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 Kenneth F. Tucker, MD Rita Ellerbrook, PhD Piero C. Giordano, PhD Kimberly R. Russell, MT (ASCP), MBA Chairperson Member Member Member Member

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

III

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

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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 CenterHouston Medical School 6431 Fannin Street, MSB, 2.290 Houston, TX 77030 USA

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

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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 Childrens Research Hospital Memphis, Tennessee USA Andrew N Young, MD, PhD Department of Pathology & Laboratory Medicine Emory University School of Medicine Atlanta, GA 30303 USA

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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

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 Love Serve

God People 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 Zia Uddin, PhD

XII

Table of Contents

Chapter 1

Hemoglobin
1.1 1.2 1.3 1.4

Thomas E. Burgess, PhD Hemoglobin Structure Hemoglobin Function Hemoglobin Synthesis Hemoglobin Variants

Chapter 2

Diagnostic Laboratory Methods

2.1 Basic Concepts 10
Jayson Miedema, MD and Christopher R. McCudden, PhD 2.1.1 2.1.2 2.1.3 2.1.4 2.1.5 2.1.6 Unstable Hemoglobins Altered Affinity Hemoglobins Sickle Solubility Test Serum Iron, TIBC, Transferrin, Ferritin Soluble Transferrin Receptor 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 2.3.1 2.3.2 2.3.3 2.3.4 2.3.5 2.3.6 Introduction Deletions Associated with the HPFH Phenotype Non-Deletion Forms of HPFH HPFH Unlinked to the -Globin Gene Cluster Conclusion Hemoglobin F Quantification

28

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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 -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

Rita Ellerbrook, PhD, and Zia Uddin, PhD 2.5.1 Introduction

61

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 2.6.1 2.6.2 2.6.3 2.6.4

73

Introduction Basic Principle Application of CZE in Diagnostic Hemoglobinopathies Interpretation of CZE Results XIV

2.7

Isoelectric Focusing
David Hockings, PhD

83

2.7.1 IEF of Normal Adult Hemoglobin: HbA (Adult), HbF ( Fetal), HbA2 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

2.8

High Performance Liquid Chromatography

Zia Uddin, PhD 2.8.1 Introduction 2.8.2 Basic Principle 2.8.3 Illustrations

95

Chapter 3

Globin Chain Analysis

3.1 Solid Phase Electrophoretic Separation
Zia Uddin, PhD 3.1.1 Cellulose Acetate Electrophoresis (Alkaline and Acid pH)

102

3.2

Reverse Phase High Performance Liquid Chromatography

Zia Uddin, PhD, and Rita Ellerbrook, PhD

106

3.3 Globin Chain Gene Mutations: DNA Studies

Joseph M. Quashnock, PhD 3.3.1 Introduction 3.3.2 Genotyping-PCR Methodology 3.3.3 Mutations

115

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3.4

Electrospray Ionization-Mass Spectrometry

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

132

3.5

PCR and Sanger Sequencing

Elaine Lyon, PhD 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

147

Chapter 4

Alpha and Beta Thalassemia

Herbert L. Muncie, MD. 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 Epidemiology Pathophysiology Alpha Thalassemia Beta Thalassemia Diagnosis Treatment Complications 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

157

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Chapter 5

Neonatal Screening for Hemoglobinopathies 178

Zia Uddin, PhD 5.1 Introduction 5.2 Methodologies 5.3 Laboratory Reports Format & Interpretation 5.4 Examples of Neonatal Screening 5.4.1. 5.4.2 5.4.3 5.4.4 Capillary Zone Electrophoresis Isoelectric focusing Isoelectric focusing and High Performance Liquid Chromatography 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 A1c
Zia Uddin, PhD 7.1 7.2 Introduction

232

HbA1c Diagnostic Role in Diabetes Mellitus, and Glycemic Control in Adults Measurement of HbA1c Factors Affecting the Accuracy of Hb A1c Assay

7.3 7.4

XVII

Case Studies Introduction

Case # 1 Case # 2 Case # 3 Case # 4

244

Normal Adult

247 252 258

Hemoglobin S trait

Hemoglobin S homozygous

Hemoglobin S with hereditary persistence of fetal hemoglobin (HPFH) 264 Hemoglobin G-Philadelphia trait Hemoglobin S-G Philadelphia Hemoglobin G-Coushatta trait Hemoglobin C trait 293 299 272 279 287

Case # 5 Case # 6 Case # 7 Case # 8 Case # 9 Case # 10

Hemoglobin C homozygous

Hemoglobin C with hereditary persistence of fetal hemoglobin (HPFH) 306 Hemoglobin S-C disease 312 319

Case # 11 Case # 12 Case # 13

Hemoglobin D-Los Angeles (D-Punjab) trait Hemoglobin S-D disease 326

XVIII

Case # 14

Hemoglobin E and Associated Disorders Case # 14 a Hemoglobin E trait 339

333

Case # 14 b Hemoglobin E homozygous Case # 14 c Hemoglobin S-E disease Case # 15 Case # 16 Case # 17 Case # 18 Case # 19 Case # 20 Case # 21 Case # 22 Case # 23 Case # 24 Case # 25 Case # 26 Case # 27 Case # 28 Hemoglobin S-Korle Bu (G-Accra) Hemoglobin O-Arab trait -Thalassemia trait
+

344 350

356

362

368 374 381

Hemoglobin S- - thalassemia Hemoglobin C- thalassemia Hemoglobin Hasharon trait Hemoglobin Zurich trait Hemoglobin Lepore trait Hemoglobin J-Oxford trait Hemoglobin J-Baltimore trait Hemoglobin Malmo trait Hemoglobin Koln trait Hemoglobin Q-India trait Hemoglobin Dhofar trait 421 432 441 454 387
o

394 400 408 415

XIX

Chapter 1
Hemoglobin
Thomas E. Burgess, PhD 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 edited by Steinberg, Forget, Higgs and Nagel should be consulted.
1

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 (), beta (), gamma () or delta (). In addition, during embryonic life in utero, zeta () and epsilon () chains are present in the first several weeks of life, being rapidly converted to alpha, beta and gamma chains as development occurs.

Figure 1. Globin chains concentration changes in embryonic, fetal and post-natal 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, 2nd Edition, 2006).

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 and 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 . 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.
2

Figure 2. Tertiary structure of a globin chain and the quaternary structure of hemoglobin molecule (Adopted with permission from Blackwell Publishing, Barbara J. Bain, Haemoglobinopathy Diagnosis, 2nd Edition, 2006).

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 (CO2) and nitrous oxide (NO). The transport of CO2 is facilitated by reversible carbamoylation (formation of carbamoyl moiety, i.e., H2NCO-) of the N-terminal amino acids of the globin chains and can,
4

via proton scavenging, keep CO2 in the soluble bicarbonate form . 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 pheno typic analysis and subsequent interpretation becomes ultimately important to the patient.

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.

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 variants 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, A2). 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 patients 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 patients 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. Bain, Barbara J.. in Hemoglobinopathy Diagnosis, 2nd Ed., pg. 4, Blackwell Publishing, 2006. Bain, Barbara J.. in Hemoglobinopathy Diagnosis, 2nd Ed., pg. 1, Blackwell Publishing, 2006.

2.

3.

Chapter 2 Diagnostic Laboratory Methods

2.1 Basic Concepts
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 andHbA2 and HbF may be increased. The peripheral smear often shows anisocytosis, poikilocytosis, basophilic stippling, polychromasia and, hypochromasia. Since not all unstable
10

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: (Hb4C-Hb50C)/(Hb4C)x100 Where Hb4C is the hemoglobin concentration at 4 degrees centigrade and Hb50C 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 hemoglobinopathies associated with unstable hemoglobin can be obtained from: http://medtextfree.wordpress.com/2011/12/30/chapter-48-hemoglobinopathies

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 SO2 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 Fe3+, requires reduction to Fe2+ 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 (Fe3+) 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 iron binding capacity (TIBC) is another test used to assess iron homeostasis. 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. presence of non-transferrin iron binding This method can be affected by the proteins, particularly in cases of

hemochromatosis and thalassemias. 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 (g/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 (g/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 (g/dL) / TIBC (g/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). In the blood, ferritin is generally poor in iron content and is referred to as apoferritin. 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 Iron Chronic of Disease Iron Deficiency Thalassemia Hemochromatosis or or or or or Anemia TIBC % Saturation or or Transferrin Ferritin

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. 21st 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;376:20182031. 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. 5th 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).
1,2,3,4

Table I Age Dependent Mean Hemoglobin and MCV Values Age 3 to 6 months 6 months to 2 years 2 to 6 years 6 to 12 years 12 to 18 years (female) 12 to 18 years (male) > 18 years (female) > 18 years (male) Mean Hemoglobin (g/dL) 11.5 12.0 12.5 13.5 14.0 14.5 14.0 15.75

Mean MCV (fL) 91 78 81 86 90 88 90 90

21

Iron deficiency anemia, -thalassemia trait, and -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 -thalassemia trait usually have

increased hemoglobin A2. It is pointed out that lower hemoglobin A2 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 -thalassemia trait in such patients. Conversely, patients with -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
Iron deficiency anemia Thalassemia trait Other hemoglobinopathies Lead toxicity Chronic inflammation Sideroblastic anemia Iron deficiency anemia Thalassemia trait Pregnancy Anemia of chronic disease Sideroblastic anemia

Men and non-menstruating women

Iron deficiency anemia Anemia of chronic disease Unexplained anemia Thalassemia trait

Several laboratory tests in addition to the CBC, e.g. serum iron, serum ferritin, total ironbinding 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 Serum ferritin Iron deficiency anemia Thalassemia Decreased Increased Normal to increased Anemia of chronic disease Normal to increased Normal Sideroblastic anemia Normal to increased Increased

RBC Increased distribution width (RDW) Serum iron Decreased

Normal to increased Normal

Normal to decreased Slightly increased

Normal to increased Normal

Total ironbinding capacity Transferrin saturation

Increased

Decreased

Normal to increased

Normal to slightly decreased

Normal to increased

Van Vranken 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. We have corrected this error in the figure 1, and the journal (American Family Physician) editor was also informed.
3

23

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 -thalassemia minor and a few with probably -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 bellshaped 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 -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 cows milk does not contain iron. A nave 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. 2nd 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 thalassemia and sickle cell anemia. HPFH is usually due to deletions of different sizes involving the -globin gene cluster, but nondeletion types of disorders have also been identified, usually due to point mutations in the -globin gene promoters (reviewed in refs. 1-3). Figure 1 diagrammatically illustrates the spatial organization of the -like globin genes in the -gene cluster on chromosome 11. However, as discussed later in this chapter, certain forms of nondeletion HPFH are clearly not linked to the -globin gene cluster.

28

Figure 1. Deletions of the -globin gene cluster associated with fusion proteins and HPFH. The circle 3 to the -globin gene indicates the 3 -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 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: 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 socalled F cells, as in thalassemia. Homozygotes for both conditions totally lack Hb A and Hb A2, indicating absence of - and -globin gene expression in cis to the thalassemia and HPFH determinants. Although the apparent striking qualitative

difference in cellular distribution of Hb F between HPFH and 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 -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, + and o thalassemia. Nevertheless,

heterocellular forms of HPFH, without a -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 -and -globin gene expression from the affected chromosome, is associated with large deletions in the -globin gene cluster that remove the -and -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 -like globin chain resulting from a non-homologous crossing-over event between two globin genes in the
30

-gene cluster. However, whereas the Lepore crossover occurred between the - and -globin genes, the Kenya gene resulted from crossover between the A- and -globin genes (Fig. 1). The crossover occurred in the second exons of the A and genes, between the codons for amino acids 80 to 87, and resulted in deletion of ~24 kb of DNA between the A to the gene. Unlike Hb Lepore, that is associated with a thalassemic phenotype, Hb Kenya is associated with a phenotype of pancellular G 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 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- and Kenya gene promoters of a well characterized enhancer element located in the 3' flanking DNA of the -globin gene, shown by the filled circle in Fig. 1, that becomes translocated into close proximity of the -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 gene to a point 0.7 kb 3' to the gene, deleting the 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' -gene enhancer on the -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 genes as a result of the deletion events, may influence -gene expression, in a manner analogous to the presumed influence of the 3' -gene enhancer on -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 -globin genes. A second theory for the mechanism of increased -gene expression in deletiontype 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 -thalassemia deletions, are located in the DNA between the and genes, the so-called -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 thalassemia, results in markedly impaired postnatal suppression of -gene activity in all erythroid cells (8). A number of subsequent observations have been made that support a role for the A-intergene region in the regulation of -gene expression (reviewed in ref. 9). The Corfu deletion in particular, involving the -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 -globin gene cluster. The most conclusive evidence for a functional role of the A-intergene DNA in the regulation of gene expression consists of the observations by Sankaran et al. who have extensively characterized a negative regulatory transcription factor, called BCL11A, that down-regulates -gene expression in adult erythroid cells and that binds to the A-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 -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 -cluster, including the upstream locus control region (LCR) and the intergenic region, but does not bind to the - or -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 -gene and its flanking DNA. One mutant with a more proximal breakpoint has a -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 -gene silencer element in the -gene cluster that associates with a BCL11A-containing repressor complex and that this interaction is an important factor in the suppression of -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 and
A

genes are over expressed, only one or the other 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 A

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

heterocellular expression of both genes. Because of the restricted pattern of -globin gene expression in the G and A 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 genes in these disorders.

34

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 G G A type, and the gene in cis to the -175 mutation carried the -158 C T change. The references for the individual mutations are cited in references 1 and 3.

The results of these structural analyses revealed a number of different point mutations in the promoter region of the over expressed 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 genes. The first region is located approximately 200 base pairs from
35

the "cap site" or site of transcription initiation of the 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 -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 -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 or A gene is associated with a phenotype of pancellular HPFH with high levels of Hb F (1525%). 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 -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 genes, the CCAAT box is duplicated and the mutation associated with the Greek A type of nondeletion HPFH is a G->A substitution at position -117, 2 bases upstream of the distal CCAAT box of the A-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 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 transacting 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 -gene promoters and thereby prevent the normal postnatal suppression of -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 -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 -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 -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 -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 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 -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 -globin gene cluster. Blood 1989; 74: 21782186. 6. Kosteas T, Palena A, Anagnou NP. Molecular cloning of the breakpoints of the hereditary persistence of fetal hemoglobin type-6 (HPFH-6) deletion and sequence analysis of the novel juxtaposed region from the 3' end of the beta-globin gene cluster. Hum Genet. 1997;100: 441-5. 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 gammaglobin 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):10724. 9. Bank A, O'Neill D, Lopez R, et al. Role of intergenic human - -globin sequences in human hemoglobin switching and reactivation of fetal hemoglobin in adult erythroid cells. Ann N Y Acad Sci 2005;1054:48-54.

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10. Chakalova L, Osborne CS, Dai YF, et al. The Corfu thalassemia deletion disrupts -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 -globin by BCL11A involves long-range interactions and cooperation with SOX6. Genes Dev 2010; 24:78398. 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):R21623. 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 D, Liu K, Sun CW, Pawlik KM, Townes TM. KLF1 regulates BCL11A expression and - to -globin gene switching. Nat Genet 2010; 42:742-4.

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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
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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 x105 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.

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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 Tlymphocytes. CD45 is particularly useful since it is differentially expressed on various nucleated blood cells (Fig. 1).

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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, 22) is the major hemoglobin (Hb) in the prenatal period that is largely replaced after birth by the adult Hb (HbA, 22) (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
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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 heterocellular, 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 HbFcontaining 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 -thalassemia, elevated HbF should compensate partially for the deficiency of -globin chains and reduce the excess of -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 23 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
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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 Fretics can indicate the efficacy of the drug and/or the patients 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, 50l 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 Manufacturers instructions or on a pre-performed titration) are added to 5x106 cells (5 l of the 10% suspension) and incubated for 1-hr at 370C, 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 104-105 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).

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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 -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.
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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, -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 patients life, including the ability of thalassemic women to give birth. Recently, we were confronted with a case of a possible FMH in a -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 -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
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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 hemoglobinopathies. In -thalassemia, excess -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 -thalassemia, the - and -globins, which are produced in excess, do not precipitate
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right away, but form the soluble tetramers 4 (Hb Barts) and later the 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 Nethylmaleimide, which totally blocks thiol groups, decreased the fluorescence in a dosedependent 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 ,-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).
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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 37C 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 4C. 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.

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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 H2O2. Histograms of RBC are shown.

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
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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 -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
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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 37oC with 0.25 M Calcein Acetoxymethyl Ester (CA-AM). After wash, the cells are treated with or without Deferiprone (L1, 100 M). 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.

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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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44.

45. 46. 47.

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. Dziegiel MH, Nielsen LK, Berkowicz A. Detecting fetomaternal hemorrhage by flow cytometry. Curr Opin Hematol 2006; 13(6): 490-5. Kleihauer E, Braun H, Betke K. [Demonstration of fetal hemoglobin in erythrocytes of a blood smear]. Klin Wochenschr 1957; 35(12): 637-8. 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 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 -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
1

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 (www.helena.com).

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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., HbA2, 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). Sebias 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 Helenas 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. Helenas 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 (www.sebia.com and www.helenalaboratories.com). 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.

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 presented this system of hemoglobin typing and provided a chart of the relative mobilities of all the hemoglobins fully characterized at
66
2

that time. This chart provided preciseness to the characterization not before possible. Ellerbrook and Matthews at Helena Laboratories felt that since the process was a visual one, therefore a quicker way to examine these relative mobilities was to convert them into a chart as depicted in Appendix I. It will not be out of place to mention here that for >30 years in the Hemoglobin Laboratories of Henri Mondor Hospital (Creteil, France), Professor Henri Wajcman and associates have organized a database of the electrophoretic mobility of > 400 Hb variants, using a similar format to that proposed by Schneider and Barwick. The Wajcman group included in their database the results of a) IEF on polyacrylamide gel, b) electrophoresis on cellulose acetate at alkaline pH, c) citrate agar electrophoresis, d) electrophoresis of dissociated globin chains in 6M urea at pH 6.0 and 9.0 or in the presence of Triton X-100 . An excerpt of eight hemoglobins from the chart developed by Ellerbrook and Mathews is shown in Fig 3 for instructional purposes.
3 4 3

Fig 3. Combined agarose gel (pH 8.6), and citrate agar (pH 5.6) electrophoretic pattern presentation for instructional purposes. The area labeled Alkaline on the left side of this figure depicts the mobility of
67

the named hemoglobins under alkaline conditions. The perpendicular lines represent the relative mobility of Hb C (-10), Hb S (-5.2), Hb F (-2.6), Hb A (0), Hb J Toronto (4.5) and Hemoglobin I (8.5). In Fig 3 Hb C is seen to have the least mobility in an alkaline electric field and is depicted squarely on the line. Each of the control hemoglobins (e.g. AFSC) will also place squarely on the line. Hemoglobin I and J are extremely rare and their actual presence on the gels as a control is not necessary because this grid is all about spacing. The gel will have hemoglobins A, F, S, and C on it to establish spacing. The distance between Hb S and Hb A is slightly more than the distance from Hb A to Hb J Toronto, and this distance is slightly less than the distance from Hb J Toronto to Hb I. While looking at the actual gel the mobility is not a depiction of the leading edge of the migration but rather the bulk of that hemoglobin band. Denatured hemoglobins usually run faster than the native form and therefore the leading edge may be a function of the age of the sample. The sample application in alkaline conditions is to the left of Hb C, and most hemoglobins at this pH migrate in the same direction (left side). The right side of the figure has the similar approach to the mobility under acidic conditions. The order of migration is different and the direction is reversed. Here the sample is applied between Hb S and Hb C. Under acidic conditions Hb F is the fastest moving hemoglobin. The distance from Hb A to Hb C is assigned a new relative distance of 10. Hb F is assigned the number -4.4, and Hb S is assigned +5.8. The +/sign is relative to the Hb A value of 0 and not due to distance from the application point.
68

There is no allowance for fast hemoglobins under acidic conditions because there are none. At a pH of 6.2 or less, fast hemoglobins migrate like Hb A. Looking at the left side of the chart, Hb J Baltimore migrates slower than Hb J Toronto because the bulk of the hemoglobin has not moved as far as one might expect Hb J Toronto to move. Acid electrophoresis is of no assistance in this case because these fast hemoglobins do not migrate differently, and thus all end up lined with Hb A. The mobility of Hb C Siriraj is not different from Hb C under acidic conditions, but can be differentiated under alkaline pH. In this case since the alkaline separation would have been done first the only apparent observation would be the presence of an abnormal hemoglobin band migrating between Hb F and Hb S. Very few hemoglobins migrate like hemoglobin S so this second test is very useful in narrowing down the possible identity of this variant. The chart in Appendix I contains the relative mobilities of 165 hemoglobins. The most common variants were discovered first so this chart should encompass the relative mobilities of most of the hemoglobins found.

2.5.6 Requirements for the identification of complex hemoglobinopathies Age, sex, ethnicity, ethnic background of biological parents, blood transfusion (past three months), CBC with differential, serum iron, TIBC, ferritin, treatment status (immunotherapy), laboratory results of AGE and AE electrophoresis, capillary zone electrophoresis, high pressure liquid chromatography, isoelectric focusing, quantitative results of Hb A2, and Hb F, hemoglobin stability, and globin chain analysis. The significance of all these parameters shall be obvious from the case studies mentioned later on in the book.
69

References 1. Pauling L, Itano HA, Singer SH, Wells IC. Sickle cell anemia, a molecular basis disease. Science 1949; 110: 53. 2. Barwick RC, Schneider RG. The computer-assisted differentiation of hemoglobin variants, in Human Hemoglobins and Hemoglobinopathies: A review to1981. Texas Reports on Biology and Medicine 1980-81; 40: 143-156 3. Helena Laboratories, Beaumont, Texas, USA 4. Wajcman H. Electrophoretic Methods for Study of Hemoglobins. Methods in Molecular medicine, vol 82: Hemoglobin Disorders: Molecular methods and Protocols, Edited by: Ronald L. Nagel, Humana Press Inc., Totowa, NJ.

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Appendix 1

Appendix 1 continued next page

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Appendix 1 continued

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Chapter 2 Diagnostic Laboratory Methods

2.6 Capillary Zone Electrophoresis
Zia Uddin, PhD

2.6.1.1

Introduction

During the last five decades separation science has witnessed unparallel growth. Chromatography and electrophoresis are the main techniques that are routinely used worldwide for the separation, identification, and quantification of analytes in clinical laboratories. Capillary zone electrophoresis (CZE) played a significant role in the completion of the human genome project. Introduction of CZE instruments by BeckmanCoulter, Sebia, and Helena Laboratories not only automated but also increased the sensitivity, specificity and reproducibility of the clinical laboratory procedures (e.g., serum protein electrophoresis, immunotyping, hemoglobin variant identification for both the adult and newborn). Besides references listed at the end of this section, the interested reader is advised to also review the online literature on CZE (e.g., Righetti, PG, and Guttman, A. 2012 Capillary Electrophoresis. eLS.)

2.6.2

Basic Principle In simple terms CZE is a liquid flow electrophoresis in which buffer has replaced

the solid support medium (e.g., agarose gel), and the separation occurs due to the interaction of the analyte with the pH of the buffer. For this reason initially CZE was also called Free Solution Capillary Electrophoresis. In Figure 1, a pictorial illustrat ion of CZE principle is presented.
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Figure 1. Capillary Zone Electrophoresis Principle

In CZE two independent phenomena occur, i.e., a) migration of negatively charged ions toward the positively charged electrode, and b) interaction of the positive charges from the buffer and the negative charges from the capillary wall leading to electro-osmotic flow (EOF) from the anode to the cathode. Both of these two processes (electrophoretic mobility and EOF) can take place at the same time working in opposite direction thus providing greater resolution.

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Automated CZE instrument (Figure 2) consists of the following: a) b) c) d) e) f) g) h) Cathode Anode Power supply to generate high voltage (10,000 volts) Catholyte (buffer solution at the cathode end) Anolyte (buffer solution at the anode end) Capillary facilitated with a cooling device Detector (415 nm for hemoglobins) Computer for data handling and storage

Figure 2. Capillary Zone Electrophoresis Instrument Components

2.6.3 Application of CZE in Diagnostic Hemoglobinopathies

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Hemoglobin variants can be separated on CZE as is the case with other proteins. This method is the most advanced and dedicated alternative to the classic alkaline and acid electrophoresis and the more sophisticated IEF. Chromatography, the separation alternative on column, has developed from separation on size, charge and hydrophobic interaction to the modern dedicated high performance liquid chromatography (HPLC), as we know today. Both of these dedicated methods (CZE and HPLC) have the advantage of using minimal amounts of material, of providing a separation in a matter of minutes, with high reproducibility and sensitivity and above all they are able to measure virtually all fractions including those present at low levels but essential for the diagnosis or hemoglobinopathy. In addition these two methods may complement each other up to a certain extent compensating for specific errors.

2.6.4 Interpretation of CZE Results The migration time of the hemoglobin variant (since the inception of the injection of the sample and the moment a specific molecule is detected) is divided into fifteen (15) zones (Table 1). It is obvious that > 1000 hemoglobin variants cannot be separated in 15 zones. However, the most common one (e.g., Hb S, C, E, and D) will be putatively identified by their zone with a specificity >90%. Table 1 shows that there is an overlap of hemoglobin variants in a particular zone (Z1 Z15). This limitation of CZE is similar to that experienced with other techniques employed for the identification of hemoglobin variants, e.g., HPLC (Szuberski J, Oliveira JL, Hoyer JD. A comprehensive analysis of hemoglobin variants by high performance liquid chromatography. Int J Lab Hematol 2012; 34: 594-604).
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In Figure 3 we have presented a CZE scan of the most commonly used AFSC control in the clinical laboratory, which illustrates the position of HbA, HbF, HbS, and HbC peaks corresponding to their respective zones. Later on in this book (case studies) we have also presented the CZE scan of the hemoglobin variant for each case. One drawback of CZE is the assignment of the migration position of the hemoglobin bands into Z1-Z15 (Table 1) in cases when the HbA / Hb A2 are absent in the specimen of interest, e.g. Hb S-C disease. This drawback is due to the shifting of band positions in the absence of Hb A / Hb A2. This limitation of CZE is avoided by mixing (1:1 ratio) the specimen devoid of HbA/HbA2 with a specimen containing Hb A, and performing the CZE test thus achieving the relevant migration position and zoning (Z1-Z15).

