface this

8

may appear as a problem, the SG phenotype is no more of a clinical issue than a

simple AS trait. Without the exact identification of the AG trait, the interpretation and

action taken by attending clinicians may be very different.

References

1. Steinberg, MH, Forget, BG, Higgs, DR and Nagel, RL., Disorders of Hemoglobin, Cambridge University Press, 2001.

2. Bain, Barbara J Publishing, 2006.

3. Bain, Barbara J Publishing, 2006.

in Hemoglobinopathy Diagnosis, 2 nd Ed., pg. 4, Blackwell

in Hemoglobinopathy Diagnosis, 2 nd Ed., pg. 1, Blackwell

9

2.1 Basic Concepts

Chapter 2 Diagnostic Laboratory Methods

Jayson Miedema, MD, and Christopher R. McCudden, PhD

2.1.1 Unstable Hemoglobins

Unstable hemoglobins are characterized by disorders in globin production which

affect the lifespan of the hemoglobin molecule and subsequently the cell leading to

decreased cell stability and increased cell turnover. There are a large number of specific

variants which can result in abnormal hemoglobin production, the most commonly

reported of which is Hb Koln. Many of these abnormal globin chains are a result of

single mutations in the form of deletions (e.g. Hb Gun Hill), insertions (e.g. Hb

Montreal), or substitutions (e.g. Hb Koln) and can result in weakened heme-globin

interactions, subunit interactions, or abnormal folding. These disorders are most

commonly expressed in the heterozygous form, most homozygous situations result in

preterm lethality.

Clinically, these patients often present with symptoms of hemolytic anemia which

can be of varying severity. Symptoms of hemolytic anemia include hyperbilirubinemia,

jaundice, splenomegaly, hyperbilirubinuria or pigmenturia as well as the formation of

Heinz bodies. This pheonotype can present or be exacerbated by infections as well as

certain types of drugs. Specifically sulfonamides, pyridium, and antimalarials are known

to cause exacerbation. Parvovirus can also induce aplastic crisis andHbA 2 and HbF

may be increased. The peripheral smear often shows anisocytosis, poikilocytosis,

basophilic

stippling,

polychromasia

and,

10

hypochromasia.

Since

not

all

unstable

hemoglobins will give abnormal results on HPLC or electrophoresis and/or these results

can be somewhat non-specific, more definitive testing is often performed.

Testing for unstable hemoglobins relies on their decreased stability in heat or

isopropanol alcohol. While normal hemoglobins should be relatively stable in these

conditions, hemoglobins with mutations causing instability tend to be less so and will

precipitate out of solution in these environments. In the context of heat stability testing,

the amount of unstable hemoglobin in a sample is given by the following equation:

(Hb4°C-Hb50°C)/(Hb4°C)x100

Where Hb4°C is the hemoglobin concentration at 4 degrees centigrade and Hb50°C is

the concentration of hemoglobin at 50 degrees centigrade.

False positives may result from samples greater than 1 week in age as well as from

samples with large amounts of fetal hemoglobin. Additional technical and clinical

information

on

obtained from:

hemoglobinopathies

associated

with

unstable

hemoglobin

can

be

2.1.2 Altered Affinity Hemoglobins

Similar to how certain types of mutations can cause instability of the hemoglobin

molecule, other mutations can cause hemoglobins to have altered affinity for oxygen.

These mutations can be single point mutations, insertions, deletions, elongation,

deletion/insertion mutations and are often named after the city in which they were

discovered (Chesapeake, Capetown, Syracuse, etc.). Both alpha-chain variants, e.g. Hb

Chesapeake, and beta-chain variants, e.g. Hb Olser, Hiroshima, Andrew-Minneapolis,

11

etc., are known in the literature for altered affinity for oxygen. Many of these are

probably

clinically

insignificant

but

when

significant

most

commonly

present

phenotypically as an increase in oxygen affinity often times resulting clinically in

polycythemia (secondary to the bodies perceived lack of oxygen and subsequent

increase in erythropoietin). Measurement of hemoglobin affinity (p50) is critical to the

diagnosis. Conversely and less frequently described, a decreased affinity for oxygen

can lead to clinical cyanosis.

Testing for altered affinity hemoglobins relies on subsequent changes to the

oxygen dissociation curve and the partial pressure of oxygen at which hemoglobin is

50% saturated, the p50. Because most types of altered affinity hemoglobins cause an

increase in oxygen binding, a left shift in the oxygen dissociation curve results.

Automated systems are available for recording the oxygen dissociation curve and rely

on a Clarke electrode to measure oxygen tension while oxyhemoglobin fraction is

measured by dual wavelength spectrophotometer. Abnormal oxygen dissociation curves

are primarily caused by altered affinity hemoglobins but can also be caused by such

factors

as

pH,

temperature,

pCO2,

and

2,3-diphosphoglycerate

(2,3-DPG).

Measurement of pO2, pCO2, pH and SO 2 allows for an estimation of p50 to be

calculated.

2.1.3 Sickle Solubility Testing

Sickle cell anemia is a disease resulting in anemia and painful crises, seen

almost exclusively in African Americans. These crises are caused by inappropriate

aggregation of deformed blood cells in small blood vessels. Widely believed to have

12

thrived in the gene pool because of its protective effects against malaria, it affects a

large number of people of African descent in its homozygous and clinically significant

form. An even greater number of people have sickle cell trait (approximately 8-10% of

African Americans), the heterozygous form, which is largely insignificant from a clinical

standpoint.

Sickle cell testing can be performed in a variety of ways and is currently most

commonly tested via hemoglobin electrophoresis when necessary. However, another

form of testing is known as sickle solubility testing which relies on the property of

increased cell fragility as a result of the glutamic acid to valine substitution at the 6 th

position of the beta globin gene, the most common genetic abnormality of sickle cell

anemia. Sickled red blood cells are soluble when oxygenated but upon deoxygenation

tend

toward

sickling,

polymerization,

and

precipitation.

The

addition

of

sodium

metabisulfite reagent to a sample with hemoglobin S promotes deoxygenating and cell

lyses, creating turbidity in the solution. This turbidity makes it difficult to read a card

through the test tube. A negative test is one in which a card can be read through the

tube, a positive test is one in which the card cannot be read.

Several types of hemoglobins can cause false positives (for example some

types of hemoglobin C) so results should be confirmed by electrophoresis; in other

words, when used, solubility testing should be used as a screening test. The test also

fails to differentiate sickle cell trait (a single copy of the sickle cell gene, heterozygous)

from true sickle cell anemia (both copies are sickle cell, homozygous). Samples with low

hemoglobin concentration (<8%) should be doubled as this low concentration can lead

to false negatives. False positives can occur in the settings of lipemia or samples with

13

monoclonal proteins (dysproteinemia). Both positive and negative controls should be

used as results can be somewhat subjective

2.1.4 Serum Iron, TIBC, Transferrin, and Ferritin

Iron is essential for numerous metabolic functions in the body through its

incorporation into proteins involved in oxygen delivery (hemoglobin, myoglobin) and

electron transport and exchange (cytochromes, catalases). While a detailed description

of iron metabolism is beyond the scope of this compendium (interested readers should

seek the references below), it is worth considering the major mechanisms of iron

homeostasis in the context of erythropoiesis. Iron intake in the diet occurs either as free

iron or as heme. Free iron, in the form of Fe 3+ , requires reduction to Fe 2+ by enzymes

and transporters to cross the intestinal mucosa; heme iron is absorbed directly by

mucosal cells where it is split from heme intracellularly. Once absorbed by the GI tract,

iron is either stored in association with ferritin or transported into the circulation in the

ferric (Fe 3+ ) form. Because of the toxicity of ferric iron, it is transported in the circulation

bound to transferrin. The main target of transferrin-bound iron is erythroid tissue, which

takes up iron through receptor-mediated endocytosis.

As dietary absorption accounts

for <20% of the daily requirement, iron recycling plays an essential role in maintaining

iron

stores.

During

recycling,

senescent

red

blood

cells

are

phagocytosed

by

macrophages in the spleen, liver, and bone marrow.

Macrophages store some iron

(bound to ferritin), but most is returned to red cell precursors via transferrin.

Unlike

dietary absorption, iron excretion is largely unregulated, where losses occur via

epithelial cell sloughing in the skin and GI tract or through menstrual bleeding in

14

premenopausal women. Accordingly, body stores depend on controlling iron uptake in

the GI tract and recycling.

Disorders of iron homeostasis fall into diseases of excess or deficiency.

Iron

deficiency is common, particularly in women, and may result from inadequate intake,

blood loss, and pregnancy; in chronic disease iron deficiency is also common.

Iron

excess may occur in hemochromatosis or as a result of repeated transfusions.

Clinically, iron status is assessed by measurement of serum iron, ferritin, transferrin,

and total iron binding capacity (TIBC).

Serum or plasma iron levels can be directly measured using several different

methods.

Most commonly, a colorimeteric reaction scheme is used in which iron is

separated from transferrin at low pH (~4) and then reduced to Fe 2+ for dye binding; the

color-complex is detected between 530-600 nm spectrophotometrically.

Although iron

is typically increased in cases of iron excess and decreased in cases of deficiency,

serum iron measurement by itself is not particularly useful for diagnosis of iron

homeostasis disorders because of the high intra-individual variation in circulating iron

levels.

Total

homeostasis.

iron

binding

capacity

(TIBC)

is

another

test

used

to

assess

iron

TIBC can be measured or calculated.

TIBC is measured by adding

excess iron to saturate transferrin (usually transferrin is 30% saturated). Unbound iron

is chelated and removed and then the remaining transferrin-bound iron is measured as

described above yielding the total capacity.

This method can be affected by the

presence

of

non-transferrin

iron

hemochromatosis and thalassemias.

binding

proteins,

particularly

in

cases

of

Alternatively, TIBC may be calculated based on

15

the stoichiometric relationship between transferrin and iron (2 molecules of iron are

bound to each molecule of transferrin).

TIBC is calculated from measured transferrin

using the following equation: TIBC (µg/dL) = 1.43 × transferrin (mg/dL). Conversely, the

concentration of

transferrin may be calculated from measured TIBC as follows:

Transferrin (mg/dL) = 0.7 × TIBC (µg/dL).