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Table 1. Hemoglobin Zones of CZE: Z1 Z15

N zone Hb Variants Library Software release 8.60 Hb Santa Ana free alpha chain, Hb Mizuho (minor peak), Hb delta A'2, Hb alpha A2, Hb T-Cambodia, "Savaria" Hb A2 variant, "Chad" Hb A2 variant, "Arya" Hb A2 variant, "Hasharon" Hb A2 variant, "Fort de France" Hb A2 variant, "Ottawa" Hb A2 variant, "Shimonoseki" Hb A2 variant, "Stanleyville II" Hb A2 variant, "O-Indonesia" Hb A2 variant, "G-Norfolk" Hb A2 variant, "San Antonio" Hb A2 variant, 1 "Handsworth" Hb A2 variant, "Matsue-Oki" Hb A2 variant, "Memphis" Hb A2 variant, "Q-Iran" Hb A2 variant, "G-Waimanalo" Hb A2 variant, "Russ" Hb A2 variant, "Q-India" Hb A2 variant, "Montgomery" Hb A2 variant, "Watts" Hb A2 variant, "G-Pest" Hb A2 variant, "Winnipeg" Hb A2 variant, "Queens" Hb A2 variant, "Inkster" Hb A2 variant, "Chapel Hill" Hb A2 variant, "Q-Thailand" Hb A2 variant, "Park Ridge" Hb A2 variant Hb C, Hb F-Hull, Hb F-Texas-I, Hb Constant Spring, Hb C-Harlem (C-Georgetown), "Boumerdes" Hb A2 variant, "Bassett" Hb A2 variant, "Tarrant" Hb A2 variant, "Manitoba I" Hb A2 variant, "St. Luke's" Hb A2 2 = Z(C) variant, "Setif" Hb A2 variant, "Sunshine Seth" Hb A2 variant, "Titusville" Hb A2 variant, "Swan River" Hb A2 variant, "Manitoba II" Hb A2 variant, "Val de Marne" Hb A2 variant Hb A2, Hb Chad (E-Keelung), Hb O-Arab, Hb E-Saskatoon, "Dallas" Hb A2 variant*, "Toulon" Hb A2 variant*, "Bonn" Hb A2 variant*, "Chicago" Hb A2 variant*, "Fontainebleau" Hb A2 variant*, "Hekinan" 3 = Z(A2) Hb A2 variant*, "Mosella" Hb A2 variant*, "Aztec" Hb A2 variant*, "Frankfurt" Hb A2 variant*, "MBoston" Hb A2 variant*, "Owari" Hb A2 variant*, "Twin Peaks" Hb A2 variant*, "Conakry" Hb A2 variant*, "Gouda" Hb A2 variant*, "Jura" Hb A2 variant, "Nouakchott" Hb A2 variant Hb E, Hb Seal Rock, Hb Kln (Ube-1), Hb Buenos Aires (minor peak), Hb M-Saskatoon (minor peak), Hb 4 = Z(E) A2-Babinga, Hb G-Siriraj, Hb Agenogi, Hb Sabine, Hb Santa Ana, Hb Savaria, "M-Iwate" Hb A2 variant, "Wayne" Hb A2 variant (peak 1), Denatured Hb C Hb S, Hb Arya, Hb Hasharon (Sinai), Hb Dhofar (Yukuhashi), Hb Shimonoseki (Hikoshima), Hb OIndonesia (Buginese-X), Hb Ottawa (Siam), Hb Fort de France, Hb Montgomery, Hb G-Copenhagen, Hb 5 = Z(S) S-Antilles, Hb Handsworth, Hb S-Oman (peak 2), Hb Hamadan, Hb Russ, Hb Stanleyville II, "Lombard" Hb A2 variant, "Tatras" Hb A2 variant, "Cemenelum" Hb A2 variant, "Jackson" Hb A2 variant, "Hopkins-II" Hb A2 variant, "J-Broussais" Hb A2 variant (alpha 2), Denatured Hb O-Arab Hb D, Hb Memphis, Hb Leiden, Hb Muravera, Hb D-Bushman, Hb G-Norfolk, Hb S-Oman (peak 1), Hb Matsue-Oki, Hb Osu Christiansborg, Hb D-Punjab (D-Los Angeles), Hb G-Waimanalo (Aida), Hb Muskegon, Hb D-Ibadan, Hb Buenos Aires (minor peak), Hb Q-India, Hb Lepore (Lepore-BW), Hb Q-Iran, Hb Summer Hill, Hb G-Philadelphia, Hb D-Ouled Rabah, Hb Yaizu, Hb San Antonio, Hb Watts, Hb Ferrara, Hb Kln (Ube-1), Hb Fort Worth, Hb Korle-Bu (G-Accra), Hb G-Taipei, Hb D-Iran, Hb St. Luke's, Hb G-Coushatta (G-Saskatoon), Hb Inkster, Hb Winnipeg, Hb Zrich, Hb G-Pest, Hb Queens (Ogi), Hb 6 = Z(D) Setif, Hb P-Nilotic, Hb Sunshine Seth, Hb Titusville, "Le Lamentin" Hb A2 variant, "J-Meerut" Hb A2 variant, "J-Rajappen" Hb A2 variant, "J-Anatolia" Hb A2 variant, "J-Oxford" Hb A2 variant, "Ube 2" Hb A2 variant, "J-Broussais" Hb A2 variant (alpha 1), "J-Toronto" Hb A2 variant, "Mexico" Hb A2 variant, "JTongariki" Hb A2 variant, "Neuilly-sur-Marne" Hb A2 variant, "J-Paris-I" Hb A2 variant (alpha 2), "JHabana" Hb A2 variant, "J-Paris-I" Hb A2 variant (alpha 1), "Wayne" Hb A2 variant (peak 2), Denatured Hb E Hb F, Hb Willamette, Hb Alabama, Hb Chapel Hill, Hb Park Ridge, Hb Porto Alegre, Hb Q-Thailand (G7 = Z(F) Taichung), Hb Sabine, Hb Bassett, Hb Rampa, Hb G-San Jos, Hb Barcelona, Hb Geldrop Santa Anna, Hb Richmond, Hb Boumerdes, Hb Swan River, Hb Burke, Hb Tarrant, Hb Presbyterian, Hb Manitoba II, Hb

78

Manitoba I, Hb Port Phillip, "J-Rovigo" Hb A2 variant, Denatured Hb S, Denatured Hb D-Punjab Hb Lansing, Hb Hinsdale, Hb Ypsilanti (Ypsi - peak 1), Hb Alberta, Hb Val de Marne (Footscray), Hb Kempsey, Hb Shelby (Hb Leslie), Hb Atlanta, Hb Ypsilanti (Ypsi - peak 2), Hb Rainier, Hb Athens-GA (Waco), Hb Debrousse Hb A, Hb Olympia, Hb Gorwihl (Hinchingbrooke), Hb Phnom Penh*, Hb Silver Springs*, Hb La Coruna*, Hb Bougardirey-Mali*, Hb Dallas*, Hb Toulon*, Hb Austin*, Hb Bonn*, Hb Buenos Aires (Bryn Mawr, major peak)*, Hb Chicago*, Hb Okayama*, Hb Fontainebleau*, Hb Raleigh*, Hb Hekinan*, Hb Mosella*, Hb Aztec*, Hb Little Rock*, Hb Frankfurt*, Hb Bethesda*, Hb M-Boston (M-Osaka)*, Hb Brisbane (Great Lakes)*, Hb Mizuho*, Hb Grange Blanche*, Hb San Diego*, Hb M-Saskatoon (main peak)*, Hb Malm*, Hb Minneapolis Laos*, Hb Owari*, Hb Rhode Island (Southwark)*, Hb Twin Peaks*, Hb Wood*, Hb Conakry*, Hb Coimbra (Ingelheim)*, Hb Linkping (Meilahti)*, Hb Templeuve*, Hb Alzette*, Hb Ty Gard*, Hb Gouda*, Hb Syracuse*, Hb Fort Dodge, Hb Camperdown, Hb Jura Hb Nouakchott, Hb Wayne (peak 1), Hb M-Iwate (M-Kankakee), Hb Camden (Tokuchi), Hb Hope Hb Vaasa, Hb Providence (X-Asn peak), Hb Tacoma, Hb Corsica, Hb K-Woolwich, Hb Lombard, Hb Andrew Minneapolis, Hb Fannin Lubbock, Hb Kaohsiung (New York), Hb Osler (Fort Gordon), Hb Himeji, Hb Jackson, Hb Tatras, "I (I-Texas)" Hb A2 variant Hb Bart's, Hb Cemenelum, Hb Wayne (peak 2), Hb Hopkins-II, Hb J-Calabria (J-Bari), Hb J-Tongariki, Hb Providence (X-Asp peak), Hb J-Meerut (J-Birmingham), Hb J-Broussais (Tagawa-I - alpha 2), Hb JRajappen, Hb Grady (Dakar), Hb Le Lamentin, Hb J-Anatolia, Hb Hikari, Hb J-Broussais (Tagawa-I - alpha 1), Hb J-Chicago, Hb J-Toronto, Hb J-Oxford (I-Interlaken), Hb Ube-2, Hb J-Meinung (J-Bangkok), Hb Neuilly-sur-Marne, Hb Mexico (J-Paris-II), Hb J-Paris-I (J-Aljezur - alpha 1), Hb J-Habana, Hb J-Baltimore (N-New Haven), Hb J-Paris-I (J-Aljezur - alpha 2), Hb K-Ibadan Hb N-Baltimore (Hopkins-I), Hb J-Rovigo, Hb J-Norfolk (Kagoshima), Hb J-Kaohsiung (J-Honolulu) Hb N-Seattle Hb I (I-Texas)

9 = Z(A)

10 11

12

13 14 15

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Figure 3. CZE scan of AFSC control

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References 1. Borbely N, Phelan L, Szydlo R, Bain B. Capillary zone electrophoresis for haemoglobinopathy diagnosis. J Clin Path 2013; 66: 29-39. 2. Greene DN, Pyle AL, Chang JS, Hoke C, Lorey T. Comparison of Sebia Capillarys Flex Capillary electrophoresis with the BioRad Variant II high pressure liquid chromatography in the evaluation of hemoglobinopathies. Clinica Chimica Acta 2012; 413: 1232-1238 3. Keren DF, Shalhoub R, Gulbranson R, Hedstrom D. Expression of Hemoglobin Variant Migration by Capillary Electrophoresis Relative to Hemoglobin A2 Improves Precision. Am J Clin Path 2012; 137: 660-664 4. Sae-ung N, Sriyorakun H, Fucharoen G, Yamsri S, Sanchaisuriya K, Fucharoen S. Phenotypic expression of hemoglobin A2, E and F in various hemoglobin E related disorders. Blood Cells, Molecules, and Diseases 2012; 48: 11-15. 5. Sangkitporn S, Sangkitporn SK, Tanjatham S, Suwannakan B, Rithapirom S, Yodtup C, Yowang A, Duangruang S. Multicenter Validation of Fully Automated Capillary Electrophoresis Method for Diagnosis of Thalassemias and Hemoglobinopathies in Thailand. Southeast Asian J Trop Med Public Health 2011; 6(5):1224-1232.[PubMed] 6. Fucharoen G, Srivorakun H, Singsanan S, Fucharoen S. Presumptive diagnosis of Hemoglobinopathies in Southeast Asia using a capillary electrophoresis system. Int. Jnl. Lab. Hem. 2011; 33: 424-433. 7. Wajcman H, Moradkhani K. Abnormal haemoglobins: detection & characterization. Indian J Med Res 2011;134 (4): 538-546 8. Liao C, Zhou J-Y, Xie X-M, Li J, Li DZ. Detection of Hb Constant Spring by a Capillary Electrophoresis Method. Hemoglobin 2010; 34(2): 175-178. 9. Cotton F, Nalaviolle X, Vertongen F, Gulbis B. Evaluation of an Automated Capillary Electrophoresis System in the Screening for Hemoglobinopathies. Clin Lab 2009; 55: 217-221. 10. Van Delft P, Lenters E, Bakker-Verweij M, De Korte M, Baylan U, Harteveld CL, Giordano PC. Evaluating five dedicated automatic devices for haemoglobinopathy dianostics in multi-ethinic populations. Int Jnl Lab Hem 2009; 31: 484-495 11. Winichagoon P, Svasti S, Munkongdee T, Chaiya W, Boonmongkol P, Chantrakul N, Fucharoen S. Rapid diagnosis of thalassemias and other hemoglobinopathies by capillary electrophoresis system. Translational Research 2008, 152 (4): 178-184 12. Keren DF, Hedstrom D, Gulbransom R, Ou C-N, Bak R. Comparison of Sebia Capillarys Capillary Electrophoresis With the Primus High-Pressure Liquid Chromatography in the Evaluation of Hemoglobinopathies. Am J Clin Pathol 2008, 130: 824-831. 13. Wang J, Zhou S, Huang W, Kiu Y, Cheng C, Lu Xin, Cheng J. CE-based analysis of hemoglobin and its applications in clinical analysis. Electrophoresis 2006; 27: 31083124. 14. Louhabi A, Philippe M, Lali S, Wallenmacq, Maisin D. Evaluation of a new Sebia kit for analysis of hemoglobin fractions and variants on the Capillarys system. Clin Chem Lab Med 2006; 44(3): 340-345. 15. Chang P-L, Kuo I-T, Chiu T-C, Chang H-T. Fast and sensitive diagnosis of thalassemia
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16. 17.

18.

19.

by capillary electrophoresis. Anal Bioanal Chem 2004; 379: 404-410. Jenkins M, Ratnaike S. Capillary Electrophoresis of Hemoglobin. Clin Chem Lab Med 2003; 41(6): 747-754. Gulbis B, Fontaine B, Vertongen F, Cotton F. The place of capillary electrophoresis techniques in screening for hemoglobinopathies. Ann Clin Biochem 2003; 40: 659-662 Gerritsma J, Sinnige D, Drieze C, Sittrop B, Houtsma P, Ulshorst-Jansen NH, Huisman W. Quantitative and qualitative analysis of hemoglobin variants using capillary zone electrophoresis. Ann Clin Biochem 2000; 37 (3): 380-389. Castagnola M, Messana I, Cassiano L, Rabino R, Rossetti DV, Giardina B. The use of capillary electrophoresis for the determination of hemoglobin variants. Electrophoresis 1995; 16(1): 1492-1498. http://www72.homepage.villanova.edu/frederick.vogt/ppt/2007/Capillary_Electrop horesis.ppt

20.

21. http://chemwiki.ucdavis.edu/Analytical_Chemistry/Instrumental_Analysis/Capillar y_Electrophoresis?highlight=capillary+zone+electrophoresis

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Chapter 2 Diagnostic Laboratory Methods

2.7 Isoelectric Focusing
David R. Hocking, PhD 2.7.1 Introduction Isoelectric focusing (IEF), also known as electrofocusing and isoelectricfocusing electrophoresis, is a separation method that resolves complex mixtures of proteins by their isoelectric points (pI). IEF is a type of electrophoresis that forms a pH gradient during the run. The technique is capable of extremely high resolution. The formation of a pH gradient is accomplished by blending a mixture of small molecular weight carrier ampholytes into a support matrix, or gel, usually of purified high -grade agarose. An anolyte solution (i.e. acetic acid) and a catholyte solution (i.e. ethanolamine) are saturated onto paper electrode wicks then are placed directly on opposite ends on the surface of the agarose gel. Proteins (i.e. hemoglobins) that are to be separated are placed near the cathode wicks using a clear plastic with rectangular wells cut out. The protein solution (i.e. hemoglobin hemolysate) is then pipetted in the defined wells and allowed to diffuse into the gel. An electric current is then passed through the medium. The proteins move through the changing pH gradient until it reaches a point in which the pH of that molecules pI is reached. At this point the protein no longer has an electric charge and becomes neutral, or isoelectric (due to the protonation or de-protonation of the associated functional amino and carboxyl groups) and as such will not proceed any further within the gel. The proteins become focused into sharp stationary bands with

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each protein positioned at a point within the newly formed pH gradient corresponding to its pI. Note: All the IEF figures in this compendium were obtained after agarose gel electrophoresis on the Wallac Resolve Hemoglobin System (Perkin Elmer), and the scans were procured using the Wallac WS-1010 IsoScan Imaging System (Perkin Elmer).

2.7.2 IEF of Normal Adult Hemoglobins: HbA (Adult), HbF (Fetal), HbA2 Normal adult hemoglobins are comprised of , , and globin chains paired as ~96% HbA (2 2), ~3% HbA2 (2 2) and <2% HbF (2 2) tetramers (Figure 1). One can usually find the glycated form of HbA, or HbA1c , anodal to it as shown in the Figure 2.

Figure 1.

84

Figure 2.

85

Aging bands, HbA3, are also anodal to HbA and are the result of post-translational modifications such as acetylation and glutathione attachment. It should be noted that beta-chain variants such as HbS, HbE, HbD, etc., will also display glycated forms anodal to the variant (HbS1c, HbE1c, HbD1c). This observation is critical to note should the patient exhibit symptoms of diabetes where their blood glucose values are documented to be high. The percentage can be upwards of 10-20% in cases of uncontrolled blood glucose levels.

2.7.3 IEF of Normal Newborn Hemoglobins: HbF (Fetal) and HbA (Adult) Normal newborn hemoglobins are comprised of ~ 60-85% HbF (2 2) and 1540% of HbA (2 2). It is very rare to see HbA2. About 10% of HbF is partially acetylated HbFac, which results in higher oxygen affinity, an important property needed for newborns. Aging hemoglobin bands, or HbF3 are always anodal to HbFac, and are the result of glutathione (an antioxidant, preventing damage to important cellular components caused by reactive oxygen species) attachment. Representative patterns of newborn hemoglobin are shown in Figure 3. Fresh cord blood is shown in channel 3a. A sample that was collected and stored using a filter paper is shown in channel 3b. Note the increased levels of HbF3 in the stored blood collected on filter paper.

86

Figure 3. IEF of normal newborn hemoglobins: HbF (fetal) and HbA (adult)

2.7.4 IEF of Beta-Chain Variant Hemoglobins

It is customary to report hemoglobins in the order of greatest concentrations. Heterozygotes are usually expressed as HbAX where X represents the beta-chain variant i.e. Sickle Cell (S), C, D, E or name of the variant. Examples are HbAS, HbAE or HbAD-Punjab. A few examples are shown (note percentages).

87

Figure 4. Hemoglobin A-S Trait

Figure 5. Hemoglobin D-Punjab Trait

88

Figure 6. Hemoglobin A-E Trait

Figure 7. Hemoglobin A-C Trait

Figure 8. Hemoglobin A-O Trait

Figure 9. Hemoglobin S/+-thal

89

These examples show the beta-chain mutation along with the Relative Charge Value (RCV) change as a result of the substitution of the normal amino acid found in HbA. In each case the HbA is in greater concentration than the beta variant (HbX). This pattern is true for all positive RCV values. Background on Hb O-Arab: This rare hemoglobin variant emerged about 2,000 years ago on a singular haplotype, characteristic of the Greek Pomaks. Its frequency increased as a consequence of high genetic drift within this population, and it was dispersed throughout the Mediterranean basin and Middle East with minor variations of its haplotypic pattern. (Haematologica. 2005 Feb;90(2):255-7. HbO-Arab mutation originated in the Pomak population of Greek Thrace. Papadopoulos V, Dermitzakis E, Konstantinidou D, Petridis D, Xanthopoulidis G, Loukopoulos D). The example in Figure 9 shows adult hemoglobin that has more HbS than HbA. The patient has reduced HbA, an increase in HbA2 and shows >than 3% of HbF. These findings indicate that the patient has a beta thalassemia (reduced amount of beta-globin chains from one parent) along with sickle cell hemoglobin (HbS) from the other parent. Note the aging bands from sample storage.

2.7.5

IEF of Alpha-Chain Variant Hemoglobins An individual inherits two sets of alpha globin genes, 1 and 2, from each

parent. If one of the alpha genes has a mutation, then one out of the four, or ~25% of the hemoglobin, will be the variant, not the typical 50% from a beta-chain variant. The affected alpha globin chain will form dimers with the non-alpha globin chains. HbGPhiladelphia is a common alpha-chain variant that is shown below (Figure 10).
90

Note that the percentage of HbG-Philadelphia (Figure 10) relative to HbA is less than what is seen in the beta variant HbD or HbD-Punjab. This is a clue to suspecting an alpha variant. Additionally you should also observe that there should be another band cathodal to HbA2. This is due to the variant alpha globin chain combining with the delta chain.

Figure 10. Hemoglobin G-Philadelphia Trait

Figure 11. Hemoglobin ASG-Philadelphia

91

The example in Figure 11 is a rare combination of the beta HbS variant and the alpha HbG-Philadelphia variant. Note the presence of four prominent bands: HbA, HbGPhiladelphia, HbS and the hybrid, HbSG-Philadelphia, the tetramer formed by the dimers of -GPhiladelphia and S. Also note the HbA2 variant that resulted from the G and dimers. It will be seen cathodal (negative electrode) to the hybrid.

2.7.6 IEF of Thalassemias A typical +-thalassemia is shown in Figure 12. Note that the percentage of HbA is reduced (95%) and the amount of circulating HbA2 is increased (>3.5%). Beta thalassemias occur in persons of Mediterranean origin, and to a lesser extent, Chinese, other Asians, and African Americans. +-thalassemia is also known as Thalassemia Minor and occurs if you receive the defective beta-globin gene from only one parent. Persons with this form of the disorder are carriers of the disease, Cooleys anemia or beta thalassemia major (0), if their other partner also passes their defective gene to the baby.

Figure 12. Hemoglobin +-thal

92

The pattern in Figure 13 is typical of those individuals presenting with a severe form of Sickle Cell disease. In this example, the patient inherited the HbS from one parent and is missing the beta globin gene from the other parent. The patient, though missing a beat globin gene, has compensated for the missing beta-globin gene with the persistence of making HbF from the gamma-globin gene.

93

Conclusion

IEF can be an important tool in assiting the laboratorian in the dection and interpretation of hemoglobin variants. The technique offers improved resolution over traditional electrophoretic methods and is useful for both adult and newborn patients. By careful observation, one can determine if the variant is either a or variant or combination. One can also correctly interpret -thalassemias.

References: 1. David R. Hocking. The Separation and Identification of Hemoglobin Variant by Isoelectric Focusing Electrophoresis (May 2004), Catalog # HC-60, Perkin Elmer Life and Analytical Sciences, Wallac Oy, P.O. Box 10, FIN-20101 Turku, Finland. Tel. 358-2-2678111 Fax. 358-2-2678357 Web site: www.perkinelmer.com 2. Additional information about the IEF procedures and instruments can be solicited from: a) Petra Furu, Ph.D., Global Business Manager, Specialty Diagnostics, Perkin Elmer , Mustionkatu 6 / 20750 Turku / Finland. e-mail: petra.furu@perkinelmer.com Tel. 358 2 267 8497 b) William R. Fisher, Technical Support Specialist, Specialty Diagnostics, Perkin Elmer, 520 South Main Street, Akron, OH 44311, USA e-mail: william.fisher@perkinelmer.com Tel. 330-564-4883

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Chapter 2 Diagnostic Laboratory Methods

2.8 High Performance Liquid Chromatography
Zia Uddin, PhD

2.8.1. Introduction In 1973 I had the privilege of attending a short course on High Performance Liquid Chromatography (HPLC), sponsored by the American Chemical Society at Virginia Polytechnic Institute, Blacksburgh, Virginia, USA. The teachers of this course were Drs. Lloyd R. Snyder and Joseph J. Kirkland. These two scientists are responsible for several advancements in HPLC, and their most significant contribution in collaboration with Dr. John W. Dolan is their latest book (Snyder LR, Kirkland JJ, Dolan JW, Introduction to Modern Liquid Chromatography, 3rd Edition, John Wiley & Sons, Hoboken, NJ 20010). Persons interested in HPLC shall find this book very helpful in understanding the theory and practice of HPLC, and the components of HPLC (solvent system, pump, injection port, column, stationary phase, detector, computer, etc.). Additional literature about HPLC can be accessed from the following Internet sites:

http://www.lcresources.com http://lchromatography.com/hplc find/index.html http://tech.groups.yaho.com/group/chrom-L/links http://userpages.umbc.edu/~dfrey1/Freylink http://www.chromatographyonline.com

95

Note: The name High Pressure Liquid Chromatography was initially used, however now the word Pressure is replaced by Performance. In this book we shall therefore use High Performance Liquid Chromatography nomenclature.

2.8.2. Basic Principle Liquid chromatography (LC) consists of a liquid mobile phase and a stationary phase and the separation is accomplished by the distribution of the solutes between these phases. Manual LC procedure is slow and needs additional steps for the identification of the compound of interest. In HPLC the separation process is expedited by forcing the mobile phase under high pressure through the column, and almost all the steps of the operation are automated (Figure 1).

96

Figure 1. Key components of a HPLC system, a) computer, b) detector, c) column, d) injection port, e) pump, f) solvent reservoir. (adopted from Snyder LR, Kirkland JJ, Dolan, JW, Introduction to Modern Liquid Chromatography, 3rd Edition. John Wiley & Sons, Hoboken, NJ 20010).

The identification of a compound of interest in HPLC is ascertained by matching its retention times (time required to separate a compound after the injection step) with a standard or control. Several kinds of detectors are employed in HPLC for detection purposes, e.g., spectrophotometric, flurometric, electrochemical, etc. Another development in the identification of a compound after HPLC is coupling it with mass spectrometry (Chapter 3.4). This technique is very useful when the retention time of the compound is not previously known. The identification is achieved by the m/z value of the ion associated with the compound of interest, e.g., globin chain of a hemoglobin variant (Chapter 3.4).

2.8.3

Illustrations a) Quantification of Hb A2, and Hb A1c : One of the widely used procedure employing HPLC is the quantification of Hb A1c and Hb A2 ( Figure 2).

97

Figure 2. Peak at 0.81 (Hb A1c) and at 3.1 (Hb A2). Adopted from the Technical Manual of D-10, Bio-Rad, Hercules, CA.

b) Hb OIndonesia in India: a rare observation The father is heterozygous for Hb OIndonesia and the mother is normal, however the daughter has an HPLC pattern similar to her father (Figure 3). Although the normal hemoglobin fractions (Hb F, Hb A, Hb A2)as well as the common variants (Hb S and Hb C) all have distinct retention times,there are less prevalent variants with similar or
98
1

identical retention times. In these cases additional laboratory procedures must be utilized for a conclusive diagnosis.

Figure 3. a) HPLC of daughter, b) HPLC of father, c) HPLC of mother

99

c) Apparent hemoglobinopathies caused by blood transfusions

Any spurious peak in HPLC can cause confusion and lead to unnecessary additional testing. It is advised that in order to reduce unwarranted commotion, the patients medical record should be examined for recent blood transfusions. Figure 4 illustrates an example of a patient with Hb SS disease on hyper-transfusion regimen who received a unit of blood from a donor heterozygote for Hb O-Arab as demonstrated by a small, but prominent peak eluting after Hb S.

Figure 4. HPLC of a Hb SS patient transfused with one unit of Hb A-O Arab blood.

100

Cited references: Chopra A, Fisher C, Khunger JM, Pati H. Hemoglobin OIndonesia in India: a rare observation. Ann Hematol 2011; 90: 353-354 2. Kozarski TB, Howantiz PJ, Howantiz JH, Lilic N, Chauhan YS. Blood transfusions leading to apparent Hemoglobin C, S, and O-Arab Hemoglobinopathies. Arch Pathol Lab Med 2006; 130: 1830-33. Additional references: 3. Szuberski J., Oliveira JL, Hoyer JD. A comprehensive analysis of hemoglobin variants by high performance liquid chromatography (HPLC). International Journal of Hematology 2012; 34: 594-604. 4. Ondei LS, Zamaro PJA, Mangonaro PH, Valencio CR, BoniniDomingos CR. HPLC determination of hemoglobins to establish reference values with the aid of statistics and informatics. Genetics and Molecular Research 2007; 6(2): 453-460. 5. Mais DD, Boxer LA, Gulbranson RD, Keren DF. Hemoglobin Ypsilanti. A High-Oxygen-Affinity Hemoglobin Demonstrated by Two Automated High-Pressure Liquid Chromatography Systems. Am J Clin Path 2007; 128: 850-853. 6. Joutovsky A, Hadzi-Nesic J, Nardi MA. HPLC Retention Time as a Diagnostic Tool for Hemoglobin Variants and Hemoglobinopathies: A Study of 60 000 Samples in a Clinical Diagnostic Laboratory. Clin Chem 2004; 50: 1736-47. 7. Ou C-N, Rognerud CL. Diagnosis of hemoglobinopathies: electrophoresis vs. HPLC. Clin Chim Acta 2001; 313: 187-94. 8. Fucharoen S, Winichagoon P, Wisedpanichkij R, et al. Prenatal and postnatal diagnosis of thalassemias and hemoglobinopathies by HPLC. Clin Chem 1998; 44: 740-748. 9. Riou J, Godart C, Hurtrel D, Mathis M, Bimet C, et al. Cation-exchange HPLC evaluated for presumptive identification of hemoglobin variants. Clin Chem 1997; 43: 34-39. 10. Huisman THJ. Review: Separation of Hemoglobins and Hemoglobin Chains By High-Pressure Liquid Chromatography. J. Chromatogr 1987; 418: 277-304 11. Colah RB, Surve R, Sawant P, DSouza E, Italia K, Phanasgaonker S, Nadkarni AH, Gorakshaker AC. HPLC studies in hemoglobinopathies. Indian J Pediatr 2007; 74(7): 657-62 12. Sachdey R, Dam AR, Tyagi G. Detection of Hb variants and hemoglobinopathies in Indian population using HPLC: Report of 2600 cases. Indian J Pathol Microbiol 2010; 53: 57-62. 13. Rao S, Kar R, Gupta SK, Chopra A, Saxena R. Spectrum of haemoglobinopathies diagnosed by cation exchange-HPLC and modulating effects of nutritional deficiency anemias from north India. Indian J Med Res 2010; 132: 513-519.
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1.

Chapter 3 Globin Chain Analysis

3.1 Solid Phase Electrophoretic Separation
Zia Uddin, PhD

In the early stages of the development of the diagnostic hemoglobinopathies, polyacrylamide gel electrophoresis (in urea, acid and non-ionic detergent Triton-X-100) and cellulose acetate electrophoresis (alkaline and acid pH) were utilized for globin chain analysis. These techniques provided information about the globin chains that contained the substitution. However, due to scientific limitations (selection of known variants as controls with mobility similar to that of the unknown), these techniques were abandoned in favor of other methods as described in this chapter. Recently capillary zone electrophoresis was also used for the separation of globin chains. For historical reasons we have briefly presented the basic features of cellulose acetate electrophoresis of globin chains.

3.1.1 Cellulose Acetate Electrophoresis (Alkaline and Acid pH) First the heme groups and the globin chains are dissociated from the hemoglobin molecule using 2-mercaptoethanol and urea. Electrophoresis at alkaline pH is performed using the tris-ethylenediaminetetraacetic acid buffer at pH 8.8-9.5. In Figure 1 the relative mobilities of globin chains at alkaline pH are presented. There is not much difference in the mobilities of globin chains between the alkaline (8.8 9.5) and acidic (6.0-6.2) pH, and in both cases the alpha chains migrate towards
102

the cathode and the beta chains towards the anode. In Figure 2 the relative mobilities of globin chains at acidic pH are presented.

Fig 1.

Relative Mobilities of Globin Chains (Cellulose Acetate Electrophoresis at pH 8.8-9.5). Adopted from Laboratory Methods for Detecting Hemoglobinopathies, Division of Host Factors, Center for Infectious Diseases, Center for Disease Control, Atlanta, GA (September 1984)
103

Fig 2.

Relative Mobilities of Globin Chains (Cellulose Acetate Electrophoresis at pH 6.0-6.2). Adopted from Laboratory Methods for Detecting Hemoglobinopathies, Division of Host Factors, Center for Infectious Diseases, Center for Disease Control, Atlanta, GA (September 1984)
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References 1. Ueda S, Schneider RG. Rapid identification of polypeptide chains of hemoglobin by cellulose acetate electrophoresis of hemolysates. Blood 1969; 34: 230. 2. Schneider RG. Differentiation of electrophoretically similar hemoglobinssuch as S,D,G and P; or A2, C,E, and O- by electrophoresis of the globin chains. Clin Chem 1974; 20(9): 1111-1115. 3. Shihabi ZK, Hinsdale ME. Simplified hemoglobin chain detection by capillary electrophoresis. Electrophoresis 2005; 26: 581-585

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Chapter 3 Globin Chain Analysis

3.2 Reverse-Phase High Performance Liquid Chromatography
Zia Uddin, PhD and Rita Ellerbrook, PhD

Conventional charge based separation techniques (electrophoresis, ion-exchange liquid chromatography, and isoelectric focusing) are sometimes ineffective in the separation of hemoglobins, when the amino acid substitution does not cause a net charge differential. Several hemoglobin variants migrate upon electrophoresis and elute upon ion-exchange liquid chromatography in the positions of hemoglobin A, S, D, A2 or C. Further clarification is necessary for newborn screening or in cases of unexplained clinical disorders. Additional testing is required to resolve this matter, e.g., DNA studies, reverse-phase chromatography (RPC), liquid chromatography-mass spectrometry (LC-mass) primarily employing the electrospray ionization (ESI) technique, and Sanger sequencing. There are three main chromatographic techniques for the separation of peptides and proteins, e.g., a) size exclusion, b) ion-exchange, and c) hydrophobic interactions. For a detailed study of the theory and practice of the liquid chromatography of peptides and proteins in general and reverse phase high pressure liquid chromatography (RP-HPLC) in particular (Chapter 13, Section 13.4), the interested reader is advised to review the 3 rd edition of Introduction to Modern Liquid Chromatography, by Lloyd L. Snyder, Joseph J. Kirkland and John W. Dolan (A John Wiley and Sons, Inc. Publication, 2010). Howard and Martin first introduced RPC in 1950, and since then, several improvements in the methodology and advancements in its application in the separation of peptides and proteins were achieved. The
1

106

recent literature on RPC can be accessed via the Internet (http://www.lcresources.com) and the specialized journals in the field. The separation of globin chains by RP-HPLC is based on the hydrophobicity of the globin chains, which is defined as a tendency of not combining with water or incapable of dissolving in water. The RP-HPLC consists of a non-polar column in combination with a polar mixture of water plus an organic solvent as a mobile phase. In this section, we shall demonstrate the usefulness of RP-HPLC in the separation of globin chains leading to the identification of hemoglobin variants. Experimental details of RP-HPLC of globin chains (hemoglobin specimen preparation, selection of column, solvent system, high pressure liquid chromatography instrumentation, temperature, retention times, detection system, etc.) were provided by the work of three research groups in this field in Italy, France and USA
2-7

A few RP-HPLC chromatograms (Fig 1-5) are shown to illustrate the application of this technique in the separation of globin chains. These chromatograms are either replicated exactly as cited in the literature (abscissa depicting actual retention times in minutes), or for comparison, as a normalized scale for the retention times. In the normalized scale, the retention time for the normal chain is 10, and for the normal chain is 20 (Fig 4). The elution window is of 0.5 units width. It is emphasized that the retention times of RP-HPLC might vary depending upon the experimental conditions, but the overall shape of the chromatogram is highly reproducible.

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Normal Cord Blood: Fetal blood obtained at 18-20 weeks of gestation age, shows the preponderance of chains (Fig 1).

Fig 1. RP-HPLC chromatogram of a normal cord blood (Leone L, Monteleone M, Gabutti V, Amione C. Reversed-Phase High Performance Liquid Chromatography of Human 2 Hemoglobin Chains. J Chromatogr. 1985, 321: 407-419) . Normal Adult Blood: The first peak (Fig 2) at 10 minutes is the heme molecule followed by two major peaks, a) chain of Hb A (31-35 minutes), and b) chain of Hb A (43-48 minutes).

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Fig 2. RP-HPLC chromatogram of a normal adult blood (Kutlar F, Kutlar A, Huisman THJ. Separation of Normal and Abnormal Hemoglobin Chains by Reverse-Phase High Performance 3 Chromatography. J. Chromatogr 1986, 357: 147-153) . Adult hemoglobin A-S trait : In hemoglobin S the variation in the chain is due to the substitution of glutamic acid by valine [6(A3)]. This is shown in Fig 3 by the separation of and chains.
S A

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Fig. 3. RP-HPLC chromatogram of an adult Hb A-S trait (Leone L, Monteleleone M, Gabutti V, Amione C. Reversed-Phase High Performance Liquid Chromatography of Human Hemoglobin 2 Chains. J. Chromatogr 1985, 321: 407-419) . Hemoglobin S interacts with Hb D-Punjab [121(GH4) GluGln] causing sickle cell disease. Hemoglonin S also interacts with Hb Korle-Bu [73(E17) AspAsn], but in the opposite direction, i.e., inhibiting sickling. Both of these hemoglobin variants (Hb D-Punjab and Hb Korle-Bu) are frequently found in the population sector dominated by Hb S. The separation of Hb D-Punjab and Hb Korle-Bu is difficult from cellulose acetate
110

electrophoresis and isoelectric focusing, however both the easily separated by RP-HPLC .
7

D-Punjab

and

Korle-Bu

chains can be

Several electrophoretic separation techniques did not distinguish Hb Camperdown [104(G6) ArgSer] from Hb Sherwood Forest [104(G6) ArgThr]. In this example th ere is no change on the charge of the two hemoglobin variants as only the hydrogen atom on serine is replaced by a methyl group of threonine. The substitution of serine by threonine on the same position of the chain changes the hydrophobicity (presumably by altering the secondary/tertiary structure of the globin chain), thus resulting in their separation by RP-HPLC (Fig 4).

Fig. 4. Normalized scale of retention times of globin chains on RP-HPLC, a) retention times of A (10), (20), G (28) and A (35), b) Hb Campertown (14.1-14.5), and c) Hb Sherwood Forest (16.1-16.5). Adopted from: Wajcman H, Riou J, Yapo AP. Globin Chain Analysis by
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Reversed Phase High Performance Liquid Chromatography: Recent Developments. 4 Hemoglobin 2002, 26: 272-284 .
8

Another interesting illustration of the usefulness of RP-HPLC was the resolution of hemoglobinopathy during newborn screening, provided by Hb Sinai-Greenspring [34(16) ValIle, GTC>ATC]. IEF showed an abnormal band (slightly anodal to HbA), and HPLC (Fig 5a) was also inconclusive except the broadening of the band due to a hemoglobin variant. RPHPLC did indicate a distinct band due to a variant Hb between the and chains (Fig 5b). Substitution of amino acid valine at position 34 of the -globin chain by isoleucine changed the hydrophobicity of the protein molecule and thus allowed the separation of two chains by RPHPLC (Fig 5b).

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Fig 5. Cation exchange HPLC chromatogram (a) of infant with Hb-Sinai-Greenspring, and RP-HPLC chromatogram (b). Adopted from: Dainer E, Wenk RE, Luddy R, Elam D, Holley L, Kutlar A, Kutlar F. Two new hemoglobin variants: Hb Sinai-Greenspring [34 (16) ValIle, GTC>ATC] and Hb Sinai-Bel Air [53 (D4) AlaAsp, GCT>GAT]. 8 Hemoglobin 2008; 32(6): 588-591

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Henri Wajcman and associates published the retention times on RP-HPLC of over 200 abnormal globin chains which were also made available on the web . Additional chromatographic and electrophoretic information about hemoglobin variants can be obtained from the database
9-11 7

References 1. 2. Howard GA, Martin JP. The separation of the C12-C18 Fatty Acids by ReversedPhase Partition Chromatography. Biochem J 1950; 46: 532-538. Leone L, Monteleone M, Gabutti V, Amione C. Reversed-Phase High Performance Liquid Chromatography of Human Globin Chains. J Chromatogr 1985; 321: 407-419. Kutlar F, Kutlar A, Huisman THJ. Separation of Normal and Abnormal Hemoglobin Chains by Reversed-Phase High-Performance Liquid Chromatography. J Chromatogr 1986; 357:147-153. Wajcman H, Riou J, Yapo AP. Globin Chain Analysis by Reversed Phase High Performance Liquid Chromatography: Recent Developments. Hemoglobin 2002; 26: 271-284. Yapo PA, Datte JY, Yapo A, Wajcman H. Separation of Adult Chains of Abnormal Hemoglobin: Identification by Reversed-Phase High-Performance Liquid Chromatography. J Clin Lab Anal 2004;18: 65-69. Zanella-Cleon I, Becchi M, Lecan P, Giordano PC, Wajcman H, Francina A. Detection of a Thalassemic -Chain Variant (Hemoglobin Groene Hart) by Reversed-Phase Liquid Chromatography. Clin Chem 2008; 54:1053-1059. Wajcman H, Riou J. Globin chain analysis: An important tool in phenotype study of hemoglobin disorders. Clinical Biochemistry 2009; 42:1802-1806. Dainer E, Wenk RE, Luddy R, Elam D, Holley L, Kutlar A, Kutlar F. Two new hemoglobin variants: Hb Sinai-Greenspring [34 (16) ValIle, GTC>ATC] and Hb Sinai-Bel Air [53 (D4) AlaAsp, GCT>GAT]. Hemoglobin 2008; 32(6): 588-591 Hardison RC, Chui DHK, Giardine B, et al. HbVar: a relational database of human hemoglobin variants and thalassemia mutations at the globin gene server. Hum Mutat 2002; 19: 225-33 (http://globin.bx.psu.edu/hbvar/smenu.html). Giardine B, van Baal S, Kaimakis P, et al. HbVar database of human hemoglobin variants and thalassemia mutations: 2007 update. Hum Mutat 2007; 28(2): 206. Patrinos GP, Giardine B, Riemer C, et al. Improvements in the Hbvar database of human hemoglobin variants and thalassemia mutations for population and sequence variation studies. Nucleic Acids Res 2004 Jan 1; 32: D537-41(Database issue).

3.

4.

5.

6.

7. 8.

9.

10.

11.

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Chapter 3 Globin Chain Analysis

3.3 Globin Chain Gene Mutations: DNA Studies
Joseph M. Quashnock, PhD

3.3.1 Introduction Hemoglobin A is the designation for the normal hemoglobin that exists after birth. Hemoglobin A is a tetramer with two alpha chains and two beta chains (22). Hemoglobin A2 is a minor component of the hemoglobin found in red cells after birth and consists of two alpha chains and two delta chains (22). Hemoglobin A2 generally comprises less than 3% of the total red cell hemoglobin. Hemoglobin F is the predominant hemoglobin during fetal development. The molecule is a tetramer of two alpha chains and two gamma chains (22). Hemoglobinopathies result from amino acid changes in the alpha or beta globin chains. Most of the mutations are single amino acid substitutions caused by a single base change, however, other amino acid mutations can be found due to various base alterations such as:

1.

More than one amino acid change e.g. the alpha chain mutation of Hb J Singapore with Asn>Asp and Ala>Gly, the beta chain mutation of Hb Poissy with Gly>Arg and Ala>Pro.

2.

Elongation of the chain due to frameshifts or insertions such as Hb Constant Spring or Hb Doha.

3. 4.

Shortened chains due to deletions such as Hb Leiden. Hybrids such as the Lepore globin gene that is a crossover of beta and
115

delta globin genes that produces hemoglobin made up of two normal alpha chains and two Hb Lepore chains.

Additionally, though much rarer, there are also changes in the gamma chains (Hb F) and delta chains (Hb A2). Over 1,000 hemoglobin mutations have been described. For a detailed list of the mutations, the reader is directed to the Globin Gene Server of Pennsylvania State University at: http://globin.cse.psu.edu/html/ and Department of Microbiology of the University of Massachusetts at: http://www.umass.edu/microbio/chime/hemoglob/index.htm. Mutations that cause diminished production of the globin molecules are termed Thalassemia. Equal numbers of alpha and beta chains are necessary for normal hemoglobin synthesis.

3.3.2 Genotyping - PCR Methodology Determining the genotype requires DNA from the subject and the synthesis of a primer and probe for the known mutation. The subjects DNA, a primer, a reporting probe, DNA bases, and DNA polymerase enzyme are incubated a number of times to amplify the mutation sufficiently to be detected with a labeled probe. However, the procedure has limitations; the first is that the mutation must be known so that a unique primer and probe can be made, secondly, a sufficient amount of sample DNA must be present to make a sufficient quantity of PCR product (amplicon) which is then detected and reported by the probe.
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Methods that have been employed over the years for identifying single mutations are:

1.

Restriction Fragment Length Polymorphism (RFLP) detection in which specific restriction enzyme digested DNA is separated by electrophoresis1.

2.

Binding of a labeled Allele-Specific Oligonucleotide (ASO) probe to amplified DNA2.

3.

Allele-Specific PCR (ASP), PCR Amplification of Specific Alleles (PASA), or Amplification Refractory Mutation System (ARMS), in which the presence or absence of a normal or mutant sequence is determined by whether the PCR products generated by specific primers can be detected through a reporting system such as electrophoresis, or a fluorescent, chemical, colorimetric, or electric signal. The signals may be read directly by the human eye (electrophoresis) or detected by instrumentation in which case they may also be quantitated3.

Some additional methods for multi-mutation detection by PCR assays include: 1. Allele-Specific Primer Extension (ASPE) assays that detect the incorporation of a labeled nucleotide that binds at a single nucleotide polymorphism (SNP) and is linked to an oligonucleotide that is bound next to the SNP site3. 2. Binding labeled multiplex ASPE products to mutation specific beads that can generate identifying signals in solution when separated by laser flow cytometry as is done by the Luminex4. 3. Oligonucleotide Ligation Assay (OLA) based on the binding and ligation of
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an allele-specific probe to a common downstream sequence reporter probe, which generates a specific fluorescent signal from the completed ligation products separated on electrophoresis5. 4. Hybridizing PCR amplification products to electrode-bound allele-specific probes (printed circuit board, microarray, chip-based) to generate electric signals6. 5. Fluorescence Resonance Energy Transfer (FRET) fluorescent signals generated by Cleavase treated PCR products7.

PCR amplification products are produced by incubating extracted DNA from the specimen with DNA primers, the substrate nucleic acid bases of adenosine, thymidine, guanosine, and cytidine, DNA polymerase, and a DNA detection probe. The mixture is repeatedly heated to ~ 95 C and cooled; each heating and cooling cycle doubles the

amount of PCR product produced; most PCR assays use 25-40 cycles. Rapid cycle PCR is based upon the low heat capacity of air and the ability to ramp through temperatures at a far greater rate than instruments using thermocyclers that rely upon heating and cooling an aluminum block. Instruments such as the

LightCycler from Roche also incorporate the improvement of using glass capillary tubes to serve as both the reaction vessel and optical cuvette. Detection is by the Fluorescence Resonance Energy Transfer (FRET) method described below, however, the time required to complete 25-40 cycles is on the order of 30-40 minutes as opposed to 3-4 hours for aluminum block thermocyclers.
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Detection of FRET probes is performed by measuring hybrid stability as modified by the introduction of base pair mismatch/es. Mismatch destabilization is measured by observing the melting temperature of the FRET probe as monitored by fluorescence output. Fluorescence is generated using two fluorophores. The first or Light -Up

fluorophore is excited at an appropriate wavelength.

The emission of the Light-Up The subsequent

fluorophore is in turn used to excite the detection fluorophore. emission of the detection fluorophore is monitored.

In order for resonance energy

transfer to occur between the Light-Up and detection fluorophores and produce a florescent signal, the two fluorophores must be in close proximity. Proximity is achieved by conjugating the fluorophores to oligonucleotides such that when the oligonucleotides are hybridized to their target in an amplicon, the fluorophores are held in proximity. The mixture is then heated and a melting curve is generated by the slow thermal denaturing of the probe-template hybrid. Melting curves are generated by monitoring the loss of fluorescence over the course of denaturation. Melting peaks are generated by plotting the inverse derivative of fluorescence verses temperature (-dF/dT) - the bigger the mismatch between the amplicon and the probe, the lower the melting temperature. Because most hemoglobinopathies are single amino acid mutations such as base substitution or base pair insertion or deletion, the ASP method is the commonly used technology. In this procedure, allele-specific primers for sequences are designed to bind to and amplify a small region surrounding the site of the known mutation. A probe of oligonucleotides, which matches the normal or abnormal sequence, binds to the PCR products. The probes incorporate a label (fluorophore) that produces a signal to show that binding has taken place and a specific sequence has been detected.
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3.3.3 Mutations Hemoglobin beta is the name of the hemoglobin gene and is abbreviated HBB. Sickle cell anemia is the most common mutation and primarily affects AfricanAmericans with a frequency of 1:400. The defect causes red cells to distort and block small capillaries. The -globin gene is located on the small (p) arm of chromosome 11 in the region of 15.5 (HBB; MIM # 141900; 11p15.5). The mutation is the replacement of an adenosine with a thymidine in the DNA that causes the substitution of valine for glutamic acid at position 6 in the beta-globin chain. The codon sequence is shown below, GAG, in the sixth position below, codes for glutamic acid; the replacement of adenosine (A) with thymidine (T) produces GTG that codes for valine.

1 3 GTG GAC CTG ACT Val Asp Leu Thr

GTG GAC CTG ACT

6 9 CCT GAG GAG AAG TCT Glu Pro Glu Lys Ser Val CCT GTG GAG AAG TCT

- - - (wildtype)

- - - (Hb S)

Hemoglobin C is a mutation in the same codon which replaces the first guanosine with adenosine (GAG becomes AAG) causing the glutamic acid to be replaced with lysine. 1 3 GTG GAC CTG ACT Val Asp Leu Thr 6 9 CCT GAG GAG AAG TCT Glu Pro Glu Lys Ser Lys CCT AAG GAG AAG TCT
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- - - (wildtype)

GTG GAC CTG ACT

- - - (Hb C)

Similar single point mutations cause other variants of hemoglobin. Hemoglobin E results when glutamic acid is replaced with the amino acid lysine at position 26 in globin (Glu>Lys) due to the same GAG>AAG mutation that causes hemoglobin C at codon 6. It is the second most common hemoglobin variant. When the hemoglobin E mutation is present with hemoglobin S, Hb SE disease, the person may have more severe signs and symptoms associated with sickle cell anemia, such as episodes of pain, anemia, and abnormal spleen function. Hemoglobin D-Punjab also known as Hb D-Los Angeles, Hb D-Chicago, Hb DNorth Carolina, Hb D-Portugal, and Hb Oak Ridge is an abnormality due to the replacement of glutamic acid with glutamine on the hemoglobin beta chain. The mutation is GAA>CAA

at codon 121 (121 Glu>Gln). Hb D is primarily found in the

Indus River Valley (Punjab) region of Pakistan and Northwestern India but is widespread, and has been observed in persons from China, England, Holland, Australia, Greece, Serbia, Bosnia Herzegovina, Macedonia, Montenegro, and Turkey. It is the fourth most frequently occurring hemoglobin variant. Heterozygotes for Hb D are normal. Homozygosity for Hb D is associated with normal hemoglobin levels, decreased osmotic fragility, and some target cells. Compound heterozygotes for Hb D and -Thalassemia have mild anemia and microcytosis. Hb D has been found in combination with Hb S, Hb C, Hb E, -

thalassemia, and in the homozygous state. Hemoglobin D has been shown to interact with the sickle hemoglobin gene S. Individuals who are compound heterozygotes for
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---------------------------------------------------------------------------

See the DNA codon table for degeneracy (redundant) codons.

Hb S and Hb D-Los Angeles (SD) have moderately severe hemolytic anemia and occasional pain episodes. Populations that have a high frequency of sickle hemoglobin (SD) disease are those of Asian and Latin American descent. Hemoglobin O-Arab is an abnormality due to the amino acid substitution of lysine for glutamic acid at the 121st position in the beta globin gene. The genetic mutation is a GAA>AAA at this codon (121 Glu>Lys). The mutation is also known as Hb Egypt and Hb O-Thrace. The mutation is found mainly in African-Americans, Gypsies, in Pomaks (a population group in the Balkan countries) and in Arabian, Egyptian, and Black families of the US and western hemisphere. Hemoglobin O-Arab is important when found with sickle syndromes. Compound heterozygotes for Hb S and Hb O-Arab have hemoglobin concentrations in the range of 7-8 g/dL with reticulocytosis, jaundice, splenomegaly, episodes of pain, and many other complications seen in Hb SS disease. manifestations. microcytosis. Heterozygote carriers have no clinical

Homozygous individuals usually present with mild anemia and Compound heterozygotes for Hb O-Arab and -thalassemia have

manifestations similar to thalassemia intermedia. Thalassemias are named by the chain that is deficient. In -Thalassemia, there is an insufficient amount of the beta subunit due to mutations such as -29A>G, -88C>T, and IVS1+6T>C. The excess alpha subunits precipitate and eventually damage the red blood cells. In severe -thalassemia, the -globin subunits begin to associate into

tetramers due to the reduced concentration of alpha chain. The tetramers of -globin do not transport oxygen. severe -thalassemia.
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No comparable tetramers of -globin subunits form with

Below are several melting curves representing the various signals obtained during an analysis. In allele specific binding assays, it is preferred that the primer and detection probe are the sequences for the mutation and not the wildtype (normal) sequence. Because the mismatch in base sequences causes the melting temperature to be lower, the use of the wild type sequence as the detection probe will indeed demonstrate a lower melting temperature when a mismatch is present, however it will not be known as to which base/s mismatch (mutation) was present. The use of the mutation as the template will always result in the specific mutation producing the highest melting temperature. The Hemoglobin S templates were used in the analysis of wildtype (normal) hemoglobin in Figure 1 and shows a melting point of 55.5 C.
o

Figure 1. Hemoglobin A (WT*) bound to Hemoglobin S probe, melting point is 55.5

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C.

* WT - Wild Type, the commonly occurring type - no mutation. Figure 2 shows a melting curve for a carrier, both hemoglobin sequences were detected. Hemoglobin S has the higher melting temperature of 62.5 C while the

wildtype melts at 55.5 C. A homozygous sickle disease individual would show only one melting point at 62.5 C.

Figure 2. Hemoglobin A (WT) and Hemoglobin S (Mutant) bound to Hemoglobin S probe. Melting temperatures: WT - 55.5 C and Mutant S - 62.5 C.

As pointed out earlier, Hemoglobin S and Hemoglobin C differ from the wild type by only one base in the same codon number 6 of the HBB gene. The Hb C mismatch causes an even lower melting temperature than Hb S or the wildtype. Figure 3 shows two melting points indicating a Hemoglobin C carrier with Hb C melting at 49.8 C and the wildtype again at 55.5 C. This detection of two mutations is an example of a
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multiplexed assay. This type of multiplexing is only useful when the bases involved in the mutation are very close, e.g. 3 bases, otherwise the energy transfer would not be very efficient and no fluorescent signal would be detected.

Figure 3. Hemoglobin C (Mutant) bound to Hemoglobin S probe. Melting temperatures: Mutant C - 49.8 C and WT - 55.5 C

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Figure 4 is an example of a non-preferred base sequence for the Hemoglobin E mutation in which the wildtype probe is used to detect the mismatch at codon 26 of GAG to AAG. Here the wildtype melts at 70.3 C and the Hb E mutation melts at 65.2 C.

Figure 4. Hemoglobin E (mutant) bound to Hemoglobin A (WT) probe. Melting temperatures: Mutant E - 65.2 C and WT - 70.3 C

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Figure 5 illustrates an analytical run in which a normal, no Hemoglobin D present patient is in red, a carrier control specimen is in blue, and the green line is the no DNA control which must not give a signal.

Figure 5. Hemoglobin A (WT) and Hemoglobin D (Mutant) bound to Hemoglobin D Probe. Melting temperatures: WT - 50.95 C and Mutant D - 63.68C.

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Figure 6 is an example of a melting curve for Hemoglobin O. Note that among the assays shown, there is no correlation of melting temperatures for the wildtype or mutations. This is because each primer and probe set is different for each specific mutation.

Figure 6. Hemoglobin A (WT) and Hemoglobin O (Mutant) bound to Hemoglobin D Probe. Melting temperatures: WT - 51.52 C and Mutant D - 61.91C .

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Degeneracy Table

Amino Acid Alanine Arginine Asparagine Aspartic acid Cysteine Glutamic acid Glutamine Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine Stop codons

DNA codons GCT, GCC, GCA, GCG CGT, CGC, CGA, CGG, AGA, AGG AAT, AAC GAT, GAC TGT, TGC GAA, GAG CAA, CAG GGT, GGC, GGA, GGG CAT, CAC ATT, ATC, ATA CTT, CTC, CTA, CTG, TTA, TTG AAA, AAG ATG TTT, TTC CCT, CCC, CCA, CCG TCT, TCC, TCA, TCG, AGT, AGC ACT, ACC, ACA, ACG TGG TAT, TAC GTT, GTC, GTA, GTG TAA, TAG, TGA

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DNA Codon Table

First TTT T TTC TTA TTG CTT C CTC CTA CTG ATT ATC A ATA ATG GTT G GTC GTA GTG

T Phe TCT TCC TCA TCG CCT Leu CCC CCA CCG ACT Ile ACC ACA Met ACG GCT Val GCC GCA GCG

C TAT Ser TAC TAA TAG CAT Pro CAC CAA CAG AAT AAC Thr AAA AAG GAT Ala GAC GAA GAG

A Tyr TGT TGC TGA TGG CGT CGC CGA CGG AGT AGC AGA Lys AGG GGT GGC GGA GGG

G Cys stop Trp

Third T C A G T Arg C A G Ser T C A Arg G T Gly C A G

Leu

stop

His

Gln

Asn

Asp

Glu

References 1. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. R. K. Saiki, S. Scharf, F. Faloona, K. B. Mullis, H. A. Erlich, and N. Arnheim, Science (1985) 230:13501354. Specific Enzymatic Amplification of DNA In Vitro: The Ploymerase Chain Reaction. K. Mullis, F. Faloona, S. Scharf, R. Saiki, G. Horn, H. Erlich, Cold Spring Harbor Symposia on Quantitative Biology (1986) LI:263-273. PCR Second Edition - The Basics. M. McPherson and S. Moller, Taylor & Francis Pub., New York (2006).
130

2.

3.

4. 5.

6.

7.

Luminex Corporation, 12212 Technology Boulevard, Austin, TX 78727. Automated DNA diagnostics using an ELISA-based oligonucleotide ligation assay. D. A. Nickerson, R. Kaiser, S. Lappin, J. Stewartt, L. Hood, and U. Landegrent, Proc. Nat. Acad. Sci. USA (1990) 87:8923-8927. Design of electrochemical biosensor systems for the detection of specific DNA sequences in PCR-amplified nucleic acids related to the catechol-Omethyltransferase Val108/158Met polymorphism based on intrinsic guanine signal. D. Ozkan-Ariksoysal, B. Tezcanli,B. Kosova, and M.Ozsoz, Anal Chem. (2008) 80(3):588-596. New Cleavase Fragment Length Polymorphism Method Improves the Mutation Detection Assay, M. C. Oldenburg and M. Siebert, BioTechniques (2000) 28:351357.

General References 1. Compound Heterozygosity Hb S/Hb Hope (136Gly>Asp): a Pitfall in the Newborn Screening for Sickle Cell Disease. R. Ducrocq, A. Bevier, A. Leneveu, M. MaierRedelsperger, J. Bardakdian-Michau, C. Badens, and J. Elion, Journal of Med Screening (1998) 5:27-30. Rapid -globin Genotyping by Multiplexing Probe Melting Temperature and Color. M. Herrmann, S. Dobrowolski, and C. Wittwer, Clinical Chemistry (2000) 46:425-428. Identification of Hb D-Punjab gene: application of DNA amplification in the study of abnormal hemoglobins. Y. T. Zeng., S. Z. Huang, Z. R. Ren, and H. J. Li, Am J Hum Genet. (1989) 44(6):886-9. The inherited diseases of hemoglobin are an emerging global health burden. D. J. Weatherall. Blood (2010) 115:4331. Percentages of abnormal hemoglobins in adults with a heterozygosity for an alpha-chain and/or a beta-chain variant. T. H. Huisman. Am J Hematol (1983) 14:393. http://www.ncbi.nlm.nih.gov/books/NBK1426/ Beta-Thalassemia, GeneReviews [Internet], A. Cao and R. Galanello. (2000 updated 2010). Construction of a Genetic Linkage Map in Man Using Restriction Fragment Length Polymorphisms. D. Botstein, R. L. White, M. Skolnick, and R. W. Davis, Am J Hum Genet (1980) 32:314-331. Specific Enzymatic Amplification of DNA In Vitro: The Ploymerase Chain Reaction. K. Mullis, F. Faloona, S. Scharf, R. Saiki, G. Horn, H. Erlich, Cold Spring Harbor Symposia on Quantitative Biology (1986) LI:263-273. High-throughput SNP genotyping. S. Jenkins and N. Gibson. Comparative and Functional Genomics (2002) 3(1):57-66.

2.

3.

4. 5.

6. 7.

8.

9.