TIBC is increased in iron deficiency and

decreased in chronic anemia of disease and in iron overload (it may be normal or

decreased in thalassemia).

From TIBC and serum iron measurement, it is also possible to calculate the %

transferrin saturation (also known as iron saturation) using a simple formula: %

saturation = serum Fe (µg/dL) / TIBC (µg/dL) ×100.

The percent saturation is usually

between 20-50%, supporting an excess capacity for iron binding.

In cases of iron

overload, the % saturation increases dramatically.

Saturation is moderately increased

in thalassemia and chronic anemia and in iron deficiency the saturation is decreased.

Ferritin is a large ubiquitous protein and the major iron storing protein in the

body.

Ferritin serves to store thousands of iron atoms/molecule in a non-toxic form

acting as an iron reserve. Ferritin is found in small amounts in the blood, where it can

be measured as an indication of overall iron reserves (1 ng/mL serum iron approximates

10 mg total storage iron).

referred to as apoferritin.

In the blood, ferritin is generally poor in iron content and is

Circulating ferritin (or apoferritin) is measured using specific

antibodies, commonly by chemiluminescent immunoassay.

Serum or plasma ferritin

levels are produced in proportion to dietary iron absorption; serum ferritin is increased

with iron overload and decreased in iron deficiency.

Serum ferritin levels change prior

to clinical and morphological manifestations of anemia (e.g. microcytosis) making it a

16

useful diagnostic marker of iron homeostasis. While considered the most useful of the

currently available tests for non-invasively assessing iron stores, ferritin is also an acute

phase reactant and may be normal or even increased when chronic infection or

inflammation

occurs

in

combination

with

underlying

iron

deficiency

anemia.

In

thalassemias, ferritin is typically elevated reflecting a state of iron overload; in contrast,

ferritin is decreased in iron deficiency making it a useful marker to differentiate causes

of microcytosis.

Transferrin is an iron transporting protein and negative acute phase reactant

produced primarily by the liver.

As with ferritin, transferrin is routinely measured by

immunoassay.

Most circulating iron is bound to transferrin, binding to Fe 3+ with very

high affinity.

Transferrin transports iron absorbed in the GI tract to cells containing

specific receptors, in particular erythroid tissue. Transferrin delivers iron to cells via the

ubiquitously distributed transferrin receptor.

Clinically, measurement of transferrin is

useful for hypochromic microcytic anemia workups.

Transferrin is increased in iron

deficiency anemias, but normal or decreased in chronic anemia of disease, iron

overload, and thalassemias.

Transferrin is decreased in cases of liver disease,

nephropathy (or other protein loss or malabsorption), and inflammation.

17

Table 1. Iron Tests in Different Disorders

Disorder

Serum

TIBC

%

Transferrin

Ferritin

Iron

Saturation

Chronic

Anemia

↔ or ↓

↔ or ↑

of Disease

Iron Deficiency

Thalassemia

↔ or ↑

↔ or ↑

↔ or ↓

↔ or ↑

Hemochromatosis

↑↑

↔ or ↓

↑↑

↓decreased; ↔ within reference interval; ↑ increased

2.1.5 Soluble Transferrin Receptor

An additional test that is useful for diagnosis of anemia is the soluble transferrin

receptor (sTfR).

The sTfR consists of the N-terminus of the membrane receptor that

can be measured in circulation.

Circulating levels reflect the activity of the erythroid

bone marrow, where sTfR levels are decreased in cases of low red cell synthesis (renal

failure and aplastic anemia) and increased in patients with hemoglobinopathies.

The

utility of sTfR measurement is that it can differentiate iron deficiency in cases of acute

inflammation because sTfR levels are not affected by inflammatory cytokines.

In

thalassemias, sTfR levels are generally increased in proportion to the severity of the

genotype. Despite the apparent advantages, sTfR testing is not widely used and is not

currently standardized.

18

2.1.6

Hepcidin

Discovered in 2000, hepcidin is a hormone involved in iron homeostasis.

Hepcidin is produced by the liver and negatively regulates iron balance by inhibiting

macrophage recycling and decreasing intestinal absorption. Thus, when iron stores are

replete, hepcidin levels are increased and when iron stores are low, hepcidin is

elevated.

Similar to ferritin, hepcidin is an acute phase reactant, making interpretation

of circulating levels in patients with inflammation more challenging.

At the

time of

writing, hepcidin testing was not available commercially. The hepcidin in human iron

stores and its diagnostic implications has been recently reviewed (Kroot JJC, Tjalsma

H,

Fleming

RE,

Swinkels

DW.

Hepcidin

in

Human

Iron

Disorders:

Diagnostic

Implications: Clin Chem 2011; 57(12): 1650-1669).

Additional Readings Fairbanks VF, Klee GG. Biochemical aspects of hematology. In Fundamentals of Clinical Chemistry. Edited by Tietz N. Saunders,1987,789-818.

Guarnone R, Centenara E, Barosi G. Performance characteristics of hemox-analyzer for assessment of the hemoglobin dissociation curve. Haematologica 1995;80:426-430.

Pincus MR and Abraham NZ. Interpreting laboratory results. In: Henry's Clinical Diagnosis and Management by Laboratory Methods (Clinical Diagnosis & Management by Laboratory Methods) Edited by McPherson RA and Pincus MR. 21 st Edition.

Higgins T, Beutler E, Doumas BT. Hemoglobin, Iron and Bilirubin. In Tietz textbook of clinical chemistry and molecular diagnostics. Edited by Burtis CA, Ashwood ER, Bruns DE. Elsevier Saunders, 2006,1165-1208.

Marengo-Rowe AJ. Structure-function relations of human hemoglobins. Proc (Bayl Univ Med Cent) 2006;19:239-245.

Mayomedicallaboratories.com/test-catalog. Accessed April 20, 2011.

19

Rees DC, Williams TN, Gladwin MT. Sickle-cell disease. The Lancet. 2010;376:2018-

2031.

Steinberg MH. Genetic disorders of hemoglobin oxygen affinity. www.uptodate.com. Accessed April 28, 2011.

Steinberg MH. Unstable hemoglobin variants. www.uptodate.com. Accessed April 28,

2011.

Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. Edited by Burtis CA,

Ashwood ER, and Bruns DE. 5

th

Edition.

Vichinsky EP. Sickle cell trait. www.uptodate.com. Accessed April 28, 2011.

20

Chapter 2 Diagnostic Laboratory Methods

2.2 Microcytosis

Diane Maennle, MD, and Kimberly Russell, MT (ASCP), MBA

Smaller-than-normal size of Red Blood Cells (RBCs) is defined as microcytosis.

This is quantified by calculating the mean corpuscular volume (MCV) using the following

formula employing the values of hematocrit and RBC count:

MCV = Hematocrit (HCT) X 10 / RBC Count (Million)

In adults, a MCV value of less than 80fL is defined as microcytosis. In pediatric

subjects, the MCV and hemoglobin range distinctly vary with age (Table I).

Table I

Age Dependent Mean Hemoglobin and MCV Values 1,2,3,4

Age

Mean Hemoglobin (g/dL)

Mean MCV (fL)

3

to 6 months

11.5

91

6

months to 2 years

12.0

78

2

to 6 years

12.5

81

6

to 12 years

13.5

86

12

to 18 years (female)

14.0

90

12

to 18 years (male)

14.5

88

> 18 years (female)

14.0

90

> 18 years (male)

15.75

90

21

Iron deficiency anemia, α-thalassemia trait, and β-thalassemia trait are the most common

causes of microcytosis. However, other clinical conditions are also associated with microcytosis

(Table II). 1,3,5,6 In addition to decreased MCV, the patients with β-thalassemia trait usually have

increased hemoglobin A 2 . It is pointed out that lower hemoglobin A 2 is also observed in patients

with concurrent deficiency of serum iron. Therefore, serum iron deficiency anemia must be ruled

out in order to correctly make the diagnosis of β-thalassemia trait in such patients. Conversely,

patients with β-thalassemia trait may acquire megaloblastic anemia or liver disease, and may

exhibit a normal range for MCV. 7

Table II Diagnostic Reasons of Microcytosis (listed in decending order of frequency)

Children and adolescents

Menstruating women

Men and non-menstruating women

Iron deficiency anemia

Iron deficiency anemia

Iron deficiency anemia

Thalassemia trait

Thalassemia trait

Anemia of chronic disease

Other hemoglobinopathies

Pregnancy

Unexplained anemia

Lead toxicity

Anemia of chronic disease

Thalassemia trait

Chronic inflammation

Sideroblastic anemia

Sideroblastic anemia

Several laboratory tests in addition to the CBC, e.g. serum iron, serum ferritin, total iron-

binding capacity (TIBC), transferrin saturation, hemoglobin electrophoresis, and the examination

of the peripheral blood smears (by a pathologist or hematologist), are employed to provide

insight and etiologies of microcytosis (Table III). 3,8

22

Table III

Laboratory Tests in the Differential Diagnosis of Microcytosis

Suggested diagnosis

Test

Iron deficiency anemia

Thalassemia

Anemia of chronic disease

Sideroblastic anemia

Serum ferritin

Decreased

Increased

Normal to increased

Normal to increased

RBC

Increased

Normal to

Normal

Increased

distribution width

increased

(RDW)

Serum iron

Decreased

Normal to

Normal to

Normal to

 

increased

decreased

increased

Total iron-

Increased

Normal

Slightly increased

Normal

binding capacity

Transferrin

Decreased

Normal to

Normal to slightly decreased

Normal to

saturation

increased

increased

Van Vranken 3 has recently suggested a protocol for diagnosing the cause of microcytosis

(Figure 1). If the cause remains unclear, the diagnosis of hemoglobinopathy by methods besides

electrophoresis alone is imperative. Note: There is a type-setting error in the presentation of

the protocol suggested by Van Vranken. 3 We have corrected this error in the figure 1, and

the journal (American Family Physician) editor was also informed.

23

24

24

Clinical observations of Kenneth F. Tucker, MD, FACP, a practicing hematologist for the last forty years:

Ordinary hemoglobin electrophoresis (cellulose acetate or agarose gel

electrophoresis) was only able to detect the more common types of thalassemias. Although

there were several other types, many of them did not have microcytosis. I had a large

number of patients, who had β-thalassemia minor and a few with probably α-thalassemia,

in which the hemoglobin and hematocrit values were relatively normal. Microcytes may or

may not be present. This diagnosis was suggested by the peripheral smear, and proven by

additional laboratory tests (IFE, globin chain analysis, etc.).

I believe that RDW, which is the average red cell width and reflects standard

deviation of red cell volumes, is a very important test. RDW normal deviation is a bell-

shaped curve. When this value is 2-3% higher, it represents red cells which have varying

widths. This certainly can be seen in patients who are iron deficient with microcytosis, but

have normal or large cells in addition to megaloblastic or dysplastic marrows, elevated

reticulocytes, vitamin B12 or folic acid deficiency, and other conditions. Despite the

availability of automated cell counters, review of the peripheral film is one of the most

informative and rewarding tests that should be done (by pathologist or hematologist) in any

case in which the cause of anemia is not obvious, e.g., bleeding, pure iron deficiency, pure

vitamin B12 deficiency, etc. It is also emphasized that the RDW test is not sensitive or

specific enough to differentiate iron deficiency and β-thalassemia trait. 9

A fairly low to extremely low ferritin is an excellent measure of iron deficiency

anemia. In my practice, regardless of what else is going on, any ferritin level of <10 ng/mL,

means there is iron deficiency. As mentioned above (Table III), elevated ferritin levels are

25

seen in refractory anemias, all types of chronic inflammatory conditions, etc. Since this test

is an acute phase reactant (similar to haptoglobin), it must not be used alone, as the ferritin

level may be normal in these clinical conditions.

Women in the second or third trimester are always anemic. This is similar to patients

who are hypervolemic because of renal or cardiac problems. Red cells in these cases are

not microcytes and when the hypervolemia is corrected, the hemoglobin and hematocrit

rises.

Severe anemia in childhood is usually due to the lack of iron in food, since cow’s

milk does not contain iron.

A naïve reader is advised to also review the “Full Color pdf of Complete Blood Count

in Primary Care,” Best Practice Journal, June 2008 (www.bpac.org.nz),

especially the section on Hemoglobin and Red Cell Indices (page 15).

References

1. Richardson M. Microcytic anemia [published correction appear in Pediatr Rev. 2007; 28(7): 275, Pediatric Rev. 2009; 30(5): 181, and Pediatr Rev. 2007; 28(4):151]. Pediatr Rev. 2007; 28(1): 5-14.

2. Beutler E, Waalen J. The definition of anemia: what is the lower limit of normal of the blood hemoglobin concentration? Blood. 2006; 107(5): 1747-1750.

3. Van Vranken ML. Evaluation of Microcytosis. Am Fam Physician. 2010; 80(9): 1117-1122.

4. RBC indices calculation and laboratory procedure (2006). St. John Health Laboratories, Warren, MI 48093.

5. Moreno Chulila JA, Romero Colas MS, Gutierrez Martin M. Classification of anemia for gastroenterologist. World J Gastroenterol. 2009: 15(37):4627-4737.

6. Guralnik JM, Eisenstaedt RS, Ferrucci L, Klein HG, Woodman, RC. Prevalence of anemia in persons 65 years and older in the United States: evidence for a high rate of unexplained anemia. Blood. 2004; 104(8): 2263-2268.

7. Bain BJ. Hemoglobinopathy Diagnosis. 2 nd ed. Malden, Mass.: Blackwell Publishing; 2006: 94-106.

26

8.

Hematologic diseases. In: Wallach J. Interpretation of Diagnostic Tests. 8 th ed. Boston, Mass.: Little Brown and Company; 2006: 385-419.

9. Ntalos G, Chatzinikolaou A, Saouli Z, et al. Discrimination indices as screening tests for beta-thalassemia trait. Ann Hematol. 2007; 86(7): 487-491.

27

Chapter 2 Diagnostic Laboratory Methods

2.3 Hereditary Persistence of Fetal Hemoglobin

Bernard G. Forget, MD

2.3.1 Introduction

Hereditary persistence of fetal hemoglobin or HPFH consists of a group of

genetic disorders characterized by the presence of a substantial elevation of fetal

hemoglobin (Hb F) in RBCs of heterozygotes, as well as of homozygotes and

compound heterozygotes for HPFH and other hemoglobinopathies. Increased levels of

Hb F can ameliorate the clinical course of hemoglobinopathies such as β thalassemia

and sickle cell anemia. HPFH is usually due to deletions of different sizes involving the

β-globin gene cluster, but nondeletion types of disorders have also been identified,

usually due to point mutations in the γ-globin gene promoters (reviewed in refs. 1-3).

Figure 1 diagrammatically illustrates the spatial organization of the β-like globin genes in

the β-gene cluster on chromosome 11. However, as discussed later in this chapter,

certain forms of nondeletion HPFH are clearly not linked to the β-globin gene cluster.

later in this chapter, certain forms of nondeletion HPFH are clearly not linked to the β

28

Figure 1. Deletions of the β-globin gene cluster associated with fusion proteins and HPFH. The circle 3’ to the β-globin gene indicates the 3’ β-globin gene enhancer. The filled vertical boxes at the 3’ breakpoints of the HPFH-1 and HPFH-6 deletions indicate the locations of DNA sequences with homology to olfactory receptor genes (adopted from reference 2). The references for the individual mutations are cited in references 1, 3 and 6.

HPFH is frequently contrasted with δβ thalassemia, which is another genetic

disorder associated with elevated Hb F levels. However, one should probably not

consider the two disorders as being unambiguously separate entities but rather as a

group of disorders with a variety of partially overlapping phenotypes that sometimes

defy classification as one syndrome or the other.

The following is a working definition

that is generally applied to the classification of these disorders: δβ thalassemia usually

refers to a group of disorders associated with a mild but definite thalassemia phenotype

of hypochromia and microcytosis together with a modest elevation of Hb F that, in

heterozygotes, is heterogeneously distributed among red cells.

In contrast, HPFH

refers to a group of disorders with substantially higher levels of Hb F, and in which there

is usually no associated phenotype of hypochromia and microcytosis.

In addition, the

increased Hb F in heterozygotes with the typical forms of HPFH is distributed in a

relatively uniform (pancellular) fashion among all of the red cells rather than being

distributed in a heterogeneous (heterocellular) fashion among a subpopulation of so-

called F cells, as in δβ thalassemia. Homozygotes for both conditions totally lack Hb A

and Hb A 2 , indicating absence of δ- and β-globin gene expression in cis to the

δβ

thalassemia and HPFH determinants.

Although the apparent striking qualitative

difference in cellular distribution of Hb F between HPFH and δβ thalassemia may be

29

due in great part to the quantitative differences in the amount of Hb F per cell and the

sensitivity of the methods used to detect Hb F cytologically, nevertheless it would

appear that the

increased amount

of

Hb

F

in

HPFH is caused

by a genetically

determined failure to suppress γ-globin gene activity postnatally in all erythroid cells,

rather than being due to selective survival of the normally occurring sub-population of F

cells such as occurs in sickle cell anemia, β + and β o thalassemia.

Nevertheless,

heterocellular forms of HPFH, without a β-thalassemia phenotype, have been clearly

defined and characterized. Therefore, in the final analysis, there is definitely some

overlap between these two sets of syndromes at the level of their clinical and

hematological phenotypes, as well as at the level of their molecular basis.

2.3.2 Deletions Associated with the HPFH Phenotype.

Classic pancellular HPFH, with absence of δ-and β-globin gene expression from

the affected chromosome, is associated with large deletions in the β-globin gene cluster

that remove the δ-and β-globin genes together with variable amounts of their 5’ and 3’

flanking DNA. At least nine different HPFH deletions of this type have been

characterized that vary in size or length from ~13 kb to ~ 85 kb (1-4), some of which are

illustrated in Fig. 1. The mechanisms by which such deletions cause marked elevation

of Hb F are not well understood, but a number of theories have been proposed.

One theory is based on the model of the proposed mechanism for the marked

elevation of Hb F associated with Hb Kenya. Hb Kenya is a structurally abnormal

hemoglobin that, like Hb Lepore, contains a "hybrid" or fused β-like globin chain

resulting from a non-homologous crossing-over event between two globin genes in the

30

β-gene cluster. However, whereas the Lepore crossover occurred between the δ- and

β-globin genes, the Kenya gene resulted from crossover between the A γ- and β-globin

genes (Fig. 1). The crossover occurred in the second exons of the A γ and β genes,

between the codons for amino acids 80 to 87, and resulted in deletion of ~24 kb of DNA

between the A γ to the β gene. Unlike Hb Lepore, that is associated with a β-

thalassemic phenotype, Hb Kenya is associated with a phenotype of pancellular G γ

HPFH: erythrocytes of affected heterozygotes have normal red cell indices and contain

7-23% Hb Kenya as well as approximately 10% Hb F, all of which is of the G γ type and

is relatively uniformly distributed among the red cells. A proposed explanation for the

HPFH phenotype associated with Hb Kenya is the influence on the G γ- and Kenya gene

promoters of a well characterized enhancer element located in the 3' flanking DNA of

the β-globin gene, shown by the filled circle in Fig. 1, that becomes translocated into

close proximity of the γ-globin gene promoters by the crossover/deletion event, resulting

in enhanced activity of these promoters.