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Chapter 3 Global Chain Analysis

3.4 Electrospray Ionization-Mass Spectrometry
Gul M. Mustafa, PhD and John R Petersen, PhD

Mass spectrometry (MS) is an analytical technique that identifies the chemical composition of a sample on the basis of the mass-to-charge ratio (m/z) of charged ions. The technique has both qualitative (structure) and quantitative (molecular mass or concentration) uses. Another way of thinking about mass spectrometry is that it can be considered as the worlds most accurate scale. Mass spectrometers can be divided into three fundamental parts, namely the ionization source, the analyzer, and the detector (Figure 1). The molecules of interest are first introduced into the ionization source of the mass spectrometer, where they are ionized to acquire positive or negative charges. This is done because ions are far easier to manipulate as compared to molecules that do not have a charge. The ions then travel through the mass analyzer and arrive at different parts of the detector according to their mass to charge (m/z) ratio. After the ions make contact with the detector, useable signals are generated and recorded via a computer. The computer displays the signals graphically as a mass spectrum showing the relative abundance of the signals according to their m/z ratio. The analyzer and detector of the mass spectrometer, and often the ionization source too, are maintained under high vacuum to allow the ions to travel from one end of the instrument to the other without colliding with air molecules which decreases the signal. The entire operation and often the sample introduction process are under complete data system control on modern mass spectrometers.
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Sample Introduction

Ionization

Analyzer(s)

Detector

Atmospheric Pressure Chemical Ionization (APCI) Chemical Ionization (CI) Electron Impact (EI) Electrospray Ionization (ESI) Matrix Assisted laser Desorption Ionization (MALDI) Photo-ionization (PI) Surface Enhanced Laser Desorption Ionization (SELDI)

Light or Charge

Magnetic mass analyzers Electrostatic mass analyzers Sector mass analyzers Quadrupole mass filter Ion trap Time-of-flight mass spectrometers (TOF) Tandem mass spectrometers (MS/MS) Fourier transform ion cyclotron resonance (FC-ICR)

Figure: 1 The method of sample introduction to the ionization source often depends on the ionization method being used, as well as the type and complexity of the sample. Many ionization methods are available and each has its own advantages and disadvantages. The ionization method used depends on the nature and type of sample under investigation and the mass spectrometer available. Figure 2 shows various ionization methods of ionization such as Atmospheric Pressure Chemical Ionization (APCI), Atmospheric Pressure Photo-Ionization (APPI), Electron Impact (EI), and Electrospray Ionization (ESI). The ionization methods used for the majority of biochemical analyses
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are Electrospray Ionization (ESI) and Matrix Assisted Laser Desorption Ionization (MALDI)

Figure: 2

Mass spectrometry using ESI is called electrospray ionization mass spectrometry (ESI-MS) or, less commonly, electrospray mass spectrometry (ES-MS). Electrospray ionization mass spectrometry was pioneered by John Bennet Fenn, who shared the Nobel Prize in Chemistry with Koichi Tanaka in 2002 for his work on the subject (1). One of the original instruments used by Dr. Fenn is on display at the Chemical Heritage Foundation in Philadelphia, Pennsylvania. This technique of ionization is especially useful in producing ions from macromolecules because it overcomes the propensity of these molecules to fragment when ionized and as such is considered a
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soft ionization technique. When analyzing biological molecules of large molecular mass, ESI-MS is very useful because it does not cause fragmentation of the macromolecules into smaller charged particles; rather it creates small droplets containing the macromolecule being ionized and solvent allowing analysis of the molecular weight of the intact macromolecule. Solvent can then be removed causing the formation of even smaller droplets, creating protonation of the macromolecules. These protonated and desolvated molecular ions are then passed through the mass analyzer to the detector, and the mass of the sample is determined (Figure: 3). This method can be performed on solid or liquid samples, and allows analysis of nonvolatile or thermally unstable molecules which means that ionization of proteins, peptides, olgiopeptides, and some inorganic molecules can be easily performed. The spectrum is shown with the mass-to-charge (m/z) ratio on the x-axis, and the relative intensity (%) of each peak shown on the y-axis. The quantitative analysis is done by considering the mass to charge ratios of the various peaks in the spectrum. Calculations to determine the unknown mass, (Mr) from the spectral data are performed using; p = m/z. The ionization mechanism first involves the liquid containing the analyte(s) of interest to be dispersed by electrospray into a fine aerosol. Because the ion formation involves extensive solvent evaporation, the typical solvents for electrospray ionization are prepared by mixing water with volatile organic compounds (e.g. methanol, acetonitrile). To decrease the initial droplet size, compounds that increase the conductivity (e.g. acetic acid) are customarily added to the solution. Large-flow electrosprays can benefit from additional nebulization by an inert gas such as nitrogen.

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Figure: 3

There are some clear advantages and disadvantages of using electrospray ionization mass spectrometry as an analytical method. It is one of the softest ionization methods available; thus it can not only analyze molecules that have high molecular masses but also has the ability to analyze biological samples that are defined by noncovalent interactions. Since the m/z ratio range of a quadrupole instrument is fairly small, the mass of the sample can be determined with a high amount of accuracy. Sensitivity of the instrument is also impressive making it useful in both quantitative and qualitative measurements. The major disadvantage of ESI-MS is that in the analysis of mixtures the results are unreliable. In addition to the difficulty in handling mixtures the multiple charges that are attached to the molecular ions can make for
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confusing spectral data. The apparatus is also very difficult to clean and has a tendency to become contaminated with residues from previous experiments. In recent years, electrospray ionization (ESI) mass spectrometry has become an increasingly important method in proteomics not only to analyze peptides but also to study proteins and protein complexes of increasing size and complexity in structural biology. The analysis of proteins and protein complexes by mass spectrometry (macromolecular mass spectrometry) has become possible because of the development of the relatively gentle ionization procedure related to ESI, which retains non-covalent interactions. The mass-to-charge (m/z) ratios of these proteins can well be over 10,000 daltons, and therefore, time-of-flight (TOF) analyzers with orthogonal injection are the most commonly used analyzers in the field of macromolecules. The m/z analysis of larger proteins and protein complexes is not a routine technique, since a careful optimization of the operating conditions is always required. Despite the theoretically unlimited mass range of TOF analyzers, most instruments have detection problems when the m/z values exceed 4,000 daltons. It has been shown that a pressure increase in the first and second vacuum chamber of the mass spectrometer is an absolute requirement for the analysis of large proteins (2-6). The increased pressure leads to collisional cooling and focusing of large ions in the ion guides and, therefore, improved transmission through the ion guides and the TOF (5). In ESI-MS, the ion signal is proportional to analyte concentration and largely independent of flow rate and injection volume used for sample introduction. The signal is linear from the limit of detection (usually pmol/L) to around 10 mol/L of analyte concentration. For quantitative measurements, it is important to incorporate an internal standard in the procedure to
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compensate for losses during sample preparation and variable detection sensitivity of the MS system. The internal standard should have a structure similar to that of the analyte and the ideal practice is to synthesize an internal standard by incorporating stable isotopes on the molecules of interest. When an ideal internal standard is not available, molecules with similar structure can also be used. Another critical issue in quantitative ESI-MS is suppression of ionization due to matrix interference. A biological sample can give significantly lower ionization signals compared to pure standard solutions with similar analyte concentrations. This phenomenon is the result of high concentrations of non-volatile materials, such as salts and lipids, present in the spray with the analyte. To overcome the matrix interference, extensive sample purification processes are required. However, these elaborate procedures are time-consuming and can cause poor recovery. A recent development is to use short Liquid Chromatography (LC) columns (or guard columns) and apply a fast High Pressure Liquid Chromatography (HPLC) purification (e.g. for 25 minutes) prior to MS analysis. The HPLC serves to separate the non-volatile compounds from the analyte. For HPLC systems with column-switching capability, the analyte in the biological sample can be purified and concentrated on separate columns before MS analysis. Unlike many other techniques which measure one analyte at a time, these techniques can measure multiple analytes (>40) at one time. In recent years the scope of testing using these techniques has expanded from toxicological purposes to newborn screening to hormones, proteins, and enzymes.

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In recent years a change in the way MS is being used in clinical laboratories has occurred. In the past MS was commonly used in conjunction with gas chromatograph (GC). Today it is not uncommon to see MS being coupled to LC in the routine clinical laboratories. Once considered too expensive and cumbersome to use except in forensic and reference settings, such systems are now used routinely to generate data for patient care. Although mass spectrometry has long been recognized as an important and powerful analytical tool, there were a number of challenges that had to be overcome to be used in the clinical setting for more than a few special applications. GCMS was introduced into the clinical laboratory more than two decades prior to LC-MS. With the advent of relatively small, inexpensive, and user-friendly LC/MS and LC tandem MS (LC/MS-MS) systems along with advances in column chemistries the door has been opened to many analyses not possible with GC/MS (7). Although the initial capital investment for LC ESI-MS equipment is substantial compared to other routine clinical laboratory analyzers, its operational costs are low. The cost-effectiveness of this technique comes from the fact that it can measure multiple analytes at the same time. This technology can be expected to exert an important influence in how analytes, both large and small, are detected and quantified in the clinical laboratory service. Since the first report on the successful measurement of large bio-molecules by ESI-MS, there has been a revolution in the identification of protein molecules in biochemical research. MS also found its way into the analysis of hemoglobin (Hb) analysis. In 1981, Wada et al. pioneered the analysis of tryptic peptides of Hb by MS. The development of the soft ionization techniques (ESI and MALDI) has made it possible to use MS to study intact globin chains. In 1990, the 1st application of ESI-MS
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involving intact Hb chains was reported by Falick et al (8). Since then ESI and MALDITOF MS has become more common in routine Hb variant analysis. Electrospray ionization is efficient in generating cluster ions for structural elucidation of macromolecules. This has fostered a new and improved approach (vs. electrophoresis) for identification and quantification of hemoglobin variants. The use of MS techniques has led to the discovery of more than 60 new mutations and even the intact Hb tetramer can be analyzed using a nano ESI-MS technique. Furthermore, MALDI-TOF MS is a highly sensitive method that enables the analysis of Hb chains from a single red blood cell. Final identification of a variant is achieved either by molecular biology techniques or by protein sequence analysis, in which MS now also occupies a key position. In variants with mutation sites close to the termini of the chain were identified by ESI-MS/MS of the intact Hb chain. With the understanding of glycohemoglobin (GHb) structure, an IFCC reference method for glycohemoglobin assay has been established using ESI-MS. It represented a significant advancement for the standardization of HbA1c in diabetic monitoring. ESI-MS has also become the preferred technique for a rapid systematic approach to definitive characterization of Hb variants. In addition, hemoglobin (Hb) variants need to be identified for the investigation of hemolytic anemia, methemoglobinemia, sickle cell disease and thalassemia. Occasionally, these variants are detected incidentally because they interfere with the measurement of GHb.

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Identification and quantification of hemoglobin variants Globin chain analysis is an important tool in phenotype study of hemoglobin disorders. The majority of hemoglobin variants result from changes in the amino acid sequence of either the or non- globin chains of hemoglobin with the majority of these changes due to a single point mutation in the globin gene. Substitution, insertion, deletion or the combination of deletion with insertion of a different amino acid than those normally present, results in changes to the amino acid sequence. Worldwide, an estimated 150 million people carry Hb variants (9) and hemoglobinopathies are the most common inherited disorders, constituting a significant healthcare problem (10). Hemoglobin (Hb) variants lead to inherited disorders with variable clinical manifestations. Therefore, reliable detection and identification methods are essential. Among more than 900 hemoglobin (Hb) variants currently described in the HbVar database of the globin Gene Server, variants with elongated chains are very rare (11,12). In this database, Hb variants leading to a charge difference are significantly over represented compared with neutral Hb variants. This result is surprising, because only 5 of the 20 amino acids contain either a basic (Lys, Arg, His) or an acidic (Asp, Glu) side chain, whereas the other 15 amino acid side chains are uncharged. Thirty-six of 141 amino acids in the -chain and 38 of 146 residues in the chain are charged residues and the rest are neutral so they cannot be detected by these traditional analytical techniques, such as ion-exchange HPLC and isoelectric focusing (IEF) on polyacrylamide gel, as these techniques depend on the presence of charge differences induced by the mutation. Also in the past, definitive characterization of Hb variants involved tedious and time-consuming analytical procedures requiring
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days and even months for completion. Recently, a strategy for rapid definitive characterization of Hb variants to identify a single mutated; inserted or deleted amino acid residue was reported using ESI-MS. In case of Hb San Martin [b6(A3)GluVal;b85(F1) PheLeu], the second mutation leads to an unstable protein causing chronic hemolytic anemia in the heterozygous carrier (13). Molecular diagnosis, achieved by DNA analyses, shows the presence of two mutations, but protein or familial studies was required to prove that the two mutations are carried by the same allele and not interacting in trans. The identification by MS methods of a new Hb variant: Hb SClichy [b6(A3)GluVal;b8(A5) LysThr], which presents a double mutation located on the same bT-1 tryptic peptide. This new variant adds the amino acid substitution of Hb Rio Grande[b8(A5) LysThr] (14) on the same b-globin chain, to that of Hb S. Difficulties encountered in structural determinations are caused by the presence of two abnormalities in the same polypeptide chain. Variants with two amino acid substitutions on the same globin chain as in Hb S-Clichy, demonstrated the importance of including MS studies. The procedure comprises the following steps: I. Molecular weight profiling of intact and globin chains by direct ESI-MS on a 500-fold dilution of the whole blood sample. The cluster ion spectrum is then deconvoluted to a true molecular weight scale using computer software that is usually supplied with the MS analyzer system. This step can detect Hb variants with molecular weight difference of more than 6 Da when compared with the wild type globin chains (15).

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II.

Overnight trypsin digestion for investigation of the amino acid substitution on the Hb variants. ESI-MS on the tryptic digest can identify the specific peptide harboring the substituted amino acid.

III.

ESI-MS/MS of the target peptide can provide the amino acid sequence of the peptide and thus the position of the substituted amino acid.

These performances can be applied at different steps of the globin variant analysis process: either as a screening method or as an additional technique to confirm the results from classical analytical methods. ESI-MS can also identify 95% of the Hb variants in over 250 samples with a turn-around-time of not more than 2 days for each sample, making it a powerful tool for Hb analysis. It must be considered that the 3-dimensional structure of the globins is determined principally by the residues that form the interhelical and helix-heme packings (16), and substitutions in these sites may lead to conformational changes in the proteins. The substitution effect also depends on the 3-dimensional position, viz. internal or external. For example, the variant Hb Sun Prairie (130Ala3Pro) is silent in IEF, whereas Hb Fontainebleu (21Ala3Pro) is detectable (12). The substituted amino acid is internal in Hb Sun Prairie and external in Hb Fontainebleu. As a very simple model, the calculation of the isoelectric points(pI)-shifts does not consider conformational changes that might alter the mobility. Therefore, mutations leading to a distinct conformational change can diverge from the predicted behavior. Furthermore, the model cannot predict reliably unstable variants. Nevertheless, pI calculations and the evaluation of the method-specific detect ability allow the prediction of the number of
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the currently undetected, silent variants. So it is now recommended that other methods that are not based on electrophoretic or chromatographic mobility should be applied in Hb variant analysis. In this regard ESI and MALDI-TOF MS are the suggested methods that enable the detection of variants when the mass difference between the abnormal and the wild-type globin chains exceeds 6 Da. This limitation in MS determination is due to the complexity between the normal and mutated globin chains which can be overcome by using high resolution instruments (FT-ICR, Orbitrap) or by special precautions on low resolution instruments. For these low mass differences between normal and variant globin chains, MS analysis of digested peptides is required. As calculated by various studies, MS method is able to detect 92% of the undetected variants. Among MS techniques for studying Hb variants, ESI-MS is the most frequently used and can be associated with peptide sequencing using tandem MS, but it often gives multiple charged fragment ions. On the other hand, MALDI-TOF MS gives single-charge peptide ions and has been used for identification of some single mutation Hb variants. Indeed, with additional MS analysis of lysate samples 3 new variants, Hb Zurich-Hottingen, Hb Zurich-Langstrasse and Hb Riccarton were detected by using ESI-MS. Neither variant had a clinical impact. These neutral variants are exclusively found by MS and are chromatographically silent. Also in an Hb Malay sample, only the MS analysis revealed the variant chain, as opposed to cationexchange HPLC which identified it as a thalassaemia. Recapitulating, 4 out of 2105 samples (0.2%) or 1% of the abnormal samples would be missed without the use of MS analysis. In ESI-MS, the sample preparation is very simple and requires only the dilution of the lysate sample. Two important drawbacks of the MS methods are worth
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mentioning. First, its insufficient resolution prevents the detection of Hb mutations with small mass differences of the globin chains. The precision of normal low-resolution mass measurements is insufficient to distinguish the wild-type chain from several chain variants, such as Hb C, D, or E. Owing to the isotopic pattern, even high-resolution MS did not separate globin chains that differed only in 1 or 2 Da from the normal chains (17). Two intact globin chains are not observed as separate entities in MS unless their masses differ from one another by more than 6 Da. Second, MS as described here is only a qualitative technique, and in particular, minor Hb fractions such as HbA1C or HbA2, which are important for diagnosis of diabetes mellitus or thalassemia, respectively, cannot be quantified. However high resolution MS enables detection of variants with low mass difference (<2 Da). Also different signals in the spectrum shows isotopic pattern.

References 1. Fenn, JB. Electrospray Ionization Mass Spectrometry: How It All Began. J. Biomol. Techl. 13:101118; 2002. 2. Sanglier S, Leize E, Van Dorsselaer A, Zal F. Comparative ESI-MS study of approximately 2.2 M Da native hemocyanins from deep-sea and shore crabs: from protein oligomeric state to biotope. J. Am. Soc. Mass Spectrom.14:419-429; 2003. 3. Schmidt A, Bahr U, Karas M. Influence of pressure in the first pumping stage on analyte desolvation and fragmentation in nano-ESI MS. Anal. Chem.73:60406046; 2001. 4. Tahallah N, Pinkse M, Maier CS, Heck A. The effect of the source pressure on the abundance of ions of noncovalent protein assemblies in an electrospray ionization orthogonal time-of-flight instrument. J. Rapid Commun. Mass Spectrom. 15:596-601; 2001. 5. Chernushevich IV, Thomson BA. Collisional cooling of large ions in electrospray mass spectrometry. Anal. Chem. 76:1754-1760; 2004. 6. Krutchinsky AN, Chernushevich IV, Spicer VL, Ens W, Standing KG. A Collisional damping interface for an electrospray ionization time-of-flight mass spectrometry. J. Am. Soc. Mass Spectrom. 9: 569-579; 1998.
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7. Plumb RS and Balogh MP. The changing face of LC-MS: from experts tousers. Current Trends in Mass Spectrometry November. 1419; 2008. 8. Falick AM, Shackleton CH, Green BN, Witkowska HE. Tandem mass spectrometry in the clinicalanalysis of variant hemoglobins. Rapid Commun Mass Spectrom. 4:396400; 1990. 9. Shimizu A, Nakanishi T, Miyazaki A. Detection and characterization of variant and modified structures of proteins in blood and tissues by mass spectrometry. Mass Spectrom Rev. 25: 686712; 2006. 10. Daniel YA, Turner C, Haynes RM, Hunt BJ, Dalton RN. Rapid and specific detection of clinically significant haemoglobinopathies using electrospray mass spectrometry-mass spectrometry. Br J Haematol. 130:63543; 2005. 11. Hardison RC, Chui DH, Giardine B, Riemer C, Patrinos GP, Anagnou N. HbVar: a relationaldatabase of human hemoglobin variants and thalassemia mutations at the globin gene server. Hum Mutat. 19:22533; 2002. 12. HbVar database. http://globin.cse.psu.edu (accessed July 2011). 13. Feliu-Torres A, Eandi-Eberle S, Calvo K, et al. Hemoglobin San Martin: A new unstable variant associated with Hemoglobin S in an Argentinean boy. Proceedings of the 49th American Society of Hematology Meeting, Atlanta, GA, December 811, Blood. 110:3806; 2007. 14. Moo-Penn WF, Johnson, MH, Mc Guffey, JE, Jue, DL, Therrell, BL, Jr. Hemoglobin Rio Grande [b8(A5) LYSTHR]: A new variant found in a Mexican American family. Hemoglobin. 7(1):9195; 1983. 15. Wild BJ, Green BN, Stephens AD. The potential of electrospray ionization mass spectrometry for the diagnosis of hemoglobin variants found in newborn screening. Blood Cells Mol Dis. 33:308-317; 2004. 16. Lesk AM, Chothia C. How different amino acid sequences determine similar protein structures: the structure and evolutionary dynamics of the globins. J Mol Biol. 136:22570; 1980. 17. Peter K, Marlis S, Karin Z, Oliver S, Markus S, Bernd R, Silke SD, Leopold U, Thomas K, Claus WH, Hannes F, and Heinz. T. Mass Spectrometry: A Tool for Enhanced Detection of Hemoglobin Variants. Clinical Chemistry. 54(1): 6976; 2008.

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Chapter 3 Globin Chain Analysis

3.5 PCR and Sanger Sequencing
Elaine Lyon, PhD

Molecular methods are commonly employed to identify hemoglobin variants. Polymerase chain reaction (PCR) exponentially amplifies regions of DNA allowing direct genotyping or targeted mutation analysis. Detecting common alpha globin deletions is accomplished by amplifying over deletion breakpoints or using quantitative methods to detect copy number changes. Sanger sequencing is considered the gold standard for mutation detection, and can confirm abnormal hemoglobinopathy and thalassemia variants. Molecular analysis confirms a diagnosis, detects carrier status, and predicts disease prenatally in highrisk pregnancies. This section will describe general methods, applications and challenges in PCR and Sanger sequencing for alpha and beta globin molecular analysis.

3.5.1 Alpha Globin Two alpha globin genes (HBA1 and HBA2) are present on each chromosome 16, resulting in a normal copy number of four genes (represented by /). One or both alpha globin genes may be deleted on a single chromosome, with the severity of disease corresponding to the overall number of deleted alpha globin genes. If two alpha globin genes are deleted (alpha-thalassemia trait), it is important to determine whether both genes are deleted on one chromosome (--/), or if each chromosome contains a single gene deletion (/-). If both parents carry a chromosome with two deletions (--/), their offspring are at risk for Hb Bart hydropsfetalis syndrome (--/--). Common deletions include 3.7kB and 4.2kB
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deletion which eliminate a single gene, while a 20.5kB deletion and the SEA, MED, FIL and THAI gene rearrangements delete portions of or completely HBA1 and HBA2. In one assay commonly used in clinical laboratories, PCR primers are designed to flank the breakpoints, amplifying a product only when the deletion is present. By multiplexing primers, any of the common deletions can be detected in a single reaction (1). Amplification products are visualized by gel electrophoresis (figure 1). Other methods to identify deletions include quantitative PCR analyses such as multiplexed-ligation probe amplification (MLPA) or high resolution (exonic level) microarray. These methods are capable of detecting known and previously unknown alpha globin deletions and alpha globin triplications (2,3). Given the frequency of alpha globin deletions, a molecular work-up for alpha thalassemia often begins with a test for deletions.

Figure 1. Gel electrophoresis for common alpha globin deletions. Each patient is tested with control primers for HBA2 and LIS genes. In a separate reaction, each patient is tested for a common deletion with a multiplex of deletion-specific primers. M: size marker, C: control primers (HBA2 and LIS1), D: deletion primers. Patient 1: no common deletions, patient 2; 3.7 kB heterozygous, patient 3; 3.7kb/SEA compound heterozygous. Note that in patient 2, the control HBA2 band is not present, as this patient has a deletion of this region on both chromsomes.
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The alpha globin genes also harbor sequence variants, and full gene sequencing is also available, although alpha globin sequencing poses challenges. Sequencing is performed on both genes (HBA1 and HBA2) that are highly homologous, and primers are designed to be specific for each gene. Sequencing the entire coding region (3 exons for each gene), intron/exon boundaries, proximal promoter regions, 5 and 3untranslated regions, and polyadenylation signals provides a comprehensive sequencing test. Sequencing should be combined with deletion analysis because deletions are not detected by sequencing, and an apparent homozygous sequence variant may be one copy of the variant with a deletion on the opposite chromosome. Samples homozygous for the 3.7kB deletion may not be able to be amplified for sequencing, resulting in a failed test. However, the common 3.7kB deletion has a single functional gene, but mutations have also been described in that fusion gene. (4,5) To be able to identify a mutation in a chromosome with the 3.7kB deletion, primers must be designed that will amplify over the deletion breakpoints.

3.5.2 Beta Globin Molecular genotyping assays targeting common beta globin mutations, (e.g. HbS and Hb C) are available to confirm the mutations for sickle cell or S/C disease. But due to the number of mutations that have been described, full gene sequencing identifies any sequence variant and complements other types of globin analysis for hemoglobinopathies and thalassemias. The mutation spectrum for beta globin is well characterized, and includes coding region mutations, splice-site mutations, regulatory mutations, and deletions. Therefore a comprehensive assay consists of sequencing the three exons of the HBB gene, the intron/exon boundaries, the proximal promoter region, the 5 and 3 untranslated regions
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(UTR), and known pathogenic deep intronic mutations (e.g. IVS-II-654, IVS-II-705 and IVS-II745). Large beta globin deletions or delta-beta fusion genes (e.g. Lepore,) will not be detected by a sequencing assay designed only for HBB, and require a different analysis. Similar to methods to detect alpha globin deletions, primers can be designed to amplify over the breakpoints. One example of this is the 619 base pair (bp) deletion found in Indian and other Asian populations (6). Recently, novel beta globin deletions have been detected by other methods, such as exonic-level microarrays or MLPA (2,7). Specific large deletions in the beta globin gene cluster is one of two molecular mechanisms that can result in HPFH. The other mechanism is point mutations in the promoters of the gamma globin genes ( HBG1 and HBG2).

3.5.3 Sequencing Sequencing assays begin with PCR for the regions to be interrogated. Primers are designed to avoid known variants at their 3 end which would prevent polymerase extension, resulting in a drop-out of that allele (8). PCR products are treated with ExoSAP (exonuclease 1 from shrimp alkaline phosphatase) to remove excess primers.

PCR primers may be tagged with a M13 sequence which allows sequencing of all amplicons from the same M13 primers. Alternatively, a second set of sequencing primers internal to the PCR primers may be used. The sequencing reaction utilizes fluorescently labeled dideoxynucleotides (ddATP, ddTTP, ddCTP and ddGTP, collectively refered to as ddNTPs) which lack a 3 hydroxyl group on the sugar residue and prevent the newly synthesized product from extending to the next base when incorporated into the product. Sanger sequencing is
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performed as a linear rather than exponential amplification, with separate sequence results each from the 5 and 3 directions (bi-directionally). After the sequencing reactions, the products are again processed to remove excess primers. Sephadex columns are often used to bind the sequencing products, which are eluted as purified products. Sequencing products are separated by capillary electrophoresis using a polymer with a singlebase resolution. The last base of each fragment is the ddNTPs with a fluorescent dye which is incorporated into the product. The sequence is visualized as an electropherogram and aligned to a reference sequence. The difference between the reference and the patient sequence are examined to determine the type of mutation present. To accurately identify the mutation, it should be identified in both the 5 and 3 direction.

3.5.4 Reporting Sequence Variants In standard nomenclature, nucleotides are numbered from the ATG of the start codon. The protein position is predicted and standard nomenclature is from the methionine of the translated product, but common or traditional nomenclature (also known as legacy nomenclature) may be from the mature protein (gene reviews for beta and alpha). For alpha and beta globin, the traditional nomenclature differs by one amino acid than the standard nomenclature, representing the cleavage of methionine. Reports should clarify if they are using the standard or the common (i.e. amino acids numbered from the mature protein). A variant is described as to whether it is structural (hemoglobinopathy) variant or quantitative (thalassemia variant). For example, a beta thalassemia mutation is classified as a B(0) [the absence of beta chains] or B(+) [reduced amount of beta chains] mutation. One copy of a thalassemia mutation is consistent with beta
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thalassemia minor, while two copies on opposite chromosomes are consistent with beta thalassemia major. On occasion, two mutations may occur on the same chromosome. Sequence analysis cant determine the phase of two mutations (whether on the same or opposite chromosomes). HPLC or parental studies may be necessary to evaluate phase. Over ten complex variants with the Glu6Val variant are listed in the globin gene server (6). One example is Hb S-Oman, with Glu6Val and Glu121Lys variants on the same chromosome. The standard nomenclature for the nucleotide changes is HBB:c.[20A>T;364G>A] (6). These two variants are also seen alone as in Hb S and Hb 0-Arab. The combination of these two mutations on opposite chromosomes is consistent with severe sickle cell disease, whereas if they are on the same chromosome, the individual is a carrier of an HBB mutation.

3.5.5 DNA Sequence Traces: Several sequencing electropherograms are presented to illustrate the application. The first shows the common alpha globin variant Constant Spring (Hb CS). This alpha + thalassemia variant changes the normal termination codon to an elongated 3 end of the protein. The second shows a sequence with two beta globin mutations. Information from family or other laboratory studies could determine if these two variants are on the same or different chromosomes.

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Figure 2.Alpha globin sequencing. An apparent homozygous Hb CS variant (c.427T>C, Term>Gln) is detected (arrows). The sequence from the patient (Forward and Reverse) are compared against a reference sequence. Differences between the reference and patient sequences are shown in the middle panes.

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Figure 3.Beta globin sequencing. A compound heterozygous genotype is detected. The first mutation is Hb S (c.20A>T, Glu6Val), while the second affects a splice site resulting in a beta(0) thalassemia mutation. (c.92+1G>A). The yellow arrow in the electropherogram indicates the exonic region. The sequence from the patient (Forward and Reverse) are compared against a reference sequence. Differences between the patient and reference sequences are shown in the middle panes.

3.5.6 Conclusion Molecular analysis confirms the familial variant in individuals who are carriers of or affected with globin gene variants. In prenatal analysis, molecular studies are often the most direct method to predict the status of a fetus. If molecular testing is used prenatally, the parents should first be tested to identify the familial mutations. In addition, amniotic fluid, amniocyte or chorionic villi cell cultures should be tested for contamination from the mother. If

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the samples show maternal cell contamination, the results may not accurately reflect the fetus genotype and a second sample should be obtained. The alpha and beta loci have complex structures that lead to a variety of molecular anomalies, such as sequence variants, and large gene rearrangements resulting in deletions or duplications. Because many mutations in HBA1, HBA2 and HBB are well understood, the

interpretations are typically straight forward. However, because these loci have complex structures that lead to a variety of molecular anomalies, molecular results should be combined with clinical, family and other laboratory findings.