Among the HPFH deletions, there is a relatively short deletion, called HPFH-5 or

Italian HPFH, that extends from a point ~3 kb 5' to the δ gene to a point 0.7 kb 3' to the

β gene, deleting the β gene but not its 3' enhancer. The molecular basis of the HPFH

phenotype associated with this deletion is presumably the influence of the translocated

3' β-gene enhancer on the γ-gene promoters, in a manner analogous to that proposed

for the basis of the HPFH phenotype of the Hb Kenya syndrome. In the case of some of

the other larger HPFH deletions, the DNA preserved at or near the 3’ breakpoint of the

deletions has been shown in various types of assays to have enhancer-like activity on

gene expression (2, 5-7). Thus, it has been proposed that the DNA sequences at the

31

HPFH 3' deletion breakpoints, that become juxtaposed to the γ genes as a result of the

deletion events, may influence γ-gene expression, in a manner analogous to the

presumed influence of the 3' β-gene enhancer on γ-gene expression in Hb Kenya and

HPFH-5. Mechanisms by which this could occur include the presence of enhancer-like

sequences in the translocated 3' breakpoint DNA or the presence in this DNA of an

active chromatin configuration that could have a spreading and activation effect on the

expression of the neighboring γ-globin genes.

A second theory for the mechanism of increased γ-gene expression in deletion-

type HPFH is the nature and function of the DNA sequences conserved at the 5’

breakpoint of the deletions. The 5’ breakpoints of the HPFH deletions, as well as many

of the δβ-thalassemia deletions, are located in the DNA between the Α γ and δ genes,

the so-called Α γδ-intergene DNA. It has long been proposed that there may exist

negative regulatory or silencer elements in this region of DNA, deletion of which in

HPFH but not in δβ thalassemia, results in markedly impaired postnatal suppression of

γ-gene activity in all erythroid cells (8). A number of subsequent observations have

been made that support a role for the A γδ-intergene region in the regulation of γ-gene

expression (reviewed in ref. 9). The Corfu deletion in particular, involving the δ-gene

and ~6 kb of upstream flanking DNA, is associated in homozygotes with a high HbF

phenotype and removes some interesting structural elements, such as a poly-pyrimidine

region that can serve as a binding site for a multi-protein chromatin remodeling complex

containing the transcription factor Ikaros, and a region of DNA that serves as a promoter

for the synthesis of an intergenic RNA transcript preferentially expressed in adult

32

erythroid cells (10). This region of DNA also appears to serve as a boundary region

between fetal and adult domains of the β-globin gene cluster.

The most conclusive evidence for a functional role of the A γδ-intergene DNA in

the regulation of γ gene expression consists of the observations by Sankaran et al. who

have extensively characterized a negative regulatory transcription factor, called

BCL11A, that down-regulates γ-gene expression in adult erythroid cells and that binds

to the A γδ-intergene DNA (11-13). BCL11A, originally identified as an important factor in

B-lymphoid cell development, is a component of a multi-protein complex that plays a

negative regulatory role in γ-gene expression. This complex has been shown to contain

GATA1 as well as all components of the nucleosome and histone deacetylase ( NuRD)-

repressive complex (14). Additional studies have shown that this complex physically

interacts with another transcription factor called SOX6 that is thought to be a repressor

of embryonic and fetal globin gene expression (15). Chromatin immunoprecipitation

(ChIP) studies have shown that BCL11A binds to a number of regions in the β-cluster,

including the upstream locus control region (LCR) and the γδ intergenic region, but does

not bind to the γ- or β-gene promoters (4, 14, 15). Sankaran et al. (4) have

characterized two important deletion mutants with nearly identical distal breakpoints but

different upstream breakpoints around the δ-gene and its flanking DNA. One mutant

with a more proximal breakpoint has a δβ-thalassemia phenotype, whereas the longer

deletion removing 3.5 kb of additional upstream DNA is associated with a HPFH

phenotype. The authors propose that this 3.5 kb region of DNA contains a silencer

element, deletion of which can cause HPFH. This hypothesis is strengthened by the fact

that the deleted region contains one of the prominent binding sites of BCL11A detected

33

in their ChIP experiments. These findings provide very strong evidence for a γ-gene

silencer element in the β-gene cluster that associates with a BCL11A-containing

repressor complex and that this interaction is an important factor in the suppression of

γ-gene expression during the perinatal switch from expression of Hb F to Hb A.

2.3.3 Nondeletion Forms of HPFH

In contrast to the deletional types of HPFH syndromes, where both linked G γ and

A γ genes are over expressed, only one or the other γ gene is usually over expressed in

the best characterized nondeletional types of HPFH associated with high levels of

pancellular Hb F expression. However, less well characterized nondeletion forms of

G γ A γ HPFH have been described that are associated with relatively low levels of

heterocellular expression of both γ genes. Because of the restricted pattern of γ-globin

gene expression in the G γ and A γ forms of nondeletion HPFH, it was assumed that the

mutations in these syndromes must be located near the affected gene and molecular

studies focused initially on the DNA sequence analysis of the over expressed γ genes in

these disorders.

34

Table 1 adopted from reference 2. The one patient studied was doubly heterozygous for Hb

Table 1 adopted from reference 2. The one patient studied was doubly heterozygous for Hb A and Hb C. About 20% of Hb F (or 8% of the total Hb) was of the

γ type, and the G γ gene in cis to the -175 A γ mutation carried the -158 C→ T change.

The references for the individual mutations are cited in references 1 and 3.

G

The results of these structural analyses revealed a number of different point

mutations in the promoter region of the over expressed γ gene in individuals with

different types of nondeletion HPFH, as listed in Table 1 (reviewed in refs. 1-3). These

point mutations have clustered primarily in three distinct regions of the 5'-flanking DNA

of the affected γ genes. The first region is located approximately 200 base pairs from

35

the "cap site" or site of transcription initiation of the γ genes (at least five different point

mutations involving single nucleotides between residues -195 to -202 from the cap site).

This region of DNA, which had not previously been suspected of playing a role in the

regulation of γ-gene expression, is very G+C rich and its sequence bears homology to

that of known control elements of other genes that contain the binding site for the

ubiquitous trans-acting protein factor called Sp1. Subsequent studies of the γ-gene

promoters have demonstrated that the -200 region is also a binding site for Sp1 and for

at least one other ubiquitous DNA binding protein.

The second region containing a mutation associated with nondeletion HPFH is

located at position -175. A point mutation (T->C) at this position of either the G γ or A γ

gene is associated with a phenotype of pancellular HPFH with high levels of Hb F (15-

25%). This region of DNA is noteworthy because it contains an octanucleotide

sequence that is present in the promoter region of a number of genes and is the binding

site of another ubiquitous trans-acting factor called OCT-1. In addition, the octamer

consensus sequence of the γ-gene promoters is flanked on either side by a consensus

sequence for the hematopoietic-specific transcription factor GATA-1. The point

mutation at position -175 affects the one nucleotide that is present in the partially

overlapping binding sites of both OCT-1 and GATA-1.

The third region affected by a point mutation in nondeletion HPFH is in the area

of a well known regulatory element of globin and other genes: the CCAAT box

sequence. In the γ genes, the CCAAT box is duplicated and the mutation associated

with the Greek A γ type of nondeletion HPFH is a G->A substitution at position -117, 2

bases upstream of the distal CCAAT box of the A γ-globin gene promoter. The base

36

change disrupts a pentanucleotide sequence, YYTTGA (Y = pyrimidine), that is highly

conserved immediately upstream of the CCAAT sequence in all animal fetal and

embryonic genes. At least two other mutations involving the CCAAT box of one or the

other γ gene have been reported in other cases of HPFH not associated with large

deletions. The CCAAT box region is known to be the binding site of a number of trans-

acting factors, including the ubiquitous factors CCAAT binding protein (CP1) and

CCAAT displacement factor (CDP) as well as the erythroid-specific factor NF-E3.

The unifying model by which these various mutations are thought to affect

hemoglobin switching proposes that these base changes alter the binding of a number

of different trans- acting factors to critical regions of the γ-gene promoters and thereby

prevent the normal postnatal suppression of γ-gene expression (reviewed in refs. 1,2).

The mutations could prevent the binding of negative regulatory factors or enhance the

binding of positive regulatory factors. Either mechanism could be operative with one

mutation or the other.

2.3.4 HPFH Unlinked to the β-Globin Gene Cluster

A number of studies have identified families in which increased levels of Hb F are

inherited due to a genetic determinant that is unlinked to the β-globin gene cluster.

Genome-wide association studies (GWAS), using co-inheritance of single nucleotide

polymorphisms (SNPs) with elevated levels of Hb F, have subsequently demonstrated

the presence of two different quantitative trait loci (QTLs), unlinked to the β-globin gene

cluster on chromosome 11, that are associated with inheritance of mildly elevated levels

of Hb F, similar to the phenotype seen in Swiss-type heterocellular HPFH (see section

37

above on Nondeletion HPFH). These loci are located on chromosome 2 and 6 (16, 17).

The locus on chromosome 2 corresponds to the site of the gene encoding BCL11A and

its identification led to the elegant studies of Sankaran and co-workers demonstrating

the role of BCL11A in the regulation of γ-gene expression. The locus on chromosome 6

is located between the genes encoding HBS1L and MYB. The mechanism by which this

locus causes elevation of Hb F is thus far poorly understood. Finally, mutations in the

gene on chromosome 19 encoding the erythroid-specific transcription factor EKLF1

have been shown to be associated with a form of HPFH (18, 19). The involved

mechanism is probably through the regulation of BCL11A levels, because it has been

demonstrated that EKLF1 binds to the promoter of the BCL11A gene and regulates the

expression of the gene (20).

2.3.5

Conclusion

Significant insights into the normal regulation of expression of the human β-

globin gene cluster have been obtained by a detailed analysis of a group of disorders

called HPFH. On the basis of this information, several important regulatory elements

have been identified for the normal functioning of the individual genes in the cluster

during the developmental switch from fetal to adult hemoglobin gene expression, as well

as for the abnormal persistent expression of the γ-globin genes in adults with HPFH.

These results provide a more sophisticated understanding of the molecular basis of

these syndromes and point to certain strategies for potential future molecular and

cellular therapies for globin gene disorders.

38

2.3.6

Hemoglobin F Quantification

Hb F can be quantified by several methods, and the most commonly used

procedures in a clinical laboratory are a) radial immunodiffusion, b) Elisa method,

c) HPLC, and d) capillary zone electrophoresis.

References

1. Bollekens JA, Forget BG. Delta beta thalassemia and hereditary persistence of fetal

hemoglobin. Hematol Oncol Clin North Am 1991;5(3):399-422.

2. Forget BG. Molecular basis of hereditary persistence of fetal hemoglobin. Ann N Y

Acad Sci 1998; 850:38-44. 3 Weatherall DJ, Clegg JB. The Thalassaemia Syndromes. 4th ed. Oxford ; Malden,

MA: Blackwell Science; 2001.

4. Sankaran VG, Xu J, Byron R, et al. Functional element necessary for fetal

hemoglobin silencing. N Engl J Med 2011; 365(9):807-14.

5. Feingold, EA, Forget BG. The breakpoint of a large deletion causing hereditary

persistence of fetal hemoglobin occurs within an erythroid DNA domain remote from the β-globin gene cluster. Blood 1989; 74: 21782186.

6. Kosteas T, Palena A, Anagnou NP. Molecular cloning of the breakpoints of the

7. Anagnou NP, Perez-Stable C, Gelinas R, et al. Sequences located 3' to the

breakpoint of the hereditary persistence of fetal hemoglobin-3 deletion exhibit enhancer

activity and can modify the developmental expression of the human fetal A gamma- globin gene in transgenic mice. J. Biol Chem 1995; 270: 10256-63.

8. Huisman TH, Schroeder WA, Efremov GD, et al. The present status of the

heterogeneity of fetal hemoglobin in beta-thalassemia: an attempt to unify some

observations in thalassemia and related conditions. Ann N Y Acad Sci 1974;232(0):107-

24.

9. Bank A, O'Neill D, Lopez R, et al. Role of intergenic human γ-δ -globin sequences in

cells. Ann N Y Acad Sci 2005;1054:48-54.

39

10.

Chakalova L, Osborne CS, Dai YF, et al. The Corfu δβ thalassemia deletion disrupts

γ-globin gene silencing and reveals post-transcriptional regulation of HbF expression. Blood 2005;105:2154-60.

11. Sankaran VG, Xu J, Orkin SH. Transcriptional silencing of fetal hemoglobin by

BCL11A. Ann N Y Acad Sci. 2010;1202:64-8.

12. Sankaran VG, Xu J, Ragoczy T, et al. Developmental and species-divergent globin

switching are driven by BCL11A. Nature 2009;460(7259):1093-7.

13. Sankaran VG, Nathan DG. Reversing the hemoglobin switch. N Engl J Med 2010;

363(23):2258-60.

14. Sankaran VG, Menne TF, Xu J, et al. Human fetal hemoglobin expression is

regulated by the developmental stage-specific repressor BCL11A. Science 2008;

322(5909):1839-42.

15. Xu J, Sankaran VG, Ni M, et al. Transcriptional silencing of γ-globin by BCL11A

involves long-range interactions and cooperation with SOX6. Genes Dev 2010; 24:783-

98.

16.

Thein SL, Menzel S, Lathrop M, Garner C. Control of fetal hemoglobin: new insights

emerging from genomics and clinical implications. Hum Mol Genet 2009;18(R2):R216-

23.

17.

Galarneau G, Palmer CD, Sankaran VG, Orkin SH, Hirschhorn JN, Lettre G. Fine

mapping at three loci known to affect fetal hemoglobin levels explains additional genetic

variation. Nat Genet 2010;42(12):1049-51.

18. Borg J, Papadopoulos P, Georgitsi M, et al. Haploinsufficiency for the erythroid

transcription factor KLF1 causes hereditary persistence of fetal hemoglobin. Nat Genet

2010;42(9):801-5.

19. Borg J, Patrinos GP, Felice AE, Philipsen S. Erythroid phenotypes associated with

KLF1 mutations. Haematologica 2011; 96:635-8.

20. Zhou

expression and γ- to β-globin gene switching. Nat Genet 2010; 42:742-4.

D, Liu K, Sun CW, Pawlik KM, Townes TM. KLF1 regulates BCL11A

40

Chapter 2 Diagnostic Laboratory Methods

2.4 Flow Cytometry Measurements of Cellular Fetal Hemoglobin, Oxidative Stress and Free Iron in Hemoglobinopathies

Eitan Fibach, MD

2.4.1 Flow Cytometry of Blood Cells

Flow cytometry (FC) is a common methodology in clinical diagnostic and

research laboratories. In hematology, it is mainly used for diagnosis, prognosis,

determining therapy efficacy and follow up of patients with leukemia or lymphoma

(1). It is also used for diagnosis of red blood cell (RBC) abnormalities such as in

Paroxysmal Nocturnal Hemoglobinuria (2) and hereditary spherocytosis (3). In

this review, I will summarize FC methodologies for analysis of RBC (and other

blood cells) from patients with hemoglobinopathies with respect to their fetal

hemoglobin (HbF) and free iron (labile iron pool, LIP) contents and parameters of

the oxidative state.

FC analyzes individual cells in a liquid medium. Most techniques use antibodies

directed against internal (following fixation and premeabilization of the

membrane) or surface antigens. The antibodies are labeled with fluorescence

probes (fluochromes) either directly or indirectly (by a secondary antibody). In

addition to antibodies, other fluorescent compounds can be used. For example,

propidium iodide, which binds stochiometrically to nucleic acids, is commonly

used for determining cell viability and their distribution in the cell cycle (4).

Following staining, the cells are analyzed by a flow cytometer; they are first

41

hydro-dynamically focused in a narrow sheath of physiological solution before

being intercepted by one or more laser beams resulting in light scatter and

fluorescence emission. Depending on the number of laser sources and

fluorescence detectors, several parameters (commonly 6, but up to 18) can be

simultaneously detected on each cell: Forward light scattering and side light

scattering provide correlates with regards to size and granularity of the cells,

respectively, and fluorescence light emission by the fluorochromes correlates

with the expression of different antigens as well as other cellular parameters (see

below).

FC is superior to other techniques in several aspects: (I) Technology is widely available

as mentioned above, most hematology and immunology laboratories use FC for both diagnosis

and research purposes. (II) Only cell-associated fluorescence is measured, excluding soluble or

particulate fluorescence. (III) Each cell is analyzed individually, but since measurement is rapid

(msec), a large number of cells can be analyzed (ranging from 0.1-10 x10 5 cells) within a few

minutes. The results are therefore statistically sound even for very small sub-populations. (IV)

Various sub-populations can be identified and measured simultaneously. (V) The method

produces mean values for each sub-population, and therefore avoids the inaccuracy of

biochemical methods that produce mean value for the whole population. This is of crucial

importance when mixed populations are studied. (VI) The procedure can be automated to permit

high throughput analysis (e.g., for screening of large libraries of compounds for inducers of

HbF). Although the FC data are expressed in arbitrary fluorescence units rather than weight or

molar concentrations, they are useful for comparative purposes.

42

FC is especially fitting for analysis of blood cells: (I) These cells which can be easily

obtained by blood drawing are present as single cells, thus in contrast to cells of solid tissues,

their use does not require harsh procedures for tissue disaggregation (e.g., trypsinization). (II)

They are present as a mixture of various cell types, including numerous subtypes (e.g.,

lymphocytes), with very large (e.g., RBC) to very small (hematopoietic stem cells)

representation. Cells of these sub-types can be identified and "gated" based on differences in

their size (forward light scattering), granularity (side light scattering) and expression of surface

antigens, and can be measured simultaneously. For measurements of various characteristics

(HbF content, oxidative stress parameters and LIP content), the blood sample is stained with

specific probes (as detailed below), and then with fluorescent reagents (usually antibodies)

against surface markers which identify a specific subpopulation. Such markers are glycophorin

A for RBC, CD61 for platelets, CD15 for neutrophils, CD19 for B-lymphocytes and CD3 for T-

lymphocytes. CD45 is particularly useful since it is differentially expressed on various nucleated

blood cells (Fig. 1).

43

PMN RBC Monocytes Lymphocytes CD45
PMN
RBC
Monocytes
Lymphocytes
CD45

Fig. 1. Flow cytometry of blood cells. A dot plot of blood cells with respect CD45 (FL3-H) and side light scatter (SSC-H).

2.4.2 Measurement of Fetal Hemoglobin-Containing Erythroid Cells

Fetal hemoglobin (HbF, α 2 γ 2 ) is the major hemoglobin (Hb) in the prenatal period

that is largely replaced after birth by the adult Hb (HbA, α 2 β 2 ) (5). In adults, less than 1%

of the Hb content is HbF which is concentrated in a few RBC, called F-cells (6). High

levels of HbF are frequently seen in hemoglobinopathies (7). Measurement of HbF (as

well as HbA, sickle hemoglobin, HbS, etc.) can assist in diagnosis and in determining

the efficacy of treatment. HbF can be measured by a variety of techniques. Most of the

techniques measure HbF in lysates prepared from RBC. These techniques include

44

spectrofluorometric measurements following treatment with alkaline (to destroy non-fetal

hemoglobins) and staining with benzidine (8), chromatography (ion-exchange HPLC for

hemoglobins and reverse-phase HPLC for globin chains) (9), as well as immunological

techniques, such as Elisa, based on antibodies against HbF (10). However, quantitative

FC measurement of RBC, fluorescently stained with antibodies to HbF (as well as for

the other hemoglobins), has several advantages. For example, in the differential

diagnosis of Hereditary Persistence of Fetal Hemoglobin (11). This condition

encompasses a heterogeneous group of disorders with marked increased levels of HbF.

Based on the cellular distribution of HbF, they are characterized as pan-cellular, where

all RBCs have increased levels of HbF, albeit not always uniformly so; and hetero-

cellular, where nearly all the HbF is confined to a minor, distinct subpopulation of RBCs.

This important distinction is most reliably ascertained by FC.