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References 1. Tan AS, Quah TC, Low PS, Chong SS. A rapid and reliable 7-deletion multiplex polymerase chain reaction assay for -thalassemia. Blood. 2001; 98(1):250251. Phylipsen M, Chaibunruang A, Vogelaar IP, Balak JR, Schaap RA, Ariyurek Y, Fucharoen S, den Dunnen JT, Giordano PC, Bakker E, Harteveld CL. Fine-tiling array CGH to improve diagnostics for - and -thalassemia rearrangements. Hum Mutat. 2012 Jan; 33(1):272-80. Galanello R, Cao A. Alpha-Thalassemia. 2005 Nov 1 [Updated 2011 Jun 7]. In: Pagon RA, Bird TD, Dolan CR, et al., editors. GeneReviews [Internet]. Seattle (WA): University of Washington, Seattle; 1993-. Available from: http://www.ncbi.nlm.nih.gov/books/NBK1435/accessed 10-04-12 Zhao P, Buller-Burckle AM, Peng M, Anderson A, Han ZJ, Gallivan MV. Secondary mutation (c.94_95delAG) in a -3.7 allele associated with Hb H disease in two unrelated African American individuals homozygous for the (3.7) deletion (-3.7/-3.7T). Hemoglobin. 2012; 36(1):103-7. Brennan SO, Chan T, Duncan J. Novel 2 gene deletion (c.349_359 del GAGTTCACCCC) identified in association with the -3.7 deletion. Hemoglobin. 2012; 36(1):93-7. Hardison RC, Chui DHK, Giardine B, et al. HbVar: a relational database of human hemoglobin variants and thalassemia mutations at the globin gene server. Hum Mutat 2002; 19: 225-33 http://globin.cse.psu.edu/accessed 1004-2012 Mikula M, Buller-Burckle A, Gallivan M, Sun W, Franklin CR, Strom CM.The importance of globin deletion analysis in the evaluation of patients with thalassemia.Int J Lab Hematol. 2011 Jun;33(3):310-7 Pont-Kingdon G, Gedge F, Wooderchak-Donahue W, Schrijver I, Weck KE, Kant JA, Oglesbee D, Bayrak-Toydemir P, Lyon E; Biochemical and Molecular Genetic Resource Committee of the College of American Pathologists. Design and analytical validation of clinical DNA sequencing assays.Arch Pathol Lab Med. 2012 Jan;136(1):41-6.

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3.

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Chapter 4
Alpha and Beta Thalassemia
Herbert L. Muncie, Jr., MD Alpha () and beta () thalassemia are hematological disorders that are the result of a decreased or absent synthesis of a globin chain.1 These genetic alterations may have been the result of selective pressure from Plasmodium falciparum malaria from which thalassemia carriers are relatively protected from invasion.2, 3 The altered globin chain synthesis can be asymptomatic or can cause severe hemolytic anemia and even death.

4.1

Epidemiology The thalassemias are prevalent in the tropical and subtropical regions of the

world and affect men and women equally. Alpha thalassemia is found more often in persons of African or Southeast Asian descent and -thalassemia occurs more often in persons of Mediterranean, African or Southeast Asian descent. Thalassemia trait can be found in 5 to 30 percent of these populations.4 An estimated 1.5% of the global population are -thalassemia carriers but only approximately 200,000 people are alive with -thalassemia major.5, 6

4.2

Pathophysiology Hemoglobin has an iron-containing heme ring and four globin chains: normally

two alpha and two nonalpha. The composition of these four globin chains determines
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the hemoglobin type. The predominant in utero hemoglobin, fetal hemoglobin (Hb F), has two and two gamma () chains (2 / 2). Adult hemoglobin A (Hb A) has two and two chains (2/2) and hemoglobin A2 (HbA2) has two and two delta () chains (2/2). The transition from -globin synthesis (Hb F) to -globin synthesis (Hb A) begins before birth. Therefore, at birth approximately 20% to 30% of hemoglobin is Hb A and the remainder is HbF.7 This transition continues and is usually completed from 6 to 24 months of age. At that time normal children will have mostly Hb A (>96%), small amounts of Hb A2 (2.0 3.4%) and very small amounts of Hb F (< 1%).8

4.3

Alpha Thalassemia Alpha thalassemia occurs when there is reduced or absent -globin chain

synthesis with subsequent excess -globin chains.9, 10 Two genes on chromosome 16 control -globin synthesis (/). Most defects are due to deletions of one or more of these genes. Since two genes on each chromosome 16 control the production of globin chains, there are four possible phenotypical presentations. With a single gene deletion (-/) the result is -thalassemia silent carrier state which is asymptomatic with normal hematological indices. With two gene deletions (-/-; --/) the result is thalassemia trait (minor) which frequently causes microcytosis without anemia. If three genes are deleted (--/-) there will be significant amounts of hemoglobin H (Hb H) consisting of four -globin chains (4). The result of significant amounts of Hb H is thalassemia intermedia (Hb H disease), which causes hemolytic anemia, microcytosis and splenomegaly. While most cases of Hb H disease are deletional, non-deletional
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forms do occur and are often more symptomatic. Hemoglobin Constant Spring is an globin chain variant that is longer than normal and produced in only small quantities. It therefore behaves in a similar manner to an alpha gene deletion.
11

When Hb Constant

Spring is inherited with a 2 alpha gene deletion, the condition may be referred to as Hb H / Constant Spring. Finally if all four genes are deleted (--/--) the result will be significant amounts of hemoglobin Barts (Hb Barts) with four gamma chains (4). With increased Hb Barts and total absence of Hb F, -thalassemia major results leading to hydrops fetalis, which is incompatible with life.
12

4.4

Beta Thalassemia Beta thalassemia occurs when there is reduced or absent -globin chain

synthesis with subsequent excess -globin chains.3, 6 Most often a mutation is the genetic defect, with more than 200 reported; a deletion is quite rare. One gene on each chromosome 11 controls the production of -globin chains (,), therefore, there are two phenotypical presentations. If a child inherits one normal gene from one parent (/) and a defective gene from the other parent (-/), the result is -thalassemia trait (minor) which causes an asymptomatic mild microcytic anemia. If both genes are defective, the result depends on the degree they are deficient in -globin chain production. If -globin chain production is severely reduced, the person will have thalassemia major (Cooley anemia). Most individuals with -thalassemia are asymptomatic at birth because of the presence of significant amounts of Hb F. As the globin chain synthesis decreases, infants may experience symptoms starting at six
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months of age. If the -globin chain synthesis is only partially reduced, the person will have -thalassemia intermedia with less severe symptoms and survival beyond 20 years of age without life-long blood transfusions.

4.5

Diagnosis Except for or thalassemia major, the diagnosis of thalassemia is usually

made incidentally when a patient is found to have microcytosis with or without anemia. The most common etiologies for microcytic anemia are iron deficiency, thalassemia, lead toxicity and sideroblastic anemia. The patients medical history, mean corpuscular volume (MCV), red cell count and the red cell distribution width (RDW) can help exclude many of these etiologies (Table 1). The MCV in -thalassemia trait is usually lower than in -thalassemia trait. In Hb H disease the MCV will be as low as 64 fl.13 Mentzer index (MCV/red blood cell count) was proposed (which is not true in children) to predict the likelihood of thalassemia. If the ratio is > 13, iron deficiency is the likely etiology whereas thalassemia is associated with a value < 13. An exact ratio of 13 would be uncertain.14 The RDW can be helpful in distinguishing thalassemia from iron deficiency and sideroblastic anemia. With iron deficiency or sideroblastic anemia the RDW is almost always elevated while it is elevated in approximately 50% of thalassemia trait patients.15 Therefore, with a microcytic anemia, if the RDW is normal the diagnosis is usually thalassemia trait. However, if the RDW is elevated additional tests to evaluate for iron deficiency and sideroblastic anemia will be needed.16

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A serum ferritin level is the best single test to rule out iron deficiency in the absence of inflammation.17 Serum iron, total iron binding capacity and transferrin may not be needed in distinguishing iron deficiency from thalassemia. A peripheral smear or bone marrow aspirate can rule out sideroblastic anemia. If lead toxicity is suspected, a serum lead level will be needed. And finally a hemoglobin electrophoresis/ HPLC can evaluate for hemoglobinopathies and may confirm the diagnosis of thalassemia. In the past the diagnosis of -thalassemia in adults was by exclusion. If a patient had a microcytic (MCV < 80 fl) hypochromic (MCH < 27 pg) anemia, normal iron studies and a normal hemoglobin electrophoresis and Hb A2 it was assumed the patient had thalassemia trait (minor). Now high-performance liquid chromatography (HPLC) can often provide an accurate diagnosis in neonates. In infants, if increased amounts of Hb H or Hb Barts are found in cord blood or neonatal blood, the diagnosis of -thalassemia is confirmed. Infants who are silent carriers may have a slightly increased amount of Hb Barts (1 2%) at birth while infants with -thalassemia trait have a moderately increased amount (5 6%).10 In adults with -thalassemia trait (minor) the HPLC or hemoglobin electrophoresis will show reduced Hb A levels (<96%), elevated Hb A2 levels (>3.5%) and often elevated Hb F levels (1.0 4.0%).3, 4 However, a normal amount of Hb A2 does not exclude thalassemia in some patients. Patients with Iron deficiency often have lower Hb A2 levels and the Hb A2 quantification may need to be repeated after iron supplement therapy.18 Genetic coinheritances may reduce Hb A2 production making it difficult to diagnosis -thalassemia. If the Hb A2 level is below normal (<2.5%) but with
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a normal Hb F level and microcytosis, the patient has -thalassemia intermedia, i.e. Hb H disease (Table 2)19. Reviews on measuring and interpreting Hb A2 and Hb F levels are available.8, 20 Beta thalassemia major is diagnosed during the infants first year of life. The infant usually displays growth retardation, pallor, irritability and later jaundice with abdominal swelling. Children who develop these symptoms after their first birthday will be diagnosed with -thalassemia intermedia.

4.6

Treatment Patients with or thalassemia trait (minor) require no treatment or regula r long-

term follow-up. While these patients have a microcytic anemia they are not iron deficient and should not be given iron supplements. If iron deficiency did develop, iron supplements would be appropriate.4, 21 Beta thalassemia major requires treatment with blood transfusions starting as early as six months of age. Transfusions correct the anemia, suppress erythropoiesis and inhibit intestinal iron absorption. Transfusions are initiated either when the hemoglobin is < 7 g/dl for more than 2 weeks (without another etiology) or if other factors such as facial changes, poor growth, bony expansion or splenomegaly occur. Without blood transfusions these patients would not survive into adulthood. They will need periodic (every 2 - 4 weeks) transfusions (lifelong) to maintain their hemoglobin higher than 9.5 g/dL.4, 22 The post-transfusion hemoglobin goal is 13 14 g/dl. Beta thalassemia intermedia patients require transfusions only when their reduced hemoglobin interferes with their quality of life or it effects their growth and development.
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Transfusions will occasionally be needed for Hb H disease depending on the severity of the condition. Patients who receive frequent blood transfusions or who have increased intestinal iron absorption will eventually develop iron overload since their bodies cannot actively eliminate excess iron. To treat the iron overload, -thalassemia major patients will require iron chelation therapy starting either around age 5, when the serum ferritin exceeds 1000 ng/ml, or after they have had 10-20 transfusions.23 A liver biopsy is the gold standard for iron overload diagnosis.24 Beta thalassemia intermedia patients will begin chelation when the serum ferritin is > 300 mcg/L.3 Deferoxamine either subcutaneously or intravenously has been the chelation treatment of choice although long-term compliance is difficult.25 An oral alternative is deferasirox (Exjade).26 Iron chelation therapy is relatively benign although it is time consuming and expensive (Table 3). The only curative therapy for patients with -thalassemia major is a bone marrow transplant. In low-risk patients with no hepatomegaly, no portal fibrosis on liver biopsy and not receiving regular chelation therapy, hematopoietic stem cell transplantation usually has excellent results.4

4.7

Complications Patients with or thalassemia trait (minor) have no complications. Patients with

Hb H disease, -thalassemia major or -thalassemia intermedia have hemolysis, growth retardation and skeletal abnormalities as a consequence of the over stimulation of the

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bone marrow and ineffective erythropoesis.21, 27 Infants with significant amounts of Hb Barts usually die in utero or shortly after birth due to autoimmune hydrops fetalis. Because of the need for multiple blood transfusions in -thalassemia major or in some cases of Hb H disease and the increased intestinal iron absorption with -thalassemia intermedia, patients develop iron overload which damages visceral organs (liver, spleen, endocrine organs) and the heart which is the primary cause of early death.28 Splenomegaly invariably develops in symptomatic thalassemia and can worsen the anemia. The risk of hepatocellular carcinoma is increased due to iron overload hepatic damage, longer survival and viral infection with hepatitis B and/or C.29 Gallstones are more prevalent with -thalassemia intermedia than with -thalassemia major. Beta thalassemia major and intermedia cause a hypercoaguable state. 30 This effect increases the risk of thromboembolic events especially after splenectomy.31 Osteoporosis was found in 51% of patients over age 12 with -thalassemia major.32

4.8

Other Treatment Issues

4.8.1

Hypersplenism Splenectomy is required for patients whose splenomegaly causes a marked

increase in their need for blood transfusions, i.e. the annual red cell requirement exceeds 180 200 ml/kg.6 Because of the importance of the spleen in clearing bacteria and preventing sepsis, the surgery is not done until the patient is at least 4 years old. One month prior to the surgery the child should be given the pneumococcal polysaccharide vaccine. They should also receive the pneumococcal conjugate vaccine
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series if they had not received it during infancy. For the first two years after the surgery patients should take penicillin 250 mg twice a day. For children the antibiotic prophylaxis continues until age 16.33 Gallbladder removal should be considered if gallstones are present34 at the time of splenectomy.

4.8.2 Endocrinopathies While growth retardation can occur with thalassemia, growth hormone therapy has limited effectiveness and is not recommended. If hypogonadism develops, hormonal therapy is effective.35 Bone mineral density has been increased with alendronate, pamidronate and zolendronate; however, studies evaluating fracture reduction are needed.36

4.8.3 Pregnancy Couples from high risk ethnic groups should be encouraged to seek preconception genetic counseling.37 Individuals with a low MCV (<80fl) and MCH (<27 pg) could be assessed with hemoglobin electrophoresis / HPLC.12 An efficient way to identify mutations is to study their parents hematology and screen them for single mutations.38 For couples, if both partners have -thalassemia trait, their child will have a one in four chance of having -thalassemia major and a two in four chance of -thalassemia trait.(Figure 1) With four genes controlling the expression of -globin chains, the inheritance pattern is more complex. If two genes are defective, the phenotype is influenced by whether the defective genes are on the same chromosome (cis) or
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different chromosomes (trans), e.g. if one parent is an -thalassemia silent carrier (-, ) and one parent has -thalassemia trait [(cis),(--,)], they have a one in four chance their child will have Hb H disease. Whereas, if the -thalassemia trait parents defect is trans (-, -), their children have no risk of Hb H disease (Figure 2). Once pregnancy occurs, patients should be counseled regarding prenatal diagnostic testing options. An amniocentesis at approximately 15 weeks gestation or a chorionic villus sample (CVS) obtained at 10 11 weeks gestation can detect point mutations or deletions utilizing polymerase chain reaction (PCR) testing. Other diagnostic options include DNA analysis of fetal cells obtained by amniocentesis and in the future analysis of maternal blood fetal cells.39, 40 If Hb Barts is detected, the mother has an increased risk of pre-eclampsia and postpartum hemorrhage. For couples using in vitro fertilization, preimplantation genetic testing is available. 41

4.8.4 Cardiac When iron overload occurs, cardiac infiltration and death are significant concerns. Serum ferritin levels have been used to predict cardiac complications with improved survival if levels are kept below 2,500 ng per ml (2500 mcg per L).42 Ferritin levels are unreliable when significant liver disease develops.43

4.8.5 Hypercoagulopathy While the risk of thromboembolic events in patients with -thalassemia major or intermedia is increased, no trials have evaluated prevention of these complications with anticoagulants. A consensus recommendation for patients with a thrombosis history is
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prophylactic treatment with low molecular weight heparin especially before surgery and during pregnancy. Because estrogen containing contraceptives may increase the risk of thrombosis, an alternative form of contraception should be recommended for these women during their reproductive years.

4.8.6

Psychosocial The impact on a patient and their family of a chronic disease such as -

thalassemia major that requires lifelong treatment is significant. Providing education regarding the inheritance patterns, the prenatal diagnostics options and the need for psychological support may help patients better manage their disease. However, based on the available evidence no specific therapy or combination of therapies can be recommended.44

4.8.7 Vitamin Deficiencies With the increase in erythropoesis some patients may develop folic acid deficiency. For these patients a 1 mg folic acid supplement daily is recommended. 34 However, patients receiving frequent transfusions rarely have this problem. While oxidative stress may contribute to the complications, the use of antioxidants has not improved the anemia nor reduced the morbidity or mortality of thalassemia. 34 If a transfusion dependent patient has proven vitamin C deficiency, supplements are recommended.

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4.8.8 Prognosis Beta thalassemia major patients live an average of 17 years and usually die by age 30. With regular blood transfusions and compliance with chelation therapy, their life can extend beyond age 40.6 Their deaths are commonly caused by cardiac complications of iron overload.28 Thalassemia trait patients have a normal life expectancy.

Sources of Additional Information: Cooley's Anemia Foundation http://www.cooleysanemia.org or http://www.thalassemia.org Thalassemia International Federation www.thalassaemia.org.cy

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Table 1 Hematologic Indices for Iron deficiency and thalassemias Indices Iron Deficiency -thalassemia -thalassemia trait MCV (abnormal <80 fl in adults; < 70 fl age 6 months 6 years; < 75 fl age 7 12 years) RDW (Adult normal 11.5 14.5%) High Normal Normal, occasionally high Ferritin (adult normal male 20 250 ng/ml; female 10 120 ng/ml) Mentzer Index Children (MCV/RBC count) Hb electrophoresis (Adult normals HbA - > 96% HbA2 2.5 -3.5% HbF - < 1%) Normal (may have reduced HbA2 before iron therapy is given) Adults : normal Newborns: cord blood may have HbH or Hb Bart Reduced HbA , increased HbA2, and increased HbF Reduced HbA, increased HbA2, and increased HbF > 13 < 13 < 13 < 13 Low Normal Normal Normal Normal, occasionally high Low Low Low -thalassemia major Low

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Table 2 Hb A2 levels with iron deficiency and thalassemia9 Diagnosis Normal Iron deficiency -thalassemia silent carrier or trait (minor) HbH disease -thalassemia trait (minor) -thalassemia major HbA2 level 2.5 3.5 % 1.6 3.2 % 2.0 3.2 % 1 2.4 % > 4.0 % > 4.0 %

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Table 3 Chelation Therapy Treatment Options Therapeutic agent Desferoxamine Route of administration Subcutaneous infusion over 8 - 12 hours Deferasirox Deferiprone (only available in the US through FDA Treatment Use Program) Oral Oral Adults - 30 50 mg/kg Children - 20 40 mg/kg 20 - 30mg/kg/day 75 100 mg/kg/day Once a day 3 times/day 5 7 days/week Dosage Frequency of therapy

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Figure 1 Beta thalassemia trait genetics

X Mother (-,) -thalassemia trait Father (-,) -thalassemia trait

Children: ( ,) Normal

(-,-) -thalassemia major or intermedia

(-,) -thalassemia trait

(-,) -thalassemia trait

Note: Shaded symbols indicate an abnormal -globin gene on chromosome 11.

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173

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References 1. Muncie HL Jr, Campbell J. Alpha and beta thalassemia. Am Fam Physician 2009; 80 (4): 339-344. 2. Mantikou E, Arkesteijn SG, Beckhoven JM, Kerkhoffs JL, Harteveld CL, Giordano PC. A brief review of newborn screening methods for hemoglobinopathies and preliminary results selecting beta thalassemia carriers at birth by quantitative estimation of the HbA fraction. Clinical Biochemistry 2009; 42(18): 1780-1785. 3. Cao A, Galanello R. Beta-thalassemia. Genetics in Medicine 2010; 12(2): 61-76. 4. Rund D, Rachmilewitz E. Beta-thalassemia. N Engl J Med. 2005; 353(11): 11351144. 5. Thalassemia International Federation. Thalassemia. Available at: http://www.thalassaemia.org.cy/index.html. Accessed 04/10, 2011. 6. Galanello R, Origa R. Beta-thalassemia. Orphanet J Rare Dis 2010; 5:11. 7. Richardson M. Microcytic anemia. Pediatr Rev 2007; 28 (1): 5-14. 8. Mosca A, Paleari R, Ivaldi G, Galanello R, Giordano PC. The role of haemoglobin A(2) testing in the diagnosis of thalassaemias and related haemoglobinopathies. J Clin Pathol 2009; 62:13-17. 9. Harteveld CL, Higgs DR. Alpha-thalassaemia. Orphanet J Rare Dis 2010; 5:13. 10. Galanello R, Cao A. Alpha-thalassemia. Genetics in Medicine 2011; 13 (2): 83-88. 11. Chen FE, Ooi C, Ha SY, et al. Genetic and clinical features of hemoglobin H disease in Chinese patients. N Engl J Med. 2000; 343(8): 544-550. 12. Leung TN, Lau TK, Chung TKH. Thalassaemia screening in pregnany. Curr Opin Obstet Gynecol 2005; 17 (2): 129-134. 13. Origa R, Sollaino MC, Giagu N, et al. Clinical and molecular analysis of haemoglobin H disease in Sardinia: Haematological, obstetric and cardiac aspects in patients with different genotypes. Br J Haematol 2007; 136(2): 326-332. 14. Mentzer WC,Jr. Differentiation of iron deficiency from thalassaemia trait. Lancet 1973; 1(7808): 882. 15. Flynn MM, Reppun TS, Bhagavan NV. Limitations of red blood cell distribution width (RDW) in evaluation of microcytosis. Am J Clin Pathol 1986; 85(4): 445-449. 16. Marsh WL Jr, Bishop JW, Darcy TP. Evaluation of red cell volume distribution width (RDW). Hematol Pathol 1987; 1(2): 117-123. 17. Guyatt GH, Oxman AD, Ali M, Willan A, McIlroy W, Patterson C. Laboratory diagnosis of iron-deficiency anemia: An overview. Journal of General Internal Medicine 1992; 7(2) : 145-153. 18. Kattamis C, Lagos P, Metaxotou-Mavromati A, Matsaniotis N. Serum iron and unsaturated iron-binding capacity in the -thalassaemia trait: their relation to the levels of haemoglobins A, A 2 , and F. J Med Genet 1972; 9(2): 154-159. 19. Van Delft P, Lenters E, Bakker-Verweij M, et al. Evaluating five dedicated automatic devices for haemoglobinopathy diagnostics in multi-ethnic populations. Int J Lab Hematol 2009; 31(5): 484-495. 20. Mosca A, Paleari R, Leone D, Ivaldi G. The relevance of hemoglobin F measurement in the diagnosis of thalassemias and related hemoglobinopathies. Clin Biochem 2009; 42(18): 1797-1801.
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21. Olivieri NF. The beta-thalassemias. N Engl J Med 1999; 341(2): 99-109. 22. Cazzola M, Borgna-Pignatti C, Locatelli F, Ponchio L, Beguin Y, De Stefano P. A moderate transfusion regimen may reduce iron loading in beta-thalassemia major without producing excessive expansion of erythropoiesis. Transfusion 1997; 37(2): 135-140. 23. Roberts DJ, Brunskill SJ, Doree C, Williams S, Howard J, Hyde CJ. Oral deferiprone for iron chelation in people with thalassaemia. Cochrane Database of Systematic Reviews 2007: 3: CD004839. 24. Angelucci E, Brittenham GM, McLaren CE, et al. Hepatic iron concentration and total body iron stores in thalassemia major. N Engl J Med 2000; 343(5): 327-331. 25. Delea TE, Edelsberg J, Sofrygin O, et al. Consequences and costs of noncompliance with iron chelation therapy in patients with transfusion-dependent thalassemia: a literature review. Transfusion 2007; 47(10): 1919-1929. 26. Deferasirox (exjade): A new iron chelator. Drugs Ther. Med Lett 2006; 48(1233): 35-36. 27. Parano E, Pavone V, Di Gregorio F, Pavone P, Trifiletti RR. Extraordinary intrathecal bone reaction in beta-thalassaemia intermedia. Lancet 1999; 354(9182): 922. 28. Modell B, Khan M, Darlison M. Survival in beta-thalassaemia major in the UK: Data from the UK Thalassaemia Register. Lancet 2000; 355(9220): 2051-2052. 29. Borgna-Pignatti C, Vergine G, Lombardo T, et al. Hepatocellular carcinoma in the thalassaemia syndromes. Br J Haematol 2004; 124(1): 114-117. 30. Eldor A, Rachmilewitz EA. The hypercoagulable state in thalassemia. Blood 2002; 99(1): 36-43. 31. Tso SC, Chan TK, Todd D. Venous thrombosis in haemoglobin H disease after splenectomy. Aust N Z J Med 1982; 126): 635-638. 32. Jensen CE, Tuck SM, Agnew JE, et al. High prevalence of low bone mass in thalassaemia major. Br J Haematol 1998; 103(4): 911-915. 33. Davies JM. Barnes R. Milligan D. British Committee for Standards in Haematology. Working Party of the Haematology/Oncology Task Force. Update of guidelines for the prevention and treatment of infection in patients with an absent or dysfunctional spleen. Clin Med 2002; 2(5): 440-443. 34. Borgna-Pignatti C. Modern treatment of thalassaemia intermedia. Br J Haematol 2007; 138(3): 291-304. 35. De Sanctis V. Growth and puberty and its management in thalassaemia. Horm Res. 2002; 58 Suppl 1: 72-79. 36. Gaudio A, Morabito N, Xourafa A, et al. Bisphosphonates in the treatment of thalassemia-associated osteoporosis. J Endocrinol Invest 2008; 31 (2): 181-184. 37. ACOG Practice Bulletin No. 78: hemoglobinopathies in pregnancy. ACOG Committee on Obstetrics. Obstetrics & Gynecology 2007; 109(1): 229-237. 38. Old JM. Screening and genetic diagnosis of haemoglobin disorders. Blood Rev. 2003;17(1): 43-53.

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39. Li Y, Di Naro E, Vitucci A, Zimmermann B, Holzgreve W, Hahn S. Detection of paternally inherited fetal point mutations for beta-thalassemia using sizefractionated cell-free DNA in maternal plasma. JAMA 2005; 293(7): 843-849. 40. Papasavva T, Kalakoutis G, Kalikas I, et al. Noninvasive prenatal diagnostic assay for the detection of beta-thalassemia. Ann N Y Acad Sci 2006; 1075: 148-153. 41. Braude P, Pickering S, Flinter F, Ogilvie CM. Preimplantation genetic diagnosis.[erratum appears in nat rev genet. 2003 feb;4(2):157.]. Nature Reviews Genetics. 2002;3:941-953. 42. Hoffbrand AV, Cohen A, Hershko C. Role of deferiprone in chelation therapy for transfusional iron overload. Blood 2003;102(1):17-24. 43. Brittenham GM, Cohen AR, McLaren CE, et al. Hepatic iron stores and plasma ferritin concentration in patients with sickle cell anemia and thalassemia major. Am J Hematol 1993; 42(1): 81-85. 44. Anie KA, Massaglia P. Psychological therapies for thalassaemia. Cochrane Database of Systematic Reviews 2001; 3: CD002890.

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Chapter 5
Neonatal Screening for Hemoglobinopathies
Zia Uddin, PhD With the technical support of Patrick Hopkins, Joseph Quashnock, Aigars Brants, Christine Moore, DAndra Morin, Rachel Lee, Mahin Azimi, and Bonifacio Dy

5.1 Introduction A gratifying achievement of my professional career was the organization of an interdisciplinary conference on Perinatal Care and Neonatal Screening in 1978 at South Macomb Hospital (now a part of St. John Providence Health System), Warren, Michigan.

CLINICAL CHEMISTRY, VOL 24, No. 7, 1978 Seven specialists eminent in the respective fields recently presented their research work to a group of obstetricians, gynecologists, pediatricians, pathologists, and clinical chemists from Michigan and Windsor (Canada) at a recent interdisciplinary conference in Warren, Michigan, sponsored by Detroit-Macomb Hospitals Association. Speakers and their topics were: Dr. Joseph Bieniarz, Amniocentesis in Perspective: Diagnostic Value of Ultrasonography and Protocol for Monitoring High Risk Pregnancy. Dr. Keith H. Marks, Elective Delivery of the Term Fetus: An Obstetrical Hazard. Dr. John. L. Kitzmiller, Management and Outcome of Pregnancy in Diabetes Mellitus. Dr. Norman F. Gant, Supine Hypertension Test and the Clinical Management of Pregnancy-Induced Hypertension. Dr. Thomas P. Foley, Neonatal Screening for Congenital Hypothyroidism and Clinical Treatment. Dr. Samuel Meites, Clinical Chemistry Laboratory in Neonatology. Mrs. Ann Bennett, Public Health and Neonatal Screening. For additional information write Zia Uddin, Ph.D., Perinatal Care & Neonatal Screening Conference, South Macomb Hospital, 11800 Twelve Mile Road, Warren, Mich. 48093.
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After this conference I started neonatal T4 screening for the four major hospitals in South Eastern Michigan. Subsequently, with the vision of the then Governor of Michigan (Honorable Mr. William Milliken), a law was passed for the establishment of a state of the art laboratory in Lansing, Michigan for the screening of inborn errors of metabolism and hemoglobinopathies. By statute, each state in the USA performs newborn screening (NBS), however, the number of tests/neonate and the methodologies utilized vary from state to state. In 2006, Honorable Senators Edward M. Kennedy, Barack H. Obama, and Hillary R. Clinton proposed a uniform standard and protocol of NBS in USA. An integral part of this proposal was to extend this program to resource-poor countries under the auspices of the United States Agency for International Development. Unfortunately, due to political events in USA and the death of Senator Edward M. Kennedy, nothing materialized in this direction. NBS in America always includes analysis for hemoglobinopathies as described by the Health Resources and Services Administration (HRSA) Maternal and Child Health Program of the U. S. Department of Health and Human Services . During the last twenty years, I had the opportunity to introduce NBS in a few developing countries (Kuwait, Iraq, Bahrain, Egypt, and Saudi Arabia) with the cooperation of Quest Diagnostics, USA and Laboratory Corporation of America, USA. PerkinElmer Genetics is the most popular private laboratory in USA that provides NBS services worldwide. Besides the popularity of PerkinElmer Genetics, several countries in Europe and Asia have instituted liquid chromatography-mass spectrometry (Applied
1

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Bioscience), high performance liquid chromatography (Bio-Rad and Trinity Biotech), and isoelectric focusing instruments (PerkinElmers Resolve, and Helenas SPIFE) for NBS.