Epidemiological studies have indicated that high levels of HbF improve the

clinical symptoms of the underlying disease. In sickle cell anemia not only do HbF-

containing cells have a lower concentration of sickle hemoglobin, but HbF inhibits

polymerization of HbS directly, accounting for the lower propensity of such cells to

undergo sickling (12). In β-thalassemia, elevated HbF should compensate partially for

the deficiency of β-globin chains and reduce the excess of α-globin chains. Several

pharmacological agents have been used to stimulate HbF production (13). Hydroxyurea

(HU) is currently the drug of choice (14). When patients are monitored during HU

treatment by measuring HbF in the hemolysate, an increase is usually observed after 2-

3 months (10). HU acts by a still unknown mechanism on the early erythroid precursors

in the bone marrow. It takes several weeks for HbF to accumulate in the peripheral

45

blood to a quantity that allows differences before and after treatment to become

apparent. Measuring differences in F-RBC by FC may be more sensitive, and

measuring F-reticulocytes (retics) may provide early indication of treatment efficacy

(15): Retics have a very short life-span (1-2 days) compared to mature RBC (120 days

in normal subjects) and therefore measuring peripheral blood F-retics more closely

characterizes the current status of HbF production in the bone marrow. Measuring F-

retics can indicate the efficacy of the drug and/or the patient’s compliance several days

after treatment initiation. Such follow up is very important since about 30% of the

patients are non-responders. It is imperative that such patients be identified as early as

possible and the treatment (that is not without potential risks) be discontinued and

replaced by treatment with another drug (e.g., butyroids).

2.4.3 Staining Protocols for F-RBC and F-Retics (15)

Heparinized blood is washed three times in phosphate buffered saline (PBS). For

fixation, 50μl of the packed cells are resuspended in 10 ml of PBS containing 4%

formaldehyde for 15-min at room temperature under constant agitation in polypropylene

tubes. For permeabilization, the cells are centrifuged for 3 min at 1,500 g, and 2 ml

methanol-acetone are added to the pellet, mixed and incubated for 1-min at room

temperature. The cells are then washed three times and resuspended in PBS to a final

volume of 0.5 ml (10% suspension).

Anti-HbF monoclonal antibodies (the amount depends on the Manufacturer’s

instructions or on a pre-performed titration) are added to 5x10 6 cells (5 μl of the 10%

suspension) and incubated for 1-hr at 37 0 C, after which the cells are washed in PBS. If

46

the antibodies are fluorochrome-conjugated, the cells are resuspended in PBS and

analyzed directly. In the case of unconjugated antibodies, a secondary antibody

(fluorochrome-conjugated rabbit F(ab’) 2 anti-mouse IgG) is added for 30-min at room

temperature. For the F-retic count, the blood cells are double labeled with

phycoerythrin-conjugated antibodies to HbF and thiazol orange, a specific nucleic acid

binding green fluorescence dye.

Following staining, the cells are washed and resuspended in PBS and analyzed by FC.

For "acquisition", the threshold is set on forward light scatter to exclude debris and

platelets. Cells are run at about 1000 cells/sec using logarithmic amplification, and data of

10 4 -10 5 cells are accumulated. RBC are gated based on their forward light scatter and

side light scatter. When the sample is also stained with thiazol orange, RBC are gated based on

their negative staining with thiazol orange, retics - based on their weak staining (because they

contain remnants of RNA) and nucleated cells (including normoblasts) based on their intense

staining; HbF is then specifically determined for each cell population (Fig. 2).

47

Fig. 2. Flow cytometry analysis of F-RBC and F-Retics. Blood cells stained with thiazol-orange (T.O)

Fig. 2. Flow cytometry analysis of F-RBC and F-Retics. Blood cells stained with thiazol-orange (T.O) and anti-HbF. A. Forward light scatter (FSC) vs. T.O. RBC (negative T.O staining) and retics (intermediate T.O staining) were gated and their HbF determined (B and C), respectively.

2.4.4 F-Cell Determination for Fetal-Maternal Hemorrhage (FMH) in Pregnant Patients

with β-Thalassemia A Single Case and General Conclusion (16)

F-cell analysis is commonly used to detect fetal-maternal hemorrhage (FMH)

where fetal RBC enter the maternal blood circulation due to fetal or maternal trauma or

a placental defect (17). These RBC of fetal origin can be distinguished from the

maternal adult RBC by their fluorescence following staining with an antibody to HbF.

48

Recently, in order to increase the sensitivity, reproducibility and accuracy of the assay,

another marker was introduced carbonic anhydrase (CA) (18). The CA isoenzymes

that are mainly represented by CAI and CAII (19) are fully expressed in the RBC only

after birth (20,21). The "Fetal Cell Count kit" manufactured by IQ Products (Groningen,

the Netherlands), which uses a combination of a murine monoclonal antibody directed

to HbF and a polyclonal antibody to the CAII isoform, has significantly improved this

assay (11,18). Most of the RBC of fetal origin do not express CA but highly express HbF

(CA - HbF ++ ), while RBC in adult blood express CA but do not express HbF (CA + HbF - ).

Some adult F-cells which express CA and HbF (CA + HbF + ) can be differentiated from

fetal F-cells (CA - HbF ++ ) present in FMH based on the extent of HbF and CA expression.

Until recently, β-thalassemia major was lethal. Improvements in treatment, such as the

introduction of blood transfusions and iron chelation, have considerably improved the life

expectancy as well as the quality of the patient’s life, including the ability of thalassemic women

to give birth. Recently, we were confronted with a case of a possible FMH in a β-thalassemic

woman. To establish the usefulness of the CA/HbF procedure, i.e. differentiating between fetal

RBC and the maternal RBC, we screened non-pregnant β-thalassemic patients (men and

women). The results demonstrated, in addition to adult non-F RBC (CA + HbF -) and adult F-RBC

(CA + HbF + ), two other sub-populations, CA + HbF ++ and CA - HbF ++ . The presence in these patients

of the latter RBC phenotype, which characterizes fetal cells, precludes the use of the CA/HbF

method for the detection of FMH in thalassemia.

2.4.5 Oxidative Stress

The oxidative status of cells is determined by the balance between pro-oxidants

and antioxidants. The reactive oxygen species (ROS) are pro-oxidants which are

49

generated in most cells mainly during energy production. Although important for various

aspects of normal physiology (e.g., signal transduction), ROS interact with and damage

various cell components when they are in excess. To protect against the deleterious

effects of ROS, cells maintain an effective antioxidant system consisting of water- or

lipid-soluble antioxidants and enzymes that remove ROS by metabolic conversion.

When the oxidant/anti-oxidant balance is tilted in favor of the oxidants, oxidative stress

ensues (22). Although oxidative stress is not the primary etiology of

hemoglobinopathies, it mediates several of their pathologies, including hemolysis which

results in chronic anemia. Hemolysis occurs both in the bone marrow, where developing

erythroid precursors undergo enhanced apoptosis (ineffective erythropoiesis) and in the

peripheral blood, where mature RBC undergo lysis in the blood vessels (intra-vascular

hemolysis). Destruction also occurs in reticuloendothelial tissues, such as the spleen,

where mature RBC undergo phagocytosis by resident macrophages (extra-vascular

hemolysis) (22).

Various factors are responsible for oxidative stress in RBC of patients with hemo-

globinopathies. In β-thalassemia, excess α-globin chains form unstable tetramers that

dissociate into monomers and then are oxidized, first to met-Hb and then to

hemichromes which precipitate intracellularly with time (23). Following the release of

heme and iron, there is deposition of the protein moiety on the plasma membrane. The

outcome of this chain of events is enhanced formation of ROS, catalyzed by free iron,

with a variety of deleterious effects on the membrane lipids and proteins, including

oxidation of the membrane protein band 4.1 and a decrease in spectrin/band3 ratio (24).

In α-thalassemia, the γ- and β-globins, which are produced in excess, do not precipitate

50

right away, but form the soluble tetramers γ 4 (Hb Bart’s) and later the β 4 (HbH), which

are less stable than HbA and have an increased susceptibility towards oxidation and

hemichrome formation (23). In sickle cell disease, met-HbS is produced at a higher rate

and is less stable than met-HbA resulting in formation of hemichromes, and release of

heme and iron, with resultant denaturation and precipitation as Heinz bodies (25).

Many approaches have been devised to quantify oxidative stress and its damage

as well as the effects of treatment with anti-oxidants (22). Most of these methods assay

the content of body fluids (mainly blood). FC can be utilized for measurements of

oxidative stress parameters in various blood cells. Although the major target of oxidative

stress in hemoglobinopathies is the RBC, other blood cells are affected as well. Thus,

defects in the abilities of polymorphonuclear cells to adhere to, engulf and lyze bacteria

may result in recurrent infections. Chronic activation of platelets may cause

thromboembolic complications (26,27). In order to study the effects of oxidative stress

on the spectrum of symptoms in hemoglobinopathies, all blood cell lineages should be

studied.

FC of oxidative stress parameters utilizes various probes: ROS can be measured

by staining cells with the non-polar compound, 2’-7-dichlorofluorescein diacetate. It

readily diffuses across the membrane and becomes deacetylated by

esterases into a polar derivative that is trapped inside the cells. When it is oxidized by

ROS (mainly peroxides), a green fluorescent product dichlorofluorescin is produced

(28). The intensity of the fluorescence is proportional to the cellular concentration of

ROS. The applicability of the method was validated by the increased fluorescence

measured following treatment with ROS-generating agents such as hydrogen peroxide

51

and t-butylhydroxyperoxide and with the catalase inhibitor sodium azide, while treatment

with ROS scavengers such as N-acetyl cysteine decreased the fluorescence. ROS can

also be measured by dihydrorhodamine 123, which freely enters into cells, and after

oxidation by ROS to rhodamine 123 emits a bright red fluorescence (29).

Reduced glutathione (GSH), the main cellular antioxidant, can be measured

using mercury orange (26), which forms fluorescent adducts with GSH via the

sulphydryl group, producing an S-glutathionyl derivative that emits red-orange

fluorescence (30). The probe reacts more rapidly with non-protein thiols, such as GSH,

compared with thiol-containing proteins, allowing specificity under controlled staining

conditions (31). The validity of this method was confirmed by demonstrating that N-

ethylmaleimide, which totally blocks thiol groups, decreased the fluorescence in a dose-

dependent manner. To ascertain that non-protein thiols are being stained, cells were

incubated with diethylmaleate, a specific non-protein thiol-depleting agent. This weak

electrophil of the α,β-unsaturated carbonyl group, which reacts with GSH only in the

presence of glutathione transferase, markedly suppressed the mercury orange

fluorescence, suggesting that GSH was the principle thiol which was stained by the dye

(32). Although there is no direct proof that the probe is specific for GSH, the assay

measures predominantly GSH, since it is the main non-protein thiol constituent of the

cellular thiol pool (33).