5.2 Methodologies Isoelectric focusing (IEF) and high performance liquid chromatography (HPLC) are the two most commonly used screening methods for hemoglobinopathies in the neonate. Recently Sebia (Evry, France) has introduced the capillary zone electrophoresis (CZE) method (Neonate Hb Fast System) for the newborn screening of hemoglobinopathies. In order to resolve abnormal results of NBS,the blood of the biological parents (and sometimes of the siblings) are analyzed by IEF, HPLC, agarose gel electrophoresis (pH 8.6 and 6.2) and capillary zone electrophoresis to ascertain genetic inheritance of the abnormality in the neonate. Finally, the diagnosis is confirmed by means of DNA mutation studies,but for Hb S the final diagnosis can be confirmed by a Sickle test, and complete blood count (CBC) with manual differential. Further confirmation, if desired, for Hb S in a newborn can be achieved by testing the blood of the parents. Electrospray ionization-mass spectrometry (Chapter 3.4), PCR and Sanger sequencing (Chapter 3.5) are also used to confirm DNA mutation. A table of screening methods by individual states in America can be obtained via internet .
2

The principle of the assay by IEF (Chapter 2.7) and HPLC (Chapter 2.8) for the screening of hemoglobinopathies in the neonate and the adult are similar. However certain adjustments are required for the neonate specimen (dried blood spot on filter paper) handling and processing. The Resolve kit and instrumentation of the
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PerkinElmer and SPIFE 2000 or 3000 instrument of the Helena Laboratories are the most commonly used methods for the screening of hemoglobinopathies by IEF. HPLC is most commonly performed employing ion-exchange chromatography by the NBS instruments manufactured by BioRad, USA., or Trinity Biotechs Ultra Resolution System. The principle of the CZE for the screening of hemoglobinopathies (Chapter 2.6) in the adults and neonates is identical, except that modifications in the automated instrument are necessary for the handling of a dried blood sample on filter paper from the neonate (Figure 1). Sebia is the main supplier of capillary zone electrophoresis instrument and reagents for the NBS of hemoglobinopathies.

Figure 1. Automated hemolysis, sample dilution and analysis instrument (Sebia)

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The hemoglobin variants in the neonate by the Sebia Capillarys Neonat Fast System separated into windows or zones (N1-N13) as illustrated in section 5.4. This method has been evaluated
3-5

and found satisfactory, however as with

IEF and HPLC methodologies for NBS, results have to be considered provisional and confirmatory procedures are always required because many rare hemoglobin variants migrate or elute on the same position of the common one and because different homozygous or hemizygous genotypes look identical with these methods.

5.3 Laboratory Reports Format and Interpretation All the NBS laboratories in America have an elaborate management system for the procurement, handling and processing of dried blood on filter paper (Guthrie card). Additional facilities are provided for confirmatory testing and the counseling of the parents. Advisory consultative services and treatment modalities are also provided by the medical staff of the NBS facilities in America. The details of all these services are available online as well as in a printed version.

Normal patterns and abbreviations: A normal newborn typically displays about 70% Hb F and 20% Hb A and perhaps traces of Hb A2. The abbreviation used to indicate normal and abnormal patterns by the laboratories are:

FA

Fetal hemoglobin is greater than adult hemoglobin. This is observed in a newborn < 3 weeks of age.
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AF

Adult hemoglobin is greater than fetal hemoglobin. This is usually observed in a newborn > 3 weeks of age unless transfused within the last eight weeks and anemia is not suspected.

Abnormal patterns and abbreviations: Fa Lower Hb A levels then expected for the gestational age usually indicate a -thalassemia carrier that could have been born from a couple at risk (50% chance). Reporting these carriers will allow the couple to consider prospective primary prevention.

BFA? The presence of Hb Barts in a further normal pattern will indicate + -thalassemia (2-5% heterozygous), (5-10% + homozygous or 0 heterozygous), (>10% could indicate Hb H disease). Hb Barts in absence of Hb F indicates hydrops foetalis. FF Absence of Hb A may indicate a delayed appearance of Hb A (early prematurity), hereditary persistence of fetal hemoglobin (HPFH), or -thalassemia major. Hb F + Hb A and Hb S indicates heterozygous Hb AS trait (sickle cell trait). Patterns with only Hb F and Hb S may indicate homozygous Hb S or Hb S--thalassemia (both resulting in sickle cell disease).

FAS

FSS

FAC Hb F, Hb A and Hb C indicates heterozygous Hb AC trait. FCC Only Hb F and Hb C indicates either homozygous Hb C or Hb C--thalassemia. FSC Hb F + Hb S and Hb C indicates compound heterozygous Hb S and Hb C (sickle cell disease). Hb F, Hb A, and Hb E indicates heterozygous Hb AE trait. Hb F and Hb E only may indicate mild Hb E homozygosity or severe Hb E--thalassemia. Hb F, Hb S and Hb E indicates Hb S/E compound heterozygosity (sickle cell disease).
Punjab

FAE FEE

FSE

FAD Hb F, Hb A and Hb D indicates heterozygous Hb AD trait (an asymptomatic condition).

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FDD Only hb F and hb D indicates Hb D homozygous or Hb D-thalassemia (both mild conditions). FDS Hb F, Hb S and Hb D indicates compound heterozygous Hb SD (sickle cell disease).
Arab Arab

FAO Hb F, Hb A, and Hb O indicates heterozygous Hb A-O (an asymptomatic condition).

trait

FSS-Barts Homozygous Hb S or Hb S--thalassemia (severe sickle cell disease condition). Earlier, laboratories used the symbol X to designate a hemoglobin variant which could not be identified by the NBS laboratory and further testing was suggested. This practice of using X for a hemoglobin variant was abandoned by some laboratories and now the symbol V is also used for this purpose. It is emphasized here that some abnormal hemoglobin designated by V is often reported in newborn screening, because the screening laboratories do not have all the diagnostic methods available. In these situations the physician is advised to have definitive diagnostic testing done at a specialized laboratory, e.g. Georgia Health Sciences University Sickle Cell Center, Augusta, Georgia (http://www.georgiahealth.edu/centers/sicklecell). The interpretation of NBS in a premature neonate is subject to a possibility of false positive results , therefore the blood is retested when the adjusted gestational age is 40 weeks and two months after transfusion if executed.
6

5.4 Examples of Neonatal Screening

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In this section, selected cases are presented to illustrate the laboratory data obtained from NBS from commonly used methods.

5.4.1 Capillary Zone Electrophoresis Capillary zone electrophoresis (CZE) scans of the most commonly expected hemoglobin variants are presented in Figures 2-9. These scans were obtained after analyzing the dried blood spot on the Sebia Capillarys Neonat Fast System. The blood sample was collected by capillary puncture between 2-5 days after birth from neonates of gestational age > 38 weeks. We have also provided the percentage of major hemoglobin bands.

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5.4.2 Isoelectric Focusing Isoelectric focusing is the most widely used NBS method for hemoglobinopathies in America. Here again, confirmatory testing is desired for accurate diagnosis of any abnormal hemoglobin. One method of confirmation, if feasible, is to include the testing of the biological parents. In Figure 10, we have presented the IEF results (Hb SC disease) of a newborn, along with that of the father and mother of the newborn. The father has Hb AS trait, and the mother has Hb AC trait, therefore there is a 25% chance of the genetic inheritance of Hb SC disease in the newborn.

Figure 10. IEF results of newborn (Hb SC disease), father (Hb AS trait), and mother (Hb AC trait).

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5.4.3 Isoelectric Focusing and High Performance Liquid Chromatography Generally speaking, it is a common practice in some laboratories in USA to further evaluate the IEF results for abnormal cases by HPLC and vice versa. In Figures 11-17, we have presented the scans for both of these methods; for the normal and a few abnormal variants in a newborn. However, absolute certainty is never achieved by these two methods and DNA sequencing is the method of choice to confirm the variant and eventually the halotype to define the prognosis, tailor the best treatment and also to allow primary prevention in case of another pregnancy (Section 5.4.4). The IEF figures provided in this section were obtained using the RESOLVE neonatal hemoglobin test kit and testing equipment (PerkinElmer). In all the IEF Figures (11-17), we have presented at the top the IEF of the traditional laboratory control AFSC. Details about this procedure can be obtained from: http://www.perkinelmer.com/CMSResources/Images/44-72976FLY_Hemoglob_12449784.pdf The high performance liquid chromatography scans provided in this section were obtained using the Trinity Biotechs Ultra Resolution System. Details about this procedure and instrumentation can be obtained from: http://www.trinitybiotech.com/HbA1c_HB/Instruments/Pages/Ultra2Variant.aspx

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Isoelectric focusing

High performance liquid chromatography

Figure 11 Normal: FA IEF of normal phenotype displays three prominent bands, Hb F, Hb A, and acetylated Hb F. Hb F is the prominent band in newborns. Hb A (the middle band) in the IEF pattern often appeared weaker in premature babies compared to full term babies. In all the HPLC separations the prominent Hb peaks (i.e. with significant concentration) eluted at specific retention times.

Isoelectric focusing

High performance liquid chromatography

Figure 12 Hb AS trait FAS

Isoelectric focusing

High performance liquid chromatography

Figure 13 Hb AE trait FAE

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Isoelectric focusing

High performance liquid chromatography

Figure 14 Hb AC trait FAC

Isoelectric focusing

High performance liquid chromatography

Figure 15 Hb SC disease FSC

Isoelectric focusing

High performance liquid chromatography

Figure 16 Hb S disease FS

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Isoelectric focusing

High performance liquid chromatography

Figure 17 Hb Barts FABarts Note: A reviewer of this chapter pointed out the possibility that the fastest band on IEF is Hb H and the second is Hb Barts, as Hb H affected babies also have fast bands on IEF. Another reviewer suggested that Hb H affected babies have three fast bands on IEF, with a Barts result on HPLC exceeds 25%, and usually greater than 30%. This case displayed < 10% Barts from HPLC and displayed the typical two -banded Barts that is observed on one and two gene deletion alpha thalassemia carriers.

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5.4.4. Isoelectric focusing, High Performance Liquid Chromatography and DNA Studies In Figure 18, we present the typical IEF result of a full-term newborn with only Hb F and Hb S, and no Hb A. This pattern suggests in order of probability the following diagnostic options: a) Hb S homozygous (both genes code for Hb S, genotype associated with severe SCD). b) Hemizygous Hb S / -thalassemia (one gene codes for Hb S and the other is not active, genotype associated with severe SCD). c) Hemizygous Hb S / deletional HPFH (one gene codes for Hb S and the other is deleted, associated with mild SCD conditions). d) Double heterozygous Hb S / Hb Lepore (a combination associated with SCD) e) Double heterozygous Hb S-like / -thalassemia (a combination not associated with SCD). f) Double heterozygous Hb S / Hb S-like (the last migrating like Hb S but causing no SCD). g) Homozygous for the same Hb S-like variant heterozygous for two Hb S-like variants This means that even the simple SCD newborn pattern comes with different diagnostic options that have to be sorted out at the DNA level.

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Figure 18. IEF of newborn

Another example of a complex interpretation is shown in Figure 19. The HPLC pattern of the newborn shows 54% of Hb F with three additional and significant bands at a retention time known for a) Hb A at 0.87 minutes (1.3%), b) Hb E/A 2 at 1.04 minutes (8.3%) and c) Hb S at 1.2 minutes (6.3%). It is emphasized that a newborn cannot be assigned Hb A2 and Hb E was not detected by IEF. Therefore, the band in HPLC at 1.04 minutes is due to a hemoglobin variant to be defined at the molecular level.

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Figure 19. HPLC of newborn

DNA sequencing revealed Hb S heterozygous mutation at codon 6 and a second point mutation at codon 43 of the -globin gene leading to a GluAla amino acid substitution known as Hb G-Galveston.Therefore the newborn was diagnosed at the molecular level as compound heterozygous Hb S / Hb G-Galveston, a combination which is not associated with SCD. It is noteworthy to mention that like Hb G-Galveston
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elutes on HPLC at the position of the common Hb E, many other variants elute at the position of Hb S, Hb D, or Hb C and therefore molecular confirmation is always needed.

References (Section 5.1 5.4) 1. Lin K, Barton M. Screening for Hemoglobinopathies in Newborns. Reaffirmation Update for the U.S. Preventive Services Task Force. Evidence Synthesis No. 52. Rockville, MD: Agency for Healthcare Research and Quality, August 2007. AHRQ Publication No. 07-05104-EF-1. Available at http://www.ahrq.gov/clinic/serfiles.htm#sicklecell http://nnsis.uthscsa.edu/xreports.aspx?xreportID=47&formid=104&fclr=1 Giordano PG. Newborn screening for hemoglobinopathies using capillary electrophoresis. Methods Mol Biol 2013; 919: 131-45. Renom G, Mereau C, Maboudov P, Perini JM. Potential of the Sebia Capillarys neonat fast automated system for neonatal screening of sickle cell disease. Clin Chem Lab Med 2009; 47(11): 1423-32. Mantikou E, Harteveld CL, Giordano PC. Newborn screening for hemoglobinopathies using capillary electrophoresis technology: Testing the Capillarys Neonate Fast Hb device. Clin Biochem 2010; 43: 1345-1350. Hustace T, Fleisher JM, Varela AMS, Podda A, Alvarez O. Increased Prevalence of False Positive Hemoglobinopathy Newborn Screening in Premature Infants. Pediatric Blood Cancer 2011; 57: 1039-1043.

2. 3. 4.

5.

6.

References related to neonatal screening experience for hemoglobinopathies: Bouva MJ, Mohrmann K, Brinkman Henri BJM, Kemper-Proper EA, Elvers B, Loeber JG, Verheul Francesco EAM, Giordano PC. Implementing Neonatal screening for haemoglobinopathies in the Netherlands. J Med Screen 2010; 17: 58-65 Michlitsch J, Azimi M, Hoppe C, Walters MC, Lubin B, Lorey F, Vichinsky E. Newborn Screening for Hemoglobinopathies in California. Pediatr Blood Cancer 2009; 52: 486-490. Kafando E, Nacoulma E, Quattara Y, Ayeroue J, Cotton F, Sawadogo, Gulbis B. Neonatal haemoglobinopathy screening in Burkina Faso. J Clin Pathol 2009; 62: 39-41.
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Streetly A, Latinovic R, Hall K, Henthorn J. Implementation of universal newborn bloodspot screening for sickle cell disease and other clinically significant haemoglobinopathies in England: screening results for 2005-7. J Clin Pathol 2009; 62: 26-30. Gulbis B, Cotton F, Ferster A, Ketelslegers O, Dresse MF, Ronge-Collard E, Minon JM, Le PQ, Vertongen F. Neonatal haemoglobinopathy screening in Belgium. J Clin Pathol 2009; 62: 49-52. Bardakdjian-Michau J, Bahuau M, Hurtrel D, Godart C, Riou J, Mathis M, Goossens M. Neonatal screening for sickle cell disease in France. J Clin Pathol 2009; 62: 31-33. Adorno EV, Couto FD, de Moura Neto JP, Menezes JF, Rego M, dos Reis MG, Goncalves MS. Hemoglobinopathies in newborns from Salvador, Bahia, Northeast Brazil. Cad. Saude Publica, Ruio de Janeiro 2005; 21(1): 292-298.

5.5 Genetic Counseling & Screening: After a careful review of the literature on the worldwide prevalence of thalassemia and hemoglobinopathies, it is my estimate that by 2050 more than 500 million individuals will be affected by these genetic disorders. During the past two decades, attempts have been made to provide premarital and prenatal genetic counseling and screening in both the endemic and non-endemic (in view of migration) countries, however achieving a thalassemia-and hemoglobinopathy free generation seems unlikely to me. Although treatment modalities for sickle cell anemia have been investigated since 1967, including the latest promising treatment with antidepressants in these individuals by increasing the concentration of Hb F, permanent cure is illusive. Impediments for the worldwide implementation of a prevention and control program are: a) financial resources, b) technical personnel, c) religious and social considerations, d) education of the entire population about the benefits of this program, and e) poor and resource-lacking population problem.
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Indeed it is very promising that various religious organizations (Muslims and Jews) have authorized screening for genetic diseases after taking into consideration the halachic concerns. Country and state specific genetic counseling and screening programs (Thailand, Cyprus, etc.) are steps in the right direction, and let us hope that these initiatives blossom into an elaborate undertaking.

References 1. 2. 3. 4. 5. 6. 7. Jewish Womens Health. http://www.jewishwomenshealth.org/article.php?article=32 Strauss BS. Genetic Counseling for Thalassemia in the Islamic Republic of Iran. Perspectives in Biology and Medicine 2009; 52(3): 364-376 Larijani B, Anaraki FZ. Islamic principles and decision making in bioethics. Nature Genetics 2008; 40(2): 123. Norton ME. Genetic screening and counseling. Current Opinion in Obstetrics and Gynecology 2009, 20: 157-163. Zlotogora J. Population programs for the detection of couples at risk for severe monogenic genetic diseases. Hum Genet 2009; 126: 247-253. Al-Ama JY. Attitudes towards mandatory national premarital screening for hereditary hemolytic disorders. Health Policy 2010; 97: 32-37. Theodoridou S, Alemayehou M, Prappas N, Karakasidou O, Aletra V, Plata E, Tsaftaridis P, Karababa P, Boussiou M, Sinopoulou K, Hatzi A, Voskaridou E, Loutradi A, Manitsa A. Carrier Screening and Prenatal Diagnosis of Hemoglobinopathies. A Study of Indigenous and Immigrant Couples in Northern Greece, over the last 5 years. Hemoglobin 2008; 32(5): 434-439. Koren A, Zalman L, Palmor H, Zamir RB, Levin C, Openheim A, Daniel Spiegel E, Shalev S, Filon D. Sickle Cell Anemia in Northern Israel: Screening and Prevention. IMAJ 2008; 11: 229-234. Yamsri S, Sanchaisuriya K, Fucharoen G, Sae-ung N, Ratanasiri T, Fucharoen S. Prevention of severe thalassemia in northeast Thailand: 16 years of experience at a single university center. Prenat Diagn 2010; 30: 540546. Tarazi I, Al-Najjar E, Lulu N, Sirdah M. Obligatory premarital tests for thalassemia in the Gaza Strip: evaluation and recommendations. Int Jnl Lab Hem 2007; 29: 111-118.
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8.

9.

10.

11.

12.

13.

14.

Al-Allawi NA, Al-Dousky AA. Frequency of haemoglobinopathies at premarital health screening in Dohuk, Iraq: implications for a regional prevention programme. Eastern Mediterranean Health Journal 2010; 16(4): 381-385. Karimi M, Jamalian N, Yarmohammadi H, Askarnejad A, Afrasiabi A, Hashemi A. Premarital screening for -thalassemia in Southern Iran: opinions for improving the programme. Journal of Medical Screening 2007; 14(2): 6266. Al-Sulaiman A, Suliman A, Al-Mishari M, Al-Sawadi A, Owaidah TM. Knowledge And Attitude Toward The Hemoglobinopathies Premarital Screening Program in Saudi Arabia: Population Based Survey. Hemoglobin 2008; 32(6): 531-538. El-Tayeb E-N H, Yaqoob M, Abdur-Rahim K, Gustavson K-H. Prevalence of -Thalassemia and Sickle Cell Traits in Premarital Screening in Al-Qassim, Saudi Arabia. Genetic Counseling 2008; 19(2): 211-218.

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Chapter 6
Prenatal Diagnosis of -Thalassemias and Hemoglobinopathies
Maria Christina Rosatelli, PhD and Luisella Saba, PhD

Abstract Prenatal diagnosis of -thalassemia was accomplished for the first time in the 1970s by globin chain synthesis analysis on fetal blood obtained by placental aspiration at 18-22 weeks gestation. Since then, the molecular definition of the -globin gene pathology, the development of procedures of DNA analysis, and the introduction of chorionic villous sampling have dramatically improved prenatal diagnosis of this disease and of related disorders. Much information is now available about the molecular mechanisms of the diseases and the molecular testing is widespread. A prenatal diagnosis has to provide an accurate, safe and early result, an efficient screening of the population and a rapid molecular characterization of the couple at risk, are necessary prerequisites. In the last decades earlier and less invasive approaches for prenatal diagnosis were developed. An overview of the most promising procedure will be done. Moreover, in order to reduce the choice of interrupting the pregnancy in case of affected fetus, Preimplantation or Preconceptional Genetic Diagnosis (PGD) has been setting up for several diseases including thalassemias.

Rosatelli MC, Saba, L. Prenatal Diagnosis of Beta-Thalassemia and Hemoglobinopathies. Mediterr J Hematol Infec. Dis. 2009; 1(1): e200911 This can also be accessed from http://www.mjhid.org/article/view/5079.
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We acknowledge all those concerned with this publication.

Introduction -thalassemias and hemoglobinopathies are among the most common autosomal recessive diseases with a high frequency in the population of the Mediterranean area, the Middle East, the Indian subcontinent, the Far East, Tropical Africa and the Caribbean [1]. However, in the last decades, the steady migratory flows have rendered these pathologies much more widespread, thus representing a general public health problem. In the '70s the set-up of globin chain synthesis analysis for the detection of a little amount of -chains in fetal blood during the 18th-22nd week of gestation [2] has allowed the development of screening programs of the general population, based on the identification of the couple at risk, and, in addition, the offer of prenatal diagnosis testing. At that time the thalassemic patients had limited lifespan and prenatal diagnosis represented the only option for the control of the disease. Such programs first started in Sardinia, Continental Italy, Cyprus, and Greece [3,4,5,6]. Prenatal diagnosis on fetal blood, even if it represented for couples at-risk an opportunity to generate healthy sons, was not easily accepted. The late gestational age in which fetal diagnosis was carried out, the risk of misdiagnosis due to a not clear cutoff between some heterozygotes and affected fetuses, the high risk of miscarriage due to the sampling procedures, made indeed the procedure difficult to accept from the couples. The continuous advances in the knowledge of the molecular pathology of the disease, the discovery of restriction fragment length polymorphisms (RFLP) linked to
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the -like globin gene, the development of methodologies for mutation detection and the application of the villocentesis for the recovery of nucleated fetal cells, allowed a fast improvement both in feasibility and acceptability of prenatal diagnosis. For a short period, in the eighties, the diagnosis of thalassemia was obtained either indirectly by linkage analysis using RFLP at the -globin cluster [7] or directly by oligonucleotide hybridization on electro-phoretically separated DNA fragments [8] or by enzymatic digestion of mutated sites. A major impulse has been given by the PCR technology that allowed the development of a number of procedures, for easier mutation detection, as well as the development of both PGD and non-invasive prenatal diagnosis procedures. Nowadays thalassemias are detected directly by the analysis of amplified DNA from fetal trophoblast or, more rarely, from amniotic fluid cells. In this review we will delineate current procedures for prenatal and preimplantation diagnosis of thalassemias as well as the most promising approaches for non-invasive prenatal diagnosis.

Prenatal Diagnosis
Detection Methods: Detection of molecular defect in both parents is a prerequisite for prenatal diagnosis of the disease. The majority of defects affecting the -globin gene are point mutations that occur in critical areas for its function, or single/few base addition/deletion that change the frame in which triplets are translated into protein. Very rarely thalassemia results from gross rearrangement in the -globin gene cluster. In spite of the marked molecular heterogeneity, a limited number of molecular defects are
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prevalent in every at risk population. This may be very useful in practice, because a panel of most frequent mutations to be searched for can be designed according the carrier's ethnic origin [9]. Known mutation detection is caried out by a number of PCRbased techniques. (ARMS, Amplification Refractory Mutation System) and the reverse oligonucleotide hybridization with specific oligonucleotide probes (RDB, Reverse Oligonucleotide-probe analysis).

Primer-specific Amplification: The method is based on the principle that a primer carrying a mismatch in its 3' region cannot anneal on its template. With this method, the target DNA fragment is amplified in two separate PCR reactions using a common primer and either of the two following primers: one complimentary to the mutation to be detected ( -thalassemia primer) and one complementary, at the same position, to the normal DNA (normal primer). Normal DNA is amplified only by the normal primer while the DNA from homozygotes only by the -thalassemia primer and DNA from heterozygotes by both primers. A different sized fragment of the -globin gene is simultaneously co-amplified as an internal control of the PCR reaction [10]. The method is very simple as it requires, for each mutation to be searched, only two PCR reactions followed by agarose gel electrophoresis. A further improvement of the methodology can be obtained by multiplexing the primers for more than one mutation. In good hands the method is very safe and particularity useful in fetal DNA analysis to search for mutations previously detected in the parents.

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Reverse Oligonucleotide Hybridization: When the spectrum of mutations to be searched is complex, ARMS is not the most appropriate method. In this case RDB results can be more informative and efficient. The method uses membrane-bound allele-specific oligonucleotide probes that hybridize to the complementary sequence of the PCR product prepared using patient DNA as starting template [11]. In this format, multiple pairs of normal and mutant allelespecific oligonucleotides can be placed on a small strip of membrane. Hybridization with PCR-amplified -globin gene is able to detect, in a single procedure, any of the mutations screened. Up to 20-30 mutations have indeed been screened in one single step and several commercial kits are available to detect the most common beta thalassemia mutations in Mediterranean population.

Other Known Mutation-detection Procedures: Several other methods have been developed to search for known mutations, i.e. oligonucleotide ligation assay [12], restriction enzyme digestion of PCR products [13]; however some of them have been abandoned in routine diagnostics as they are less informative or more complex. In recent years a real time PCR assay has been successfully applied to both carriers screening and prenatal diagnosis [14]. This is a one-step method that is based on the use of fluorescent hybridization probes followed by a melting curve analysis. This method, which allows the simultaneous multiple mutation detection, has been
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successfully applied also to the detection of maternal contamination. In spite of these advantages its use is still limited as it needs a dedicated apparatus as well as an accurate population-based design of detection probes. Technically, we can realistically predict further simplification and full automation of the procedures for the detection of the -thalassemia mutations is commercially available, which are not completely automated and quite expensive. Among them, the oligonucleotide microchip-based assays have been proposed many times for the largescale detection of mutations in genetic diseases, including -thalassemia [15]. Given the alternative features of high throughput and automation, the DNA chip has the potential to become a valuable method in future applications of mutation detection in medicine. At the moment, the technology developed several years ago is not yet transferred in the clinical practice, due to the higher costs and to the lower analytical sensitivity and specificity.

Unknown Mutations Detection: When carriers escape to the above mutation detection approaches, further investigations need to be carried out by alternative methods which uncover the presence of unknown mutations by scanning the whole gene. Denaturing gradient gel electrophoresis (DGGE) [16,17,18], Denaturing High Pressure Liquid Chromatography (dHPLC) and Single Strand Conformation Polymorphism (SSCP) [19] are the most widely used in the last years, followed by direct sequencing analysis [20] which characterizes the undefined mutation found by these methods. Nowadays, considering the small size of the -globin gene (1,8kb), the simplified technologies available and the
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reduced costs of analysis, direct sequencing, based on cycle sequencing with fluorescent dye terminators and automated capillary DNA sequencing technology, seems to be the faster and most useful approach to detect unknown thalassemia mutations. If a mutation is not detected by sequence analysis, we search for the presence of small deletions by polyacrylamide gel electrophoresis of amplicons designed for the most frequent small deletional defects of the -globin gene (gap PCR). Furthermore, the presence of larger deletions of the cluster may be identified by Southern-blotting or more recently by Multiple Ligation-Dependent Probe Amplification (MLPA) for which a commercial kit is available (SALSA MLPA KIT P102 HBB-MRC Holland). In a very limited number of cases, direct sequencing from position -600 to 60 bp downstream from the -globin gene and methods for deletion detection, failed to detect the disease causing defect. In these cases, the molecular defect may reside either in the locus control region of the -globin gene cluster, or in one of the genes, outside the -globin gene region, encoding for regulatory proteins acting in trans on the function of the -globin gene. Very recently it has been proved that the -thalassemia-like phenotype could be caused by the coinheritance of a -globin gene defect and a duplication of the -globin gene cluster, which results in an excess of chain. In these selected cases, the characterization of these -globin gene rearrangements (SALSA MLPA KIT P140-B2 HBA-MRC Holland) can be routinely carried out with success by MLPA analysis.

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Genetic Counseling of the Couple at Risk: Both members of the couple at risk are counseled in a non-directive way. The nature of the disease, the implications of being carriers and reproductive choices are analyzed, specifically those concerning birth control, including prenatal or preimplantation diagnosis and the possibility, in case of affected fetus HLA compatible to not interrupt pregnancy. As for fetal testing, detailed information is offered regarding the risk of fetal mortality, the risk of misdiagnosis, and the mortality and morbidity of an abortion in case of affected fetus.

Fetal DNA Sampling: Fetal DNA for analysis can be obtained from either amniocytes or chorionic villi. At present the most widely used procedure is chorionic villi sampling, because of the clear advantage of being carried out during the first trimester of pregnancy, generally at the 10th-12th week of gestation [21, 22, and 23]. The risk of fetal mortality associated with both methods is in the order of 1-2%. Chorionic villi may be obtained transcervically or transabdominally, the last being most widely used, mainly because it has a low infection rate and a lower incidence of amniotic fluid leakage. Moreover it is a simple procedure, largely preferred by pregnant women, which can be carried out also in late gestational age. Samples obtained by villocentesis need to be accurately dissected under inverted microscope in order to remove maternal decidua, that represent the major cause of diagnostic error in prenatal diagnosis of monogenic diseases.
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Fetal DNA Analysis: Fetal DNA is analyzed using the same methods described above for detection of known mutations during carrier molecular screening. To limit the possibility of misdiagnosis, we analyze chorionic villous DNA with two different procedures: i.e. RDB hybridization and primer-specific amplification, using distinct couple primers. Misdiagnosis may occur for several reasons: failure to amplify one copy of the target DNA fragment, mispaternity, maternal contamination, and sample exchange. Misdiagnosis for failure of DNA amplification is obviously limited by the double approach described above. To avoid misdiagnosis due to maternal contamination as well as mispaternity and/or sample exchange, a fetal DNA microsatellite analysis is usually performed to verify the presence of one allele from each parent [9]. In our hands, by the above mentioned PCR-based procedures, no misdiagnoses have occurred in more than 5000 cases. Figure 1 shows the overall results of the Sardinia prenatal diagnosis program since the beginning of 1976 up to the end of the past year.