Other parameters of oxidative stress measured by FC are membrane lipid

peroxidation by staining with fluor-DHPE (26), and externalization of

phosphatidylserine (PS) moieties, a marker of damage to the membrane lipid, by

fluorochrome-conjugated annexin-V (34).

52

2.4.6

Staining Protocols for ROS and GSH

ROS Assay Blood cells are incubated with 2'-7'-dichlorofluorescin diacetate, dissolved

in methanol, at a final concentration of 0.4 mM. After incubation at 37°C for 15 min, the cells are

washed and re-suspended in PBS to the original cell concentration.

GSH Assay - Blood cells are washed with PBS and then spun down. The pellet is incubated for

3 min. at room temperature with 40 M (final concentration) of mercury orange. A 100 M stock

solution of mercury orange is made up in acetone and stored at 4°C. In both cases, cells are

then washed and resuspended in PBS, and analyzed by FC.

Fig. 3 shows FC measurements of ROS and GSH in normal and thalassemic RBC. The

results indicate that thalassemic RBC have higher ROS but lower GSH contents than

normal RBC.

53

Fig. 3. Flow cytometry of ROS and GSH in normal and thalassemic RBC. Blood cells

Fig. 3. Flow cytometry of ROS and GSH in normal and thalassemic RBC. Blood cells derived from a normal donor (A,C) and a thalassemic patient (B,D) were stained for ROS (A,B) and GSH

(C,D) following 1-h pre-incubation with (white) or without (pink) 2 mM H are shown.

O 2 2
O
2
2

. Histograms of RBC

2.4.7 Intracellular Free Iron

Another contributor to oxidative stress in cells is excess of iron. Iron overload is

generated in thalassemic or sickle RBC as a result of Hb-instability as discussed above.

In addition, iron accumulates in these diseases as a result of increased absorption from

the intestinal mucosa and by a failure to dispose of excess iron acquired by frequent

therapeutic blood transfusions (35). Moreover, iron-containing compounds (Hb or

54

hemin) that are released during hemolysis can add to the iron load and further

aggravate the hemolysis.

Normally, iron is transported in the circulation bound to transferrin and is

transferred into cells through the surface transferrin-receptor (36). Most of the

intracellular iron is firmly bound to various components such as Hb, heme and

cytochrome C; excess is stored in ferritin (37). In iron overload, serum iron which

exceeds the binding capacity of transferrin is present in the form of non-transferrin

bound iron (38). This iron can be taken up through a transferrin-independent pathway,

to form the cellular unbound "labile iron pool" (LIP) (16). The small fraction of LIP was

suggested as a low molecular weight intermediate or transitory pool between

extracellular iron and cellular firmly-bound iron (39). LIP is redox active and it

participates in generation of free radicals by the Fenton and Haber-Weiss reactions and

consequently in cell and tissue damage (40).

Since iron overload plays an important role in the pathology of transfused

patients with β-hemoglobinopathies, the patients are commonly treated with iron

chelators. The three chelators currently in clinical use are deferioxamine, deferiprone

and deferasirox (41). Evaluation of iron overload is important for assessing its severity

and for determining the efficacy of iron chelation therapy. The parameters usually tested

are serum ferritin protein level and transferrin iron saturation. However, serum ferritin is

an acute phase reactant that may increase by iron-independent factors such as

infection, inflammation and liver disease (42). In addition, serum ferritin levels often fail

to predict impending cardiac iron overload and ensuing cardio-myopathies (43). The

advent of non-invasive proton relaxation assays (by NMR R2* or T2*) of organs has

55

provided a significant advance in monitoring iron overload, although, similarly to serum

ferritin, substantial changes in these parameters are seen only weeks to months after

the initiation of chelator treatment. In addition, these techniques require expensive

instrumentation that is not always available.FC quantification of the LIP content in

various blood cell types overcomes many of these problems.

2.4.8 Staining Protocol for LIP

Cells are washed twice with saline and incubated at a density of 1x10 6 per ml for 15 min

at 37 o C with 0.25 μM Calcein Acetoxymethyl Ester (CA-AM). After wash, the cells are treated

with or without Deferiprone (L1, 100 μM). Fig. 4 shows the results of LIP measurements in RBC.

LIP is defined as the difference between histograms of cells treated or untreated with L1.

56

Fig. 4. Flow cytometry of labile iron pool (LIP) in RBC. Blood cells were loaded

Fig. 4. Flow cytometry of labile iron pool (LIP) in RBC. Blood cells were loaded with calcein, then washed and treated with or without the iron chelator Deferiprone (L1). Distribution fluorescence (FL1-H) histograms are shown. LIP is defined as the difference between the mean fluorescence channels of histograms of cells treated or untreated with L1.

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3. Kedar PS, Colah RB, Kulkarni S, Ghosh K, Mohanty D. Experience with eosin-5'- maleimide as a diagnostic tool for red cell membrane cytoskeleton disorders. Clin Lab Haematol 2003; 25(6): 373-6.

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6. Boyer SH, Belding TK, Margolet L, Noyes AN. Fetal hemoglobin restriction to a few erythrocytes (F cells) in normal human adults. Science 1975; 188(4186): 361-3.

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11. Leers MP, Pelikan HM, Salemans TH, Giordano PC, Scharnhorst V. Discriminating fetomaternal hemorrhage from maternal HbF-containing erythrocytes by dual-parameter flow cytometry. Eur J Obstet Gynecol Reprod Biol 2007; 134(1): 127-9.

12. Benesch RE, Edalji R, Benesch R, Kwong S. Solubilization of hemoglobin S by other hemoglobins. Proc Natl Acad Sci U S A 1980; 77(9): 5130-4.

13. Gambari R, Fibach E. Medicinal chemistry of fetal hemoglobin inducers for treatment of beta-thalassemia. Curr Med Chem 2007; 14(2): 199-212.

14. Steinberg MH. Determinants of fetal hemoglobin response to hydroxyurea. Semin Hematol 1997; 34(3 Suppl 3): 8-14.

15. Amoyal I, Fibach E. Flow cytometric analysis of fetal hemoglobin in erythroid precursors of beta-thalassemia. Clin Lab Haematol 2004; 26(3): 187-93.

16. Prus E, Fibach E. Heterogeneity of F-cells in β -thalassemia. Transfusion 2012, in press.

17. Sebring ES, Polesky HF. Fetomaternal hemorrhage: incidence, risk factors, time of occurrence, and clinical effects. Transfusion 1990; 30(4): 344-57.

18. Porra V, Bernaud J, Gueret P, Bricca P, Rigal D, Follea G, Blanchard D. Identification and quantification of fetal red blood cells in maternal blood by a dual-color flow cytometric method: evaluation of the Fetal Cell Count kit. Transfusion 2007; 47(7): 1281-9.

19. Tashian RE. The carbonic anhydrases: widening perspectives on their evolution, expression and function. Bioessays 1989; 10(6): 186-92.

20. Brady HJ, Edwards M, Linch DC, Knott L, Barlow JH, Butterworth PH. Expression of the human carbonic anhydrase I gene is activated late in fetal erythroid development and regulated by stage-specific trans-acting factors. Br J Haematol 1990; 76(1): 135-42.

21. Aliakbar S, Brown PR. Measurement of human erythrocyte CAI and CAII in adult, newborn, and fetal blood. Clin Biochem 1996; 29(2): 157-64.

22. Fibach E, Rachmilewitz EA. The role of antioxidants and iron chelators in the treatment of

oxidative stress in thalassemia. Ann N Y Acad Sci 2010; 1202: 10-6.

23. Rachmilewitz EA. Formation of hemichromes from oxidized hemoglobin subunits. Ann N

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24. Advani R, Sorenson S, Shinar E, Lande W, Rachmilewitz E, Schrier SL. Characterization and comparison of the red blood cell membrane damage in severe human alpha- and beta-thalassemia. Blood 1992; 79(4): 1058-63.

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Winterbourn CC. Oxidative denaturation in congenital hemolytic anemias: the unstable hemoglobins. Semin Hematol 1990; 27(1): 41-50.

26. Amer J, Fibach E. Oxidative status of platelets in normal and thalassemic blood. Thromb Haemost 2004; 92(5): 1052-9.

27. Amer J, Fibach E. Chronic oxidative stress reduces the respiratory burst response of neutrophils from beta-thalassaemia patients. Br J Haematol 2005; 129(3): 435-41.

28. Bass DA, Parce JW, Dechatelet LR, Szejda P, Seeds MC, Thomas M. Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation. J Immunol 1983; 130(4): 1910-7.

29. Rothe G, Oser A, Valet G. Dihydrorhodamine 123: a new flow cytometric indicator for respiratory burst activity in neutrophil granulocytes. Naturwissenschaften 1988; 75(7):

354-5.

30. O'Connor JE, Kimler BF, Morgan MC, Tempas KJ. A flow cytometric assay for intracellular nonprotein thiols using mercury orange. Cytometry 1988; 9(6):529-32.

31. Hedley DW, Chow S. Evaluation of methods for measuring cellular glutathione content using flow cytometry. Cytometry 1994; 15(4): 349-58.

32. Plummer JL, Smith BR, Sies H, Bend JR. Chemical depletion of glutathione in vivo. Methods Enzymol 1981; 77: 50-9.

33. Di Simplicio P, Cacace MG, Lusini L, Giannerini F, Giustarini D, Rossi R. Role of protein - SH groups in redox homeostasis--the erythrocyte as a model system. Arch Biochem Biophys 1998; 355(2): 145-52.

34. Freikman I, Amer J, Ringel I, Fibach E. A flow cytometry approach for quantitative analysis of cellular phosphatidylserine distribution and shedding. Anal Biochem 2009; 393(1): 111-6.