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Currently, prenatal diagnosis is a widely applied and well-accepted procedure. Among the patients screened we have found an acceptability of 99.3% for early prenatal diagnosis by CVS. This data, if compared with previously utilized procedures such as fetal blood sampling, with an acceptability of 93.2%, and 96.4% by amniocentesis, demonstrates how the acceptance of the procedure depends on its precocity [22]. The screening program in the Mediterranean countries has proven to be very successful in reducing the number of thalassemia patients. In Sardinia, thalassemia major was present in 1 in 250 births, and has declined to 1 in 4000 births (Figure 1). Other countries in which such thalassemia programs have been introduced also show similar trends.

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Preimplantation and Preconceptional Genetic Diagnosis: The progress in assisted reproduction and molecular genetics techniques, particularly the advent of PCR that has made possible to analyze the genotype of a single cell, has paved the way for preimplantation genetic diagnosis (PGD) [24,25]. This technique was introduced as an option for avoiding the decision to terminate an established pregnancy diagnosed as affected by conventional approaches. The term preimplantation genetic diagnosis describes those procedures which involve the removal of one or more nuclei from oocytes (polar bodies) or embryos (blastomeres of trophectoderm cells) to test for mutation in the target gene or aneuploidy before transfer. PGD requires that couples at risk undergo in vitro fertilization (IVF) even if not infertile and for this reason a multidisciplinary approach including an appropriate genetic counseling and the referral to both a fertility clinic and to a highly specialized molecular genetics laboratory is mandatory. Counseling for couples considering PGD must include additional information regarding at least the risk associated with IVF procedures and with embryo biopsy, the technical limitations of DNA analysis, including the risk of failure of the procedure as well as that of misdiagnosis, and the need of subsequent prenatal diagnosis to confirm the result. Beyond that, the possibility that no embryos may be transferred and the dispositions of the embryos not transferred have also to be seriously considered.

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Cell Biopsy: Preimplantation may be carried out by either cleavage-stage biopsy of 1-2 blastomeres, from an eight-cell embryo three days after in vitro fertilization carried out by ICSI (Intracytoplasmatic Sperm Injection), or by the biopsy of polar bodies. For cleavage-stage biopsy the embryo is grown in vitro until it reaches a six-eight cell stage which usually occurs on the third day after insemination. Polar bodies diagnosis, pioneered by Verlinsky and his group in 2006 is based on the analysis of the first polar body of unfertilized eggs [27], and may lead to distinguish between unfertilized eggs that carry the defective gene and those without the defect. The successive sampling and analysis of the second polar body that is extruded from the oocyte after fertilization and completion of the second meiotic division, is carried out in order to avoid misdiagnosis due to the high rate of recombination that happens during the first meiosis. By fertilizing in vitro only the eggs without the defect and replacing them in the mother, a successful pregnancy with a normal fetus can be obtained. Recently a preconceptional genetic diagnosis based on the analysis of only the first polar body has been proposed for countries in which the use of PGD and manipulation of embryos is prohibited [28]. This approach although permitting to avoid the manipulation, cryopreservation and/or discard of sovranumerary and/or affected embryos, shows several problems: the need to obtain more than 10-12 oocytes, the increased risk of diagnostic error and the increased risk of the technical difficulties. Blastocyst biopsy, even if it has the advantage to provide a higher number of cells, is at present more rarely used because of the difficulties of the embryos to reach this stage in IVF programs. The cleavage-stage
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biopsy of blastomeres from an eight-cell embryo is the most frequently used PGD procedure all over the world.

Detection Methods: Methods for mutation detection in OGD are always based on multiple steps of PCR. Mutations are detected in PCR products by various methods that combine speed, analytical sensitivity and specificity. In particular, a first round of multiplex PCR is performed to amplify both the -globin gene region including the mutation and one or more polymorphic loci. Secondly, two separated nested PCR reactions are performed to amplify the two or more selected genomic regions. Finally, the polymorphic alleles are directly detected by capillary electrophoresis of the amplified fragment, while the presence of -globin gene mutations are identified by the subsequent mini sequencing reaction [29]. This approach is expressly designed to detect the presence of the globin gene mutations and to monitor, in the same sample, the presence of contamination as well as the eventual allele drop-out that represent the most frequent causes of error in PGD.

Quality Control: For both techniques a prenatal diagnosis by villocentesis is recommended in order to avoid diagnostic errors. Successful pregnancies following the transfer of human embryos in which the -globin gene defect has been excluded, occur only in 2025% of cases and the birth rate of a child is even lower. Due to the low birth rate most women have to undergo PGD several times in order to give birth to a healthy child [30].
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Transfer of no more than 1-2 embryos is strongly recommended in order to avoid multiple pregnancies [31]. Elective Single Embryo Transfer (eSET) is in fact a wellestablished procedure which has demonstrated to ensure a better prognosis of IVF patients [32}.

PD or PGD? Among clinical geneticists there has been much discussion about the main goal of PD. Some have argued that the main aim is to avoid the birth of an affected child. Others have emphasized the reproductive confidence and the purpose of informing the couples at risk about the status of the fetus. Several studies indicate that if there is no PD option, a large proportion (up to 50%) of the couples at high risk of an affected child refrain from pregnancy despite their wish to reproduce. When PD is possible many more at-risk couples dare to embark on a pregnancy. Most experts consider PGD as an additional option for couples at risk and not as a replacement for conventional prenatal diagnosis. PGD is still considered a highly specialized experimental procedure with limited results, mainly dedicated to couples against abortion for ethical and religious reasons and to a small proportion of couples who have experienced repeated abortion, that ask for referral for this procedure. At present its use in routine monitoring of pregnancies at risk is precluded by the technical demand for these procedures, the difficulty in organizing the service, and the high costs.

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Simplification of preimplantation and preconception genetic diagnosis, together with an increase in the pregnancy rate may lead to a more extensive use of the procedure in the future.

Non-Invasive Prenatal Diagnosis (NIPD):

Analysis of Fetal Cells in Maternal Blood: In the sections below the most significant studies, which have been carried out in this field of research, are briefly summarized. The mot relevant results have been grouped in three different sections, according to the different cell type in which they have been acquired. A separate section is dedicated to NIPD of -thalassemia. Trophoblasts: The first evidence that fetal cells circulate in maternal peripheral blood dates back to 1893 when George Schmorl observed the presence of placentally derived trophoblasts in the lungs of 17 autopsied women affected by severe eclampsia [33]. In 1959 Douglas [34] established that migration of trophoblasts is a normal process during pregnancy and twenty-five years later, Covone et al [35] demonstrated that these cells could be detected in healthy pregnant women as early as six weeks gestation. They also found that an increased concentration of trophoblast cells were frequently presenting in women affected by preeclampsia. Further studies have established that trophoblasts are entrapped in the maternal lungs and rapidly removed from the pulmonary circulation [36].

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Tropoblast-specific cell-surface antigens have not yet been characterized and several experimental evidences have shown that the H315, initially described as the specific antigen for trophoblasts, is indeed absorbed in maternal leucocytes [37]. These are some of the reasons why, in recent years, trophoblasts are no longer considered as the best target cells for non-invasive prenatal diagnosis. Nevertheless, this line of research has not yet been completely abandoned as the characterization of trophoblast-specific antigens is one of the objectives of the SAFE (Special Non-Invasive Advances in Fetal and Neonatal Evaluation) Network (for more information please visit www.safenoe.org). Lymphocyte: Fetal lymphocytes are the second cell type which has been extensively studied as a possible source of fetal DNA. In 1969 Walknowska et al [38] detected for the first time 46, XY karyotype cells in maternal peripheral blood of women bearing male fetuses. Ten years later Herzenberg and colleagues described the use of FACS (Fluorescent Activated Cell Sorting) as a method for the enrichment of fetal lymphocyte expressing the HLA-A2 paternal antigen [39]. Detection of Y chromosome was then obtained in the enriched cells deposited directly onto microscope slides, thus confirming their fetal origin. Unfortunately other groups have failed to replicate these results with success, even when cytogenetic analysis was carried out in fetal cells that were flow sorted on the basis of several HLA differences and by using monoclonal antibodies. In the same years further studies demonstrated that lymphocytes were not removed from maternal circulation after delivery. One of the earliest studies provided
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the first evidences that fetal lymphocytes persist in maternal circulation one year after delivery was published in 1974 by Bianchi et al [40]. Several years later Bianchi et al described the presence of fetal progenitor cells 27 years after delivery [41]. For these reasons also lymphocytes, as trophoblasts, became an unattractive candidate for noninvasive prenatal diagnosis. Erythroblasts: One of the main advantages to study fetal erythroid cells is that they are nucleated, terminally differentiated short-lived cells and for this reason they do not persist in maternal circulation for a long time after delivery. Furthermore, first primitive erythroblasts appear in the embryonic bloodstream around the four-five week gestations so they can be detected early during gestation. Nevertheless, their isolation from maternal peripheral blood is still problematic because of their rarity and the lack of a fetal specific antibody. In 1990 Bianchi [41] first described a method for fetal nucleated erythroid cells CD71 transferrin receptor, highly expressed in erythroid cells. Two years later Ganshirt-Ahlert et al [43] obtained similar results by using a new detection system called MACS (Magnetic Cell Sorting) which is based on the use of antibodies labeled with magnetic beads. Since then, both systems have been extensively improved and used, by several groups, following different approaches which can consist in the positive selection of CD71 and/or glycophorin-A fetal cells and/or in the negative depletion of CD45 maternal cells. Usually, in both cases, a previous density (Ficoll or Histopaque) gradient centrifugation step is carried out to remove non-nucleated maternal erythrocytes. A
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schematic workflow resuming one of the strategies used for isolating fetal NRBCs from maternal peripheral blood is represented in Figure 2. Finally both MACS and FACS sorted cells are labeled with fluorescent antibodies which recognize embryonic (, ) or fetal () hemoglobin chains and are eventually subjected to FISH analysis for chromosome Y detection. An example of positive labeling with the antibody for gammaglobin conjugated with FITC is shown in Figure 3. Molecular characterization can eventually be carried out in positive fluorescent cells isolated by laser microdissection. Even with the high progress made in the last twenty years in this field, the methods for erythroblasts enrichment are still limited as they mostly result in the recovery of fetal samples with low yield (FACS) and scarce purity (MACS), being variably contaminated by maternal cells. For these reasons in recent years several studies have been addressed to the proteomic field with the attempt to characterize novel fetal erythroblast cell-specific surface markers. For example, bi-dimensional electrophoresis coupled with mass spectrometry has allowed the identification of 2 proteins, differentially expressed in sickle erythrocytes in comparison to healthy erythrocytes, and the detection of proteins up-or downregulated in fetal erythroid cells in comparison to their adult counterparts. Some of these results have been published as a full-patent application and the data concerning the new antibodies developed against these new targets expect to be validated in large samples of maternal blood [44]. In addition, further developments in

fetal cell recovery are expected to be obtained through the application of micro-fluidic rare-cell capture technologies [45] which are being developed to detect not only fetal but also cancer as well as other rare cells in biologic fluids.
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Analysis of Fetal Cells in Maternal Blood and Non Invasive Prenatal Diagnosis (NIPD) of -Thalassemia: Despite the difficulties encountered to find the best target cell and the best method for their enrichment and isolation, several attempts have been made in the last twenty years, to transfer the results of these researches into clinical practice. Unfortunately the lack of reproducibility of experiments hardly makes the isolation of fetal cells from maternal blood as a first choice method of NIPD of monogenic disorders. Below the most significant results obtained in NIPD of -thalassemia are briefly summarized. The first example of non invasive prenatal diagnosis of hemoglobinopathies was described in 1990 by Camaschella et al [46]. The genetic test was carried out in three selected couples where the mother was a carrier of thalassemia and the father of the Hb Lepore-Boston trait. The absence/presence of the paternal trait was successfully detected in PCR amplified samples DNA extracted from T-cell samples were obtained by incubating Ficoll-separated cells of the mother with the CD 3-specific MoAb Leu 4 and then separating the positive cells with goat-anti-mouse immunoglobulin G (1gG)-coated immunomagnetic beads. In those years most of the studies were addressed to couples carrying different mutations and only aimed to the exclusion of the paternal allele in the enriched fetal cells, as most of the times they were contaminated from maternal cells. In subsequent years, even if the fetal cells enrichment and selection methods have been greatly improved, other IP diagnosis have been carried out but with fluctuating results. Below are described three significant examples of NIPD realized, with different levels of success, by using single or pooled erythroid cells.
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In 1996 the group of Y.W.Kan47 reported the successful identification of two fetal genotypes by using fetal nucleated erythroid cells selected by MACS, anti- globin immunostaining and then isolated by microscopy and cell scraping. The presence/absence of sickle cell and beta thalassemia mutations of both parents were finally detected by Reverse Dot Blot in PCR amplified samples constituted by pools of fetal dissected cells. A few years later the group of Di Naro [48] replicated these results using a slightly different procedure for erythroblast enrichment which was carried out by Percoll and Gastrografin multiple gradient centrifugation. Mutation detection was then obtained by automated sequencing of single cells amplified by PCR. According to authors, even if the risk of allele drop out is higher when amplifying single cells, however the possibility to study several individual, instead of pooled, cells guarantees an accurate diagnosis of the fetal DNA. More recently the group of Kolialexi [49] has hardly tried to replicate these results. In this study, NIPD was performed through magnetic cell sorting (MACS) and microdissection of single NRBCs with a laser micromanipulation system. Single-cell genotyping was achieved by nested real-time PCR for genotyping -globin gene mutations; a multiplexed minifingerprinting was used to confirm the origin of the isolated cells and to exclude their possible contamination. A total of 224 cells were isolated but only half of them were successfully amplified. In the majority (n=80) of these cells minifingerprinting was not informative because of allele dropout or homozygosity. In the rest of the samples, 22 cells resulted to be of fetal origin, 26 maternal while 80 were non informative.
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Analysis of Fetal DNA in Maternal Plasma and Non Invasive Prenatal Diagnosis (NIPD) of Thalassemia: The existence of cell-free nucleic acids within the human plasma was firstly reported in 1948 by Mendel and Metais [50] which described their presence both in normal subjects and in individuals affected by various diseases. Some decades later other studies have confirmed the presence of circulating DNA as well as of RNA in several pathological conditions (pancreatitis, inflammatory diseases, cancer, diabetes, etc) [51]. In 1997 Lo et al discovered for the first time that a fetus may release cell-free fetal DNA (cffDNA) into maternal plasma, thus providing an alternative to fetal cells for noninvasive prenatal diagnosis [52]. In recent years more information has been acquired about the concentration, the origin and the characteristics of the cell-free fetal DNA and several procedures have been developed in order to use it in prenatal diagnosis. The cell-free DNA is constantly present in peripheral blood of non pregnant women and its concentration increases during pregnancy. The cell-free fetal DNA represents the 3-5% of the DNA present in maternal plasma from which, after delivery, it is rapidly cleared. Recent studies carried out by microfluidic digital PCR have revealed that cffDNA can be present at even higher concentrations which can reach up to 10-20% of total DNA in maternal plasma [53]. Nevertheless, because of the high background of maternal DNA, an enrichment step is needed to obtain highly purified fetal DNA samples suitable for non invasive prenatal diagnosis.
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Size-fractionation agarose gel electro-phoresis is one of the methods developed for fetal DNA enrichment and consists in the isolation of short-length DNA fragments (<300 bp of length) which is the medium length of the cffDNA. This method coupled with the peptide-nucleic-acid clamp (PNA) PCR, which selectively suppresses the amplification of maternal alleles, and with the Allele-specific Real-Time PCR for mutation detection, has been used with success by Li et al [54] to detect mutations of paternal origin in fetuses at risk for -thalassemia. More recently [55] the same group has described a new procedure, still based on size fractionation method, but coupled with MALDI-TOF mass-spectrometry, a mediumthroughput platform which detects with high sensitivity the presence of known and unknown point mutations. In this case no suppression of maternal allele was caried out and the molecular diagnosis was addressed to the exclusion of the paternal mutated allele. The analysis by MALDI-TOF preceded by size fractionation has given improved results, in comparison to the absence of enrichment, in the detection of the codon 39 thalassemia paternal allele. Nevertheless, for eventual future diagnostic application the protocol needs to be validated in larger samples, even if the high cost of the instrumentation required makes this platform difficult to apply in routine diagnostics and the size fractionation is considered an enrichment method more susceptible to maternal contamination. The use of peptide-nucleic-acid clamping to suppress the amplification of normal maternal alleles was first described by Cremonesi in 2004 [56]. Peptide nucleic acid is artificially synthesized polymers similar to nucleic acids and able to hybridize DNA sequences. The PNA/DNA hybrids are more stable than equivalent DNA/DNA hybrids
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but less stable in the presence of single-pair mismatches. In that paper their ability to clamp wild type -globin sequences was proved in artificial mixture of DNA samples enriched with increased amounts of wild type alleles, by using a microchip platform to detect the -thalassemia mutations. Four years later [56] the efficacy of PNA was evaluated with success in 41 non invasive prenatal diagnosis of -thalassemia and in combination with three different techniques (microelectronic chip, pyro sequencing and direct sequencing) to detect fetal DNA mutations in maternal plasma. Despite its successful application, this strategy, as the other above described technologies, is still restricted to couples which carry different mutated alleles and aimed to the detection of mutated paternal alleles. Another method recently described for NIPD of -thalassemia is called APEX namely Arrayed Primer Extension. This is a mutation detection system which is based on the combined use of the microchip technology and the single nucleotide base extension method. This system has been recently described by the group of Papasavva [57] and used to characterize the presence of the paternal -thalassemia mutations and associated -globin gene SNPs, in cffDNA isolated from maternal plasma. The possibility to study the polymorphisms associated to the mutated alleles represent a feature of great value since it would give the possibility to extend NPID to couples which carry the same mutated allele. Prerequisite for its application is to find informative SNPs associated with parental mutations which can help to discriminate the paternal mutated allele and to characterize the haplotype inherited from the fetus. The authors of the paper described the correct application of this methodology in the NIPD
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of six out of seven couples at risk for -thalassemia, carried out in the Cypriot population. Future Perspective: As previously reported, one of the major problems which still limits the application of the described protocols in clinical practice is the impossibility to obtain highly purified fetal, cellular as well as cffDNA, samples which could allow the detection of parental alleles, even when they are identical. Few clinical applications of NIPD are actually restricted to the detection of the Y chromosome, for fetal sex determination, or the Rhesus D gene, in Rhesus D negative women, or, in general, of genetic loci which are absent in the maternal genome. In recent years a great improvement has been obtained in the field of the technologies which can explore the presence of sequence variations even in single molecules of DNA. The concept of "Digital PCR" was firstly introduced in 1992 by Sykes [58] who described a method to determine the number of starting DNA templates by doing Poisson statistical analysis of PCR results obtained in limiting dilutions. The more recent development of the emulsion PCR (emPCR) have further enhanced the possibility to study single molecules of DNA by using a small volume of reactions, water-oil emulsions and microfluidic as well as high-throughput platforms (for a review of both methods and application to NIPD please see Zimmermann et al [59]. Recent applications of these technologies in the field of NIPD, and in particular in the diagnosis of aneuploidies and monogenic disorders, have shown that these methodologies may find useful application in the near future, even if several drawbacks

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need to be solved and wider validation studies should be carried out before transferring their use in routine diagnostics.

References
1. Wetherall DJ, Clegg JB. The Thalassemia Syndromes. Wiley-Blackwell; 2008. 2. Kan YW, Golbus MS, Klein P, Dozy AM. Successful application of prenatal diagnosis in a pregnancy at risk for homozygous -thalassemia. New Engl J of Med. 1975; 292:1096-9. [PubMed] 3. Cao A. Results of programs for antenatal detection of thalassemia in reducing the incidence of the disorder. Bool Rev. 1987; 1:169-176. [PubMed] 4. Cao A, Rosatelli MC, Galanello R. Control of -thalassemia by carrier screening, genetic counseling and prenatal diagnosis: the Sardinian experience Variation in the human genome. Chirchester, England: Wiley; 1996. pp 137-155. Ciba Foundation Symposium 197. 5. Loukopulos D. Current states of thalassemia and sickle cell syndromes in Greece. Semin Hematol. 1996; 33: 76-86. [PubMed] 6. Angastiniotis M, Kyriakidou S, Hadjiminas M. The Cyprus Thalassemia Control Program, White Plains. Vol.23. New York: March of Dimes Birth Defects Foundation; 1988. pp.417-432. Original Article Series. [PubMed] 7. Kan YW, Lee KY, Furbetta M, Angius A, Cao A. Polymorphism of DNA sequence in New Engl J of Med. 1980; 302: 185-8. [PubMed] 8. Pirastu M, Kan YW, Cao A, Conner BJ, Teplitz RL, Wallace RB. Prenatal diagnosis of -thalassemia. Detection of a single nucleotide mutation in DNA. New Engl J of Med. 1983; 309: 284-7. [PubMed] 9. Rosatelli MC, Tuveri T, Scalas MT, Leoni GB, Sardu R, Faa V, Meloni A, Pischedda MA, Demurtas M, Monni G, et al. Molecular screening and fetal diagnosis of thalassemia in the Italian population. Hum Genet. 1992; 83: 590-2. [PubMed] 10. Newton CR, Graham A, Hepteinstall Le, Powell SJ, Summers C, Kalsheker N, Smith JC, Markham AF. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS) Nucleic Acid Research. 1989; 17:2503-16. [PMC free article] [PubMed} 11. Saiki RK, Walsh PS, Levenson CH, Erlich HA. Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes. Proc. Natl. Acad. Sci (USA) 1989; 86:6230-4. [PMC free article] [PubMed] 12. Nickerson DA, Kaiser R, Lappin S, Stewart J, Hood L, Landegren U. Automated DNA diagnostic using an Elisa-based oligonucleotide ligation Assay. Proc. Natl. Acad. Sci (USA) 1990; 87: 8923-7. [PMC free article] [PubMed]

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28. Fiorentino F, Biricik A, Nuccitelli A, De Palma R, Kahraman S, Sertyel S, Karadayi H, Cottone G, Baldi M, Caserta D, Moscarini M. Rapid protocol for pre-conception genetic diagnosis of single gene mutations by first polar body analysis: a possible solution for the Italian patients. Prenat Diagn. 2008 Jan; 28: 62-4. [PubMed] 29. Monni G, Cau G, Usai V, Perra G, Lai R, Ibba G, Faa V, Incani F, Rosatelli MC. Preimplantation genetic diagnosis for beta-thalassemia: the Sardinian experience. Prenat Diagn. 2004 Dec 15; 24(12): 949-54. [PubMed] 30. Goossens V, Harton G, Moutou C, traeger-Synodinos J, Van Rij M, Harper JC. ESHRE PGD Consortium data collection IX: cycles from January to December 2006 with pregnancy follow-up to October 2007. Hum reprod. 2009 Aug; 24: 1786810. [PubMed] 31. Cutting R, Morroll D, Roberts SA, Pickering S, Rutherford A, BFS and ACE Elective single embryo transfer: guidelines for practice British Fertility Society and Association of Clinical Embryologists. Hum Fertil (Camb) 2008 Sep; 11: 131-46. [PubMed] 32. Schmorl G. Pathologisch-anatomische Untersuchungen ueber Publer eklampsie. Vogel; Leipzig: 1893. 33. Douglas GW, Thomas L, Carr M, Cullen NM, Morris R. Trophoblasts in the circulating blood during pregnancy. Am J Obstet Gynecol. 1959 Nov; 78: 960-73. [PubMed] 34. Covone AE, Johnson PM, Mutton D, Adinolfi M. Trophoblast cells in peripheral blood of pregnant women. Lancet. 1984; ii: 841-843. [PubMed] 35. Attwood HD, Park WW. Embolism to the lungs by trophoblasts. J. Obstet. Gynaecol. Br. Commonw. 1960; 68: 611-617. [PubMed] 36. Bertero Mt, Camaschella C, Serra a, Bergui L, Caligaris-Cappio F. Circulating, trophoblast cells in pregnancy have maternal genetic markers. Prenat Diagn. 1988; 8: 585-90. [PubMed] 37. Walknoska J, Conte FA, Grumbach MM. Practical and theoretical implication of fetal/maternal lymphocyte transfer. Lancet. 1969; i: 1119-1122. [PubMed] 38. Herzenberg LA, Bianchi DW, Schroder J, Cann HM, Iverson GM. Fetal cells in the blood of pregnant women: detection and enrichment by fluorescence activated cell sorting. Proc. Natl. Acad. Sci USA. 1979; 76: 1453-1455. [PMC free article] [PubMed] 39. Schroder J, Tilikainen A, de la Chapell A. Fetal leucocytes in maternal circulation after delivery. Transplantation. 1974; 17: 346-360. [PubMed] 40. Bianchi DW, Zickwolf GK, Weil GJ, Sylvester S, DeMaria MA. Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum. Proc. Natl. Acad. Sci. USA. 1996; 93: 705-708. [PMC free article] [PubMed] 41. Bianchi DW, Flint AF, Pizzimenti M, Knoll JH, Latt SA. Isolation of fetal DNA from nucleated erythrocytes in maternal blood. Proc. Natl. Acad. Sci. USA. 1990; 87: 3279-3283. [PMC free article] [PubMed] 42. Ganshirt-Ahlert D, Burschyk M, Garritsen HSP, Helmer L, Miny P, Horst J, Schneider HP, Holzgreve W. Magnetic cell sorting and the transferrin receptor as potential means of prenatal diagnosis from maternal blood. Am. J. Obstet. Gynecol. 1992; 166: 1350-1355. [PubMed]
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43. Avent ND, Plummer ZE, Madgett TE, Maddocks DG, Soothill PW. Post-genomics studies and their application to non-invasive prenatal diagnosis. Semin Fetal Neonatal Med. 2008; 13:91-8. Review. [PubMed] 44. Huang R, Barber TA, Schmidt MA, Tompkins RG, Toner M, Bianchi DW, Kapur R, Flejter WL. A microfluidics approach for the isolation of nucleated red blood cells (NRBCs) from the peripheral blood of pregnant women. Prenat Diagn. 2008 Oct; 28: 892-9. [PubMed] 45. Camaschella C, Alfarano A, Gottardi E, Travi M, Primignani P, Caligaris Cappio F, Saglio G. Prenatal diagnosis of fetal hemoglobin Lepore-Boston disease on maternal peripheral blood. Blood. 1990; 75: 2102-2106. [PubMed] 46. Cheung MC, Goldberg JD, Kan YW. Prenatal diagnosis of sickle cell anemia and thalassemia by analysis of fetal cells in maternal blood. Nat Genet. 1996 Nov; 14: 264-8. [PubMed] 47. Di Naro E, Ghezzi F, Vitucci A, Tannoia N, Campanale D, D'Addario V, Holzgreve W, Hahn S. Prenatal diagnosis of beta-thalassemia using fetal erythroblasts enriched from maternal blood by a novel gradient. Mol Hum Reprod. 2000 Jun; 6: 571-4. [PubMed] 48. Kolialexi A, Vrettou C, Traeger-Synodinos J, Burgemeister R, Papantoniou N, Kanavakis E, Antsaklis A, Mavrou A. Noninvasive prenatal diagnosis of betathalassemia using individual fetal erythroblasts isolated from maternal blood after enrichment. Prenat Diagn. 2007 Dec; 27: 1228-32. [PubMed] 49. Mendel P, Metais P. Les acides nucleiques du plasma sanguine chez l'homme. C. R. Acad. Sci. Paris. 1948; 142: 241-243. 50. Gahan PB, Swaminathan R. Circulating nucleic acids in plasma and serum. Recent developments. Ann N Y Acad. Sci. 2008 Aug; 1137: 1-6. [PubMed] 51. Lo YM, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, Wainscoat JS. Presence of fetal DNA in maternal plasma and serum. Lancet. 1997 Aug 16; 350: 485-487. [PubMed] 52. Microfluidics digital PCR reveals a higher than expected fraction of fetal DNA in maternal plasma. Lun FM, Chiu RW, Allen Chan KC, Yeung Leung T, Kin Lau T, Dennis Lo YM. Clin Chem. 2008 Oct; 54: 1664-72. [PubMed] 53. Detection of paternally inherited fetal point mutations for beta-thalassemia using size-fractionated cell-free DNA in maternal plasma. Li Y, Di Naro E, Vittuci A, Zimmerman B, Holzgreve W, Hahn S. JAMA. 2005 Feb 16; 293: 843-9. [PubMed] 54. Li Y, Di Naro E, Vitucci A, Grill S, Zhong XY, Holzgreve W, Hahn S. Size Fractionation of Cell-Free DNA in Maternal Plasma Improves the Detection of a Paternally Inherited beta-Thalassemia Point Mutation by MALDI-TOF Mass Spectrometry. Fetal Diagn Ther. 2009 Jun 5; 2: 246-249. [PubMed] 55. Cremonesi L, Galbiati S, Foglieni B, Smid M, Gambini D, Ferrari A, Viora E, Campogrande M, Pagliano M, Travi M, Piga A, Restagno G, Ferrari M. Feasibility study for a microchip-based approach for noninvasive prenatal diagnosis of genetic diseases. Ann N Y Acad. Sci. 2004 Jun; 1022: 105-12. [PubMed]

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56. Galbiati S, Foglieni B, Travi M, Curcio C, Restagno G, Sbaiz L, Smid M, Pasi F, Ferrari A, Ferrari M, Cremonesi L. Peptide-nucleic acid-mediated enriched polymerase chain reaction as a key point for noninvasive prenatal diagnosis of beta-thalassemia. Haematologica. 2008 Apr; 93: 610-4. [PubMed] 57. Papasavva T, Kalikas I, Kyrri A, Kleanthous M. Arrayed primer extension for the noninvasive prenatal diagnosis of beta-thalassemia based on detection of single nucleotide polymorphisms. Ann N Y Acad. Sci. 2008 Aug; 1137: 302-8. [PubMed] 58. Sykes PJ, Neoh SH, Brisco MJ, Hughes E, Condon J, Morley AA. () Quantitation of targets for PCR by use of limiting dilution. Biotechniques. 1992; 13: 444-449. [PubMed] 59. Zimmermann BG, Grill S, Holzgreve W, Zhong XY, Jackson LG, Hahn S. Digital PCR: a powerful new tool for noninvasive prenatal diagnosis? Prenat Diagn. 2008 Dec; 28: 1087-93. [PubMed]

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Chapter 7
Hemoglobin A1c
Zia Uddin, PhD

7.1 Introduction The term glycated hemoglobin refers to the non-enzymatic, irreversible, covalent bonding of glucose at one or both N-terminal valine residues of the hemoglobin -chain, and the N-terminus of the -chains, or the -amino groups of lysine residues (Figure 1).