35. Rund D, Rachmilewitz E. Beta-thalassemia. N Engl J Med 2005; 353(11): 1135-46.

36. Richardson D R, Ponka P. The molecular mechanisms of the metabolism and transport of iron in normal and neoplastic cells. Biochimica et Biophysica Acta 1997; 1331(1): 140.

37. Konijn AM. Iron metabolism in inflammation. Baillieres Clin Haematol 1994; 7(4): 829-49.

38. Breuer W, Hershko C, Cabantchik ZI. The importance of non-transferrin bound iron in disorders of iron metabolism. Transfus Sci 2000; 23(3): 185-92.

39. Jacobs A. Low molecular weight intracellular iron transport compounds. Blood 1977; 50(3): 433-9.

40. Cabantchik ZI, Kakhlon O, Epsztejn S, Zanninelli G, Breuer W. Intracellular and extracellular labile iron pools. Advances in Experimental Medicine and Biology 2003; 509:

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41. Cappellini MD, Piga A. Current status in iron chelation in hemoglobinopathies. Curr Mol Med 2008; 8(7): 663-74.

42. Kalantar-Zadeh K, Kalantar-Zadeh K, Lee GH. The fascinating but deceptive ferritin: to measure it or not to measure it in chronic kidney disease? Clin J Am Soc Nephrol 2006; 1 Suppl 1: S9-18.

43. Wood JC. Cardiac iron across different transfusion-dependent diseases. Blood Rev 2008;22 Suppl 2: S14-21.

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44.

Davis BH, Olsen S, Bigelow NC, Chen JC. Detection of fetal red cells in fetomaternal hemorrhage using a fetal hemoglobin monoclonal antibody by flow cytometry. Transfusion 1998; 38(8): 749-56.

45. Dziegiel MH, Nielsen LK, Berkowicz A. Detecting fetomaternal hemorrhage by flow cytometry. Curr Opin Hematol 2006; 13(6): 490-5.

46. Kleihauer E, Braun H, Betke K. [Demonstration of fetal hemoglobin in erythrocytes of a blood smear]. Klin Wochenschr 1957; 35(12): 637-8.

47. Navenot JM, Merghoub T, Ducrocq R, Muller JY, Krishnamoorthy R, Blanchard D. New method for quantitative determination of fetal hemoglobin-containing red blood cells by flow cytometry: application to sickle-cell disease. Cytometry 1998; 32(3): 186-90.

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Chapter 2 Diagnostic Laboratory Methods

2.5 Solid Phase Electrophoretic Separation

Rita Ellerbrook, PhD, and Zia Uddin, PhD

2.5.1 Introduction

Electrophoresis is defined as the movement of charged molecules (e.g. proteins)

under an electrical field, either through a solution (moving boundary electrophoresis) or

through a semi-solid material embedded in a buffer (zone or solid phase

electrophoresis). Historically, the first hemoglobin variant (HbS) identification using

moving boundary electrophoresis was achieved by Professor Linus Pauling 1 in 1949 at

the University of Chicago, Chicago, Illinois. Subsequently the moving boundary

electrophoresis due to experimental difficulties was replaced by solid phase

electrophoretic methods, e.g., cellulose acetate, agarose, and agar, etc.

In view of the convoluted three-dimensional structure of the hemoglobin

molecule, even a single genetic mutation, resulting in the substitution of an amino acid

in the globin chain (e.g. the substitution of the amino acid valine for glutamic acid in the

sixth position of the β-chain of hemoglobin molecule) may result in the change of the

secondary/tertiary structure of the hemoglobin molecule/the net charge on the molecule.

This change in the shape/net charge of the hemoglobin molecule is sufficient to modify

its electrophoretic mobility (movement under an electric field), and thus is

advantageously employed for the separation and identification of the hemoglobin

variants. The migration and the identification of hemoglobin variants in solid phase

61

electrophoretic methods are accomplished at alkaline pH (8.6) and acid pH (5.6), and

the commonly used solid phases for this purpose are described here.

2.5.2 Cellulose Acetate Electrophoresis (alkaline pH)

Cellulose upon treatment with acetic anhydride converts into cellulose acetate by

virtue of the acetylation of the hydroxyl groups. The separation characteristic of

cellulose acetate depends on the degree of acetylation reaction and other variables,

e.g., additives used, prewashing procedure utilized by the manufacturer, pore size,

thickness of the membrane, etc. Historically, cellulose acetate electrophoresis (CAE)

was used worldwide in view of the speed of separation, ability to make the membrane

transparent for the quantification of bands by densitometry, ability to store the

transparent membranes for longer periods (plastic backed cellulose acetate plates), no

need for controlled lower temperature for the electrophoresis, low cost, etc. Under the

electrophoretic conditions of pH 8.6, the ionizable groups (e.g. carboxyl group) are

negatively charged thus rendering a negative charge on the hemoglobin molecule. The

relative migration of the hemoglobin towards the anode is dependent on the net

negative charge on the hemoglobin molecule.

CAE laboratory procedure and information about the required hardware and

consumables can be obtained from Helena Laboratories, Beaumont, Texas, USA

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Fig 1. Computer simulated cellulose acetate electrophoresis of adult hemoglobins (pH 8.6) In Figure 1,

Fig 1. Computer simulated cellulose acetate electrophoresis of adult hemoglobins (pH 8.6)

In Figure 1, separation of a few hemoglobin variants by CAE is illustrated. This is a

computer simulation of the separation of hemoglobins. Generally in all electrophoretic

separations, a commercially prepared “AFSC” control is used to designate the migration

position of the unknown. Hb S, Hb D, Hb Lepore and Hb G migrate in approximately the

same position, therefore further confirmation of the hemoglobin variant is achieved by

additional laboratory tests, e.g., solubility test and citrate agar electrophoresis at pH 5.6

(see below). In case the hemoglobin variant is not identified by these preliminary

laboratory tests, the laboratory employs other procedures, e.g., HPLC, IEF, and DNA

63

studies. The same procedure is also followed about the co-migration of Hb C, Hb E,

and Hb O-Arab upon CAE.

2.5.3 Agarose Gel Electrophoresis (alkaline pH)

Agar is a gelatinous material prepared from certain marine algae, and is a

mixture of agarose and sulfated polysaccharides contaminants called

agaropectin. The highly purified agar (neutral fraction of agar) that is almost free

of agaropectin (ionizable groups like sulfate and carboxylic) is called agarose.

Agarose gel electrophoresis (AGE) at alkaline pH 8.6 is the widely used clinical

laboratory method for the identification of hemoglobin variants. The reason for the

popularity of AGE is due to the lower affinity of agarose for proteins, ability to exhibit

decreased endosmosis, and also the transparency of the film after drying which allows

quantification of the hemoglobin molecule by densitometry. It is emphasized that

hemoglobinopathy is never determined alone by AGE (alkaline pH 8.6), as is the case

with CAE. The resolution of atypical bands or a band co-migrating at the positions of

commonly encountered bands upon AGE (e.g., HbA 2 , HbS, etc.) is accomplished by

additional laboratory tests.

Currently the AGE reagents, separation gels, and Peltier cooling device (which

cools the gel during electrophoresis) are supplied by two major manufacturers (Sebia,

France, and Helena Laboratories, USA). Sebia’s hemoglobin AGE kit (Hydragel) is used

in conjunction with their semi-automated HYDRASYS System. Helena Laboratories,

USA is a pioneer in supplying AGE kits for >35 years. The Helena’s QuickGel method

available in manual mode is ideal for smaller volume clinical laboratories, and the same

64

plate form is used in the semi-automated instruments (SPIFE 2000 and SPIFE 3000) for

handling a larger volume of testing. Helena’s fully automated instrument (SPIFE 4000)

utilizes a different plate form than QuickGel. Detailed information about AGE

procedures of these two manufacturers can be obtained from their web site

In Fig 2 we have presented the computer simulation of the electrophoretic

mobilities of the commonly used “AFSC” control and few hemoglobin bands obtained

from AGE at alkaline pH.

and few hemoglobin bands obtained from AGE at alkaline pH. Fig 2. Computer simulation of hemoglobin

Fig 2.

Computer simulation of hemoglobin agarose gel electrophoresis bands

2.5.4

Agar Electrophoresis (acid pH)

Agar electrophoresis (AE) at acid pH (5.6-6.2) for the identification/confirmation

of hemoglobins has been widely used for > 40 years. Agarose and agaropectin are the

two main components of agar. Both the electrophoresis and electroendosmotic flow

65

principles are involved in the separation of hemoglobins by AE. Citrate buffer is usually

used for the electrophoretic purpose (Beckman-Coulter uses maleate buffer in their

Paragon kit), therefore it is also called citrate agar electrophoresis. Commercially the AE

kits (plates, reagents, consumables, etc.) are also available from Sebia, France

(HYDRAGEL ACID HEMOGLOBIN) and Helena Laboratories, USA (Titan Gel and

QuickGel). In both cases, hemoglobin “AFSC” control is used to confirm the

electrophoretic mobility of the unknown (i.e. Hb S, Hb C, Hb E, etc.). Quantification of

the bands is not required and the electrophoregrams are evaluated visually. Laboratory

procedures for AE by Sebia and Helena Laboratories can be obtained from their web

sites (www.sebia.com and www.helenalaboratories.com). In Fig 3, we have presented a

computer simulation of an electrophoregram of the AE.

2.5.5 Interpretation of Hemoglobin Agarose Gel (pH 8.6) and Agar Gel (pH 5.6) Electrophoresis

The commercially available control that consists of a mixture of Hb A, Hb F, Hb

S, and Hb C serves to set the framework upon which the various hemoglobin variant

mobilities are compared. This combination of hemoglobins is run on each

electrophoretic plate and the interpretation is aided by comparing the mobility of the

variant to these hemoglobins in the control material. By assigning the distance from

HbA to HbC an arbitrary distance unit of 10 (under either acid or alkaline conditions), a

relative number may be assigned to any hemoglobin.

Schneider and Barwick 2 presented this system of hemoglobin typing and

provided a chart of the relative mobilities of all the hemoglobins fully characterized at

66

that time. This chart provided preciseness to the characterization not before possible.