Figure 1. Non-enzymatic glycation of hemoglobin

The term normal hemoglobin phenotype beyond the neonatal period involves a major fraction due to Hb A (22), and a minor fraction of Hb A2 (22). Occasionally a very minor fraction of Hb F (22) is also detected. Further chromatographic analysis of
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Hb A showed that it contains a number of minor hemoglobins, e.g., Hb A1a1, Hb A1a2, Hb A1b and Hb A1c.These four minor fractions of Hb A were collectively referred as 1) Hb A1, 2) fast hemoglobins, 3) glycosylated hemoglobins, 4) glycated hemoglobins , or 5) glycohemoglobins. To provide more consistency in nomenclature the Joint Commission on Biochemical Nomenclature of the International Union of Pure and Applied Chemistry has recommended the term glycated hemoglobin instead of the above mentioned five names used in the literature. The Hb A1a1 and Hb A1a2 fractions that are covalently bonded to Glucose-6phosphate account for 10% of the glycated hemoglobin. In Hb A 1b the N-terminus of the -chain is covalently bonded to pyruvic acid instead of glucose molecule, and this also accounts for 10% of the glycated hemoglobin. Hb A1c is a specific species of glycated hemoglobin resulting from covalent
1 bonding of glucose to the N-terminal valine of the hemoglobin -chain. Hb A1c

accounts for 80% of the glycated hemoglobin, and more importantly it is the only portion of the glycated hemoglobin that is elevated in diabetes. Since hemoglobin remains in the red blood cell during its entire life span ( 120 days), the constantly changing glucose level in the cell will directly effect the formation of Hb A 1c. Therefore, the measurement of Hb A1c is directly proportional to the time averaged glucose levels.

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2 The fast moving forms of Hb A were separated in the late 1950s and recognized as 3

being associated with diabetes in the late 1960s

. Since Hb A1a1, Hb A1a2 and Hb A1b

are not elevated in diabetes, the clinical focus has been solely on Hb A 1c. For clinical testing purposes the term Hb A1c analysis is referred to as A1c or the A1c test with the word hemoglobin omitted as a matter of convenience.

7.2 Hb A1c Diagnostic Role in Diabetes Mellitus, and Glycemic Control in Adults After three decades of investigation and evaluation of numerous proposals by various scientific and clinical organizations, Hb A1c found its status as a diagnostic test for diabetes mellitus. One of the four criteria for the diagnosis of diabetes mellitus are:
4

Hb A1c > or = 6.5% (48 mmol/mL) Fasting plasma glucose > or = 126 mg/dL (7.0 mmol/L) 2-h plasma glucose > or = 200 mg/dL (11.1 mmol/L) during an Oral Glucose Tolerance Test Symptoms of hyperglycemia and casual plasma glucose > or = 200 mg/dL (11.1 mmol/L)

Several investigators have recently suggested the combined use of fasting glucose and Hb A1c for the diagnosis of diabetes mellitus.
5-7

Beyond diagnosis, modification of medical

treatment for diabetics is now being performed based on the laboratory test results of Hb A 1c.

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One objective would be to get glucose levels to as close to normal as possible with minimal or no hypoglycemia. American Diabetic Association (ADA) has suggested the lowering of Hb A 1c < 7% for non-pregnant adults for reducing microvascular, and neuropathic complications of the disease (type I and II). Recently a follow up study of the ACCORD (Action to Control Cardiovascular Risk in Diabetes) stipulated that the best target of Hb A1c in middle aged or older patients with cardiovascular risk factors is between 7.0 and 7.9%. Hb A1c is widely used to judge the treatment of diabetes and adjustment of the medication dose when necessary. In chronic glycemia the blood glucose is monitored more frequently (once a day or more). Since Hb A1c is measured less frequently and in percent, and is a complicated process to explain to the patient, it is convenient for the physician to relate the result to glucose concentration (in mg/dL or mmol/L) over the preceding 5-12 weeks. This derived glucose concentration from Hb A1c value is called Estimated Average Value (eAG). Patients monitor their blood glucose and their physician can relate that performance to the eAG. This way the patients can see the effect of their behavior over time on the test outcome. The only way this feat could be accomplished, if the result for Hb A1c be the same no matter where the result was run. This simple feat required the cooperation of many government agencies and all Hb A1c laboratory testing manufacturing facilities and was brought about by the determination of the Diabetes Control and Complication Trial (DCCT) Research Group and the American Diabetes Association.
8 9

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The mathematical relationship then developed between HbA1c and eAG is based on the following linear regression equation. eAG (mg/dL)
10

= (28.7 x Hb A1c %) - 46.7

eAG (mmol/L) = (1.59 x Hb A1c %) - 2.59 Table 1 provides the National Glycohemoglobin Standardization Program (NGSP) Values
11

of Hb A1c % and its corresponding eAG.

Table 1. NGSP (Hb A1c%) 5 6 7 8 9 10 11 12 eAG(mg/dL) 97 126 154 183 212 240 269 298 eAG (mmol/L) 5.4 7.0 8.6 10.2 11.8 13.4 14.9 16.5

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7.3 Measurement of Hb A1c Currently over 100 different methods are available for quantification of Hb A1c. Most available Hb A1c methods are certified by the NGSP the following techniques:
12

and are based on one of

Immunoassay Boronate affinity binding/HPLC Ion-exchange HPLC Capillary zone electrophoresis Enzymatic

Measurement of Hb A1c was recently reviewed (December 12, 2012) by David B. Sacks.
13

(also available on line)

http://care.diabetesjournals.org/content/35/12/2674.full)). It is encouraging to note that most of the commercial diagnostic manufacturers for Hb A1c test kits are now attempting to provide an acceptable Hb A1c for eAG calculation.

7.4

Factors Affecting the Accuracy of Hb A1c Assay In spite of the efficacy of Hb A1c in the diagnosis and the management of

diabetes (type I and II), several factors influence the accuracy of its laboratory results, e.g., a) hemolytic disease or other conditions with reduced red blood cell survival, b) recent blood loss, c) iron deficiency anemia, d) patients with renal failure, and e) hemoglobin variants. All these interferences cannot be easily delineated by the
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laboratory personnel and the physician. Due to the diluvial of methods, reagents, and instruments for the assay of Hb A1c , it is impossible for the laboratory to be aware of the methods limitation with respect to the presumptive interference by >1000 hemoglobin variants reported so far in the literature (http://globin.cse.psu.edu). In the case of the most common hemoglobinoathies (AS, AE, AC, AD), Hb A1c can be accurately measured if the correct method is used. The affect of these hemoglobin variants (AS, AE, AC, AD) and elevated Hb F in HPFH (not pathological) on the results of Hb A1c by the most often used methods is presented in Table 2.

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239

Table 2. Hb A1c methods: Effects of Hemoglobin Variants (Hb C, Hb S, Hb E and Hb D traits) and Elevated Fetal Hemoglobin (Hb F). Updated March 2013. (with the permission of http://www.ngsp.org/interf.asp). The methods are listed in an alphabetic order of manufacturers name. The criteria used to determine whether or not a method shows interference that is clinically significant (indicated by Yes) is > + or 7% at 6 and/or 9% Hb A1c. In the absence of data for a specific method (designated by @), it can generally be assumed that immunoassay methods do not have clinically significant interference from Hb E and Hb D because the E and D substitutions are distant from the N-terminus of the hemoglobin -chain. In the absence of data for a specific method (designated by $), it can generally be assumed that both immunoassay and boronate affinity methods show interference from Hb F levels above 10-15%.

In situations where Hb A1c cannot be reliably measured, an alternative is the assay of serum frustosamine. Fructosamine is the generic name for plasma protein ketoamines and is also known as glycated serum protein (GSP). Frustosamine provides
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evaluation of glucose status over a short period of time (2-3 weeks rather than months). Several studies have shown a correlation of Hb A1c with fructosamine and was thus recommended in patients with hemoglobinopathies.
14

References 1. Sacks DB. Carbohydrates. In Burtis CA, Ashwood ER, eds. Tietz Fundamentals of Clinical Chemistry. 5th Ed. St. Louis: W.B. Saunders 2011; 452-457. Allen DW, Schroeder WA, Balog J. Observations on the chromatographic Heterogeneity of Normal Adult and Fetal Human Hemoglobin: A Study of the Effects of Crystallization and Chromatography on the Heterogeneity and Isoleucine Content. Am J Chem Soc 1958; 80: 1628-34. Rahbar S. An abnormal hemoglobin in red cells of diabetics. Clin Chim Acta 1968; 22: 296-98 Sacks DB, Arnold M, Bakris GL, Bruns DE, Horvath AR, Kirkman MS, Lernmark A, Metzger BE, Nathan DM. Guidelines and Recommendations for Laboratory Analyzers in the Diagnosis and Management of Diabetes Mellitus. Clin Chem 2011; 57: 793-798. Inzucchi SE. Diagnosis of Diabetes. N Engl J Med 2012; 367: 542-50. Hu Y, Kiu W, et al. Combined use of fasting plasma glucose and glycated hemoglobin A1c in the screening of diabetes and impaired glucose tolerance. Acta Diabetol 2010; 47: 231-36. Heianza Y, Hara S, Arase Y, et al. Hb A1c 5.7-6.4% and impaired fasting plasma glucose for diagnosis of prediabetes and risk of progression to diabetes in Japan (TOPICS 3): a longitudinal study. Lancet 2011; 378: 147-55. American Diabetic Association Clinical Practice Recommendations: Executive Summary: Standard Methods of Care in Diabetes-2010. Diabetes Care 2010: 33, suppl. 1: S4-5. Gerstein HC, Miller ME, Genuth S, et al. ACCORD Study Group. Long term effects of intensive glucose lowering on cardiovascular outcomes. N Engl J Med 2011; 364 (9): 818-828. Nathan DM, Kuenen J, Borg R, Zheng H, Schoenfeld D, Heine RJ, and for the A1c-Derived Average Glucose (ADAG) Study Group. Translating the A1c Assay into Estimated Average Glucose Values. Diabetes Care 2008; 31: 1473-1478.
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2.

3. 4.

5. 6.

7.

8.

9.

10.

11.

12. 13. 14.

Nathan DM, Kuenen J, Borg R, Zheng H, Schoenfeld D, Heine RJ, FOR THE A1c-Derived Average Glucose (ADAG) Study Group. Translating the A1c Assay into Estimated Average Glucose Values. Diabetes Care 2008; 31: 1-6. List of NGSP Certified Methods-Hb A1c (updated 11/2012). http:/www.ngsp.org Sacks DB, Measurement of Hemoglobin A1c. A new twist on the path to harmony. Diabetes Care 2012; 35 (12): 2674-2680. http://labtestsonline.org/understanding/analytes/fructosamine/tab/test.

Additional references (not quoted above) concerning hemoglobin variant interference in the assay of Hb A1c. i) ii) Sofronescu A-G, Williams LM, Andrews DM, Zhu Y. Unexpected Hemoglobin A1c Results. Clin Chem 2011; 57:2, 153-157 Selvin E, Steffes MW, Ballantyne CM, Hoogeveen RC, Coresh J, Brancati FL. Racial Differences in Glycemic Markers: A cross-sectional Analysis of Community-Based Data. Ann Inter Med 2011; 154: 303-309 Bergman A-C, Beshara S, Byman I, Karim R, Landin B. A New -Chain Variant: Hb Stockholm [7(A4)GluAsp] Causes Falsely Low A 1c. Hemoglobin 2009; 33(2): 137-142 Williams JP, Jackson H, Green BN. Hb Belleville [10(a&)AlaThr] Affects the Determination of HbA1c by Routine Cation Exchange High Performance Liquid Chromatography. Hemoglonin 2009; 33(1): 45-50. + Zhu Y, Williams LM. Falsely elevated hemoglobin A1c due to S-beta thalassemia interference in Bio-Rad Variant II Turbo HbA1c assay. Clin Chem Acta 2009; 409(1-2): 18-20. Thevarajah M, Nadzimah MN, Chew YY. Interference of hemoglobin A1c (HbA1c) detection using ion-exchange high performance liquid chromatography (HPLC) method by clinically silent hemoglobin variant in University Malaya Medical Center (UMMC)- A case report. Clin Biochem 2009; 42: 430-434. Mongia SK, Little RR, Rohlfing CL, Hanson S, Roberts RF, Owen WE, DCosta MA, Reyes CA, Luzzi VI, Roberts WL. Effects of Hemoglobin C and S on the Results of 14 Commercial Glycated Hemoglobin Assays. Am J Clin Pathol 2008; 130: 136-140. Barakat O, Krishnan STM, Dhatariya K. Falsely low HbA1c value due to a rare variant of hemoglobin J-Baltimore. Primary Care Diabetes 2008; 2: 155-157.

iii)

iv)

v)

vi)

vii)

viii)

242

ix)

x)

xi)

Little RR, Rohlfing CL, Hanson S, Connolly S, Higgins T, Weykamp CW, DCosta M, Luzzi V, Owen WE, Roberts WL. Effects of Hemoglobin (Hb) E and HbD Traits on Measurements of Glycated Hb (HbA1c) by 23 Methods. Clin Chem 2008; 54:8, 1277-1282. Lee S-T, Weykamp CW, Lee Y-W, Kim J-W, Ki C-S. Effects of 7 Hemoglobin Variants on the Measurement of Glycohemoglobin by 14 Analytical Methods. Clin Chem 2007; 53(12): 2202-2205. Roberts WL. Hemoglobin Constant Spring can interfere with Glycated Hemoglobin Measurements by boronate Affinity Chromatography. Clin Chem 2007; 53(1): 142-43.

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Case Studies
Introduction The following case (# 1-28) studies include laboratory data representing results from five different hemoglobin separation methods commonly used in the clinical laboratory. Due to the large number of variants possible the mandate is that more than one separation method be used in identification. The question is which two methods would provide discriminative information. The results of the lab tests for each case are presented in a tabular form to assist in these choices.

The alkaline electrophoresis images are of Helena SPIFE Alkaline Electrophoretic results but identical separation results would have also been obtained using alkaline cellulose acetate, Helena Biosciences SAS alkaline hemoglobin gels, Helena Quick Gels or Sebia Hydrasys alkaline hemoglobin gels.

The acid electrophoretic images are of Helena SPIFE or QUICK Gel Acid electrophoresis. For Acid electrophoretic separation, two classes of media have been used with differing separation results. Historically, acid hemoglobin separation was done on agar using citric acid buffer. Helena SPIFE and Quick Gels are of this type. Agarose purified from agar has more recently been used by Beckman, Sebia and Helena BioSciences. The purified nature of the agarose makes these products easier to produce but historically they lacked easily available documentation of the differences in mobilities compared to the

244

historically used agar. These differences have been documented in the table associated with the attached case studies. All acid agarose data were adopted from (Variant Hemoglobins. a Guide to Identification. 1st edition, by Barbara J. Bain , Barbara J. Wild , Adrian D. Stephens, Lorraine A. Phelan . Published 2010 by Wiley-Blackwell Publishing Ltd).

All Capillary Zone Electrophoresis (CZE) data were generated using the Sebia Capillarys System. This CZE system separates hemoglobins into 15 zones and provides a list of possible variants that migrate in that zone. The operator then selects the hemoglobin variant they expect that peak to represent. The peaks in the CZE reports in the case studies have been labeled in such a fashion but a different assignment could have been made by the operator had they had information warranting the choice. Details of other vendor results would require contact with the vendor but the goal again would be to maintain equality as close as possible and the assumption would be that the order of separation would not be different.

All isoelectric focusing images are actual or simulated from actual data obtained with the Helena Isoelectric Focusing Gels either on the SPIFE or the REP systems. The Perkin Elmer Resolve (formerly IsoLab) isoelectric focusing systems would obtain the same results, because the pH range of the ampholytes are the same. These agarose gels contain acrylamide to sharpen the bands.
245

Again the end user is the final discriminator. In this case, proper selection of the controls determines the degree of discrimination possible instead of the number of Zones available. If there is Hb S control and the variant migrates anodal to Hb S, the variant might be Hb D or Hb G but you may not reliably report which, even though they are both anodal to Hb S. You only know it is not Hb S. If the control is Hb D or Hb G then you may report based on the migration compared to that control. The High Performance Liquid Chromatography (HPLC) separations were all obtained using BioRad Variant information from several sources.

This data for cases 1-28 is the cooperative effort of many institutions. Hemoglobin screening is done on neonates as well as adults. Sometimes data from these rarer hemoglobin variants may include Hb F at low levels that is ignored in the discussion because its presence is to be expected due to the patients age. In this regard, some discrepancy in the data may appear. The presence of an alpha chain variant on a newborn can be complicated by this temporary presence of gamma chains. The gamma chains compete for the variant alpha chains as well as the normal resulting in two gamma alpha possibilities. In neonates the Hb A2 is barely visible because delta chain production is just beginning. If sufficient delta chains are expressed they also would show a competition for alpha bands resulting in Hb A2 and a smaller alpha variant band. These complications will be discussed in the cases in which they are encountered.
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Case # 1

Normal Adult

71 years old male, recent medical examination showed no abnormality.

Laboratory Data:
Hemoglobin Hematocrit RBC MCV MCH MCHC RDW Platelet Hb A Hb A2 Hb F Peripheral Blood Smear: 13.5 39.6 4.7 84.3 28.8 34.1 13.5 200 98.0 1.8 0.2 No abnormality was detected. 13.5 -18.5 g/dL 38.0 - 54.0 % 3 4.6 - 6.2 Mil/mm 80 - 100 fL 27 - 34 pg 31 - 36% 11.5 - 14.5% 3 150 - 400 Th/mm 94.3 - 98.5% 1.5 - 3.7% 0.0 - 2.0%

Agarose Gel Electrophoresis (pH 8.6)

247

Case # 1 Normal Adult

Citrate Agar Electrophoresis (pH 6.2)

Isoelectric focusing

248

Case # 1 Normal Adult

Capillary zone electrophoresis

High performance liquid chromatography

249

Case # 1 Normal Adult

Interpretation & Discussion:

Agarose gel electrophoresis at alkaline pH 8.6 showed a major band (98%) in the position of Hb A, and a very faint band of Hb A2 (1.8%) in the position of Hb C. Another very faint band detected cathodal to Hb A2 is due to the enzyme carbonic anhydrase. This carbonic anhydrase band is mostly detected in fresh specimens of blood. Acid electrophoresis at pH 6.2 does not separate Hb A from Hb A 2 , therefore only one major band is shown in the position of Hb A. A smudged band cathodal to Hb A includes Hb F, and modified forms of Hb A such as HbA1c.

Isoelectric focusing showed a major band in the position of Hb A, and a very faint band in the position of Hb A2. A smudged minor band anodal to Hb A represents modified Hb A such as acetylated Hb F, denatured Hb A, and Hb A1c.

From capillary zone electrophoresis a major peak of Hb A was detected in window Z9, and a minor peak due to Hb A2 was present in window Z3. No other peaks were observed.

High performance liquid chromatography showed a major peak at a retention time of 2.42 (peak value) ascribed to Hb A and a minor peak due to Hb A2 at a retention time of 3.64 (peak value). There are 2-3 minor peaks before Hb A and after Hb F, and these peaks represent Hb A1c fraction besides fractions of other hemoglobins.
250

The term normal hemoglobin phenotype beyond the neonatal period involves a major band due to Hb A (22), and a minor band of Hb A2 (22). Occasionally a very faint band of Hb F (22) is also detected. By definition the concentration of both the Hb A2 and Hb F must be in the normal range for that method regardless of the methodology used.

Reference
Bain BJ. Hemoglobin and the genetics of hemoglobin synthesis: In: Haemoglobinopathy Diagnosis, Blackwell Publishing, second edition, 2006, pp 12-22.

251

Case # 2 Hemoglobin S trait

A 20 year old female African-American pre-nursing student in a local community college was screened for hemoglobinopathy by her family physician. Her physical examination and chemistry profile were normal.

Laboratory Data:
Hemoglobin Hematocrit RBC MCV MCH MCHC RDW Platelet Hb A Hb S Hb A2 Hb F 13.1 39.6 4.4 82.1 27.3 32.1 12.6 267 59.2 38.4 1.8 0.6 12.0 16.0 g/dL 35.0 - 48.0 % 3 4.0 5.5 Mil/mm 79-98 fL 26-34 pg 31-36% 11.5-14.5% 3 150-400 Th/mm 94.3-98.5% 1.5 -3.7% 0.0-2.0%

Peripheral Blood Smear: No abnormality was present. Solubility test for Hb S was positive.

Agarose Gel Electrophoresis (pH 8.6)

252

Case # 2 Hemoglobin S trait

Citrate Agar Electrophoresis (pH 6.2)

Isoelectric focusing

253

Case # 2 Hemoglobin S trait

Capillary zone electrophoresis

High performance liquid chromatography

254

Case # 2 Hemoglobin S trait

Interpretation & Discussion

Summary of Results Method Alk Agarose Hb A area Major band (Hb A) Major band (Hb A+ Hb A2) Major peak (Hb A) Zone 9 Major band (Hb A) Major peak (Hb A) RT=2.34 Hb S area Major band (Hb S) Major band (Hb S) Major peak (Hb S) Zone 5 Major band (Hb S) Major peak (Hb S) RT=4.26 Minor peak (Hb A2) Zone 3 Minor band (Hb A2) Minor peak (Hb A2) RT=3.65 Hb A2/C area Minor band (Hb A2)

Acid Agar/Agarose

CZE

IEF

HPLC

Since the solubility test was positive and the aberrant band fell between 35 40%, a diagnosis of Hb S trait was made. Concentrations of Hb S other than 35 40% require consideration of the effect of a transfusion, the possibility of iron deficiency, a concurrent Hb S--thalassemia (Hb S < 33%), a Hb S-thalassemia (Hb S >49%) or the possibility that the fraction may not be Hb S at all. Mutation at the 6th amino acid position of the chain [6 (A3) GluVal) causes the substitution of glutamic acid by valine that results in the formation of Hb S.
255

Since one negative charge is reduced by this mutation, Hb S migrates slower than Hb A in alkaline and acid electrophoretic procedures. There are other Hb variants that migrate in the position of Hb S in alkaline electrophoresis, but not in acid. Use of Acid electrophoresis eliminates all the other common hemoglobin variants that migrate in the Hb S alkaline area or by CZE, IEF or HPLC. Other identification methods do exist.

Individuals with Hb S should be advised that it is almost a benign and innocuous condition . However, there are exceptions and in some individuals: hematuria and aseptic necrosis of bone has been reported. If the hematuria persists for a long time and is profuse, then the possibility of bladder cancer by cystoscopy and bladder cancer markers must be evaluated.
3 2

Recently, a new sickling hemoglobin (Hb S-San Martin) was reported from an Argentinean family. Besides the usual -globin chain mutation associated with sickle cell [6(A3)GluVal, (GAGGTG)], an additional mutation on the same -globin chain [105 (G7) LeuPro (CTCCCC) ] was confirmed by the DNA studies. The electrophoretic mobility of Hb S-San Martin at both the alkaline pH (8.6) and acid pH (6.2) was identical with the Hb S. This is a rare occurrence and only ten (10) hemoglobin variants out of >1000 variants discovered so far have double mutation on the same -globin chain besides the sickle cell mutation.

256

Case # 2 Hemoglobin S trait

References
1. Bain BJ. Sickle cell haemoglobin and its interactions with other variant haemoglobins and with thalassaemias. In: Bain BJ, Ed. Haemoglobinopathy Diagnosis, 2nd edition, Blackwell Publishing; 2006:141-149. Steinberg MH. Sickle cell trait. In: Steinberg MH, Forget BG, Higgs DR, Nagel RC, eds. Disorders of Hemoglobin: Genetics, Pathophysiology and Clinical Management. Cambridge, England: Cambridge University Press; 2001: 811-830. Feliu-Torres A, Eberle SE, Bragos IM, Sciuccati G, Ojeda MJ, Calvo KL, Voss ME, Pratti AF, Milani AC, Bonduel M, Diaz L, Noguera NI. Hb S-San Martin: A new sickling hemoglobin with two amino acid substitutions [6(A3)GluVal;105(G7)LeuPro]. Hemoglobin 2010; 34(5): 500-504.

2.

3.

257

Case # 3 Hemoglobin S homozygous

A 21 years old African American female came to the emergency department of a hospital complaining abdominal and joint pain.

Laboratory Data:
Hemoglobin 7.6 RBC 2.6 MCV 80.0 RDW 21.0 Platelet 201 Hb A 4.2 Hb S 90.0 Hb A2 2.8 Hb F 3.0 (Hemoglobin fractions from HPLC) Peripheral Blood Smear: 12.0 16.0 g/dL 3 4.0 - 5.5 Mil/mm 78 - 98 fL 11.5 -14.5% 3 150 - 400 Th/mm 94.3 - 98.5% 1.5 - 3.7% 0.0 - 2.0%

2+ poly morphic, 1+ target cells, few HowellJolly bodies, sickle cells Sickle cell solubility test for Hb S: Positive.

Agarose Gel Electrophoresis (pH 8.6)

258

Case # 3 Hemoglobin S homozygous

Citrate Agar Electrophoresis (pH 6.2)

Isoelectric focusing

259

Case # 3 Hemoglobin S homozygous

Capillary zone electrophoresis

Note: The original CZE on this specimen showed no presence of Hb A, therefore the analysis was repeated after mixing the specimen 1:1 with a normal blood. This is the standard practice in cases whenever Hb A is not detected.

High performance liquid chromatography

260

Case # 3 Hemoglobin S homozygous

Interpretation & Discussion

Summary of Results Method Alk Agarose Acid Agar / Agarose CZE Hb A area Hb S area Major band (Hb S) Major band (Hb S) Major peak (Hb S) Zone 5 Major band (Hb S) Major peak (Hb S) RT=4.38 Hb A2/C area Minor band (Hb A2)

IEF

HPLC

Minor peak (Hb A2) Zone 3 Minor band (Hb A2) Minor peak (Hb A2) RT=3.6

Minor peak (Hb F) RT=1.04

Agarose gel electrophoresis (alkaline pH 8.6) and citrate agar electrophoresis (acid pH 6.2) showed only one major band in the position of Hb S. In view of the positive sickle cell solubility test a diagnosis of homozygous Hb S disease was apparent.

It must be emphasized that due to co-migrating hemoglobin variants a confirmatory discriminatory test must be run. The selection of these confirmatory tests must be done with an eye on the results, for instance CZE would not be a
261

good confirmatory test for the identification of Hb S vs Hb Dhofar following alkaline electrophoresis, because the migration is not different. Acid electrophoresis will suffice as confirmation for either of them. Other readily available tests that can be of use are HPLC and IEF. If hemoglobinopathy testing is performed within three months of a blood transfusion, the separation pattern will indicate the presence of Hb A from the transfused blood and thus complicate the interpretation of the results. Therefore, it is advised that all the laboratories obtain the blood transfusion history before interpreting hemoglobin results. Sometimes it is impossible to know the patient transfusion history, especially if the patient arrived in the emergency department of the hospital. About eight years ago, a very unusual case was observed by me in our hospital. The Hb S diseased patient without insurance and facing sickle cell crisis went to the emergency department of a large hospital in Detroit. The patient was transfused with two units of blood and then discharged. He felt a little better after blood transfusion, but two days later he went to the emergency department of another large hospital in Detroit and received a second transfusion. Two days later, this patient was examined in the emergency department of our hospital. In our laboratory, the hemoglobin assays indicated Hb A (60%), Hb S (34%), Hb A2 (2.5%), and Hb F (3.5%). These results are suggestive of Hb S trait without knowing the blood transfusion history of the patient. Therefore, in order to make a correct diagnosis of a hemoglobin variant, it is prudent to know the recent blood transfusion record.
262

Case # 3 Hemoglobin S homozygous

References
1. 2. 3. 4. Kutlar A. Sickle Cell Disease: A Multigenic Perspective of a Single Gene Disorder. Hemoglobin 2007; 31 (2): 209-224. Steinberg MH. Genetic Etiologies for Phenotypic Diversity in Sickle Cell Anemia. The Scientific World Journal 2009; 9: 46-67. Bain BJ. Sickle cell anemia, In: Bain BJ, Ed. Hemoglobinopathy Diagnosis, 2nd edition, Blackwell Publishing; 2006: 150-164. Beutler E. The sickle cell diseases and related disorders. In: Beutler E, Lichtman MA, Coller BS, Kipps TJ, Seligsohn U, eds. Williams Hematology, 6th ed. New York, NY: McGraw-Hill; 2000: 581-606. Nagel RC, Platt VS. General pathophysiology of sickle cell anemia. In: Steinberg MH, Forget BG, Higgs DR, Nagle RL, eds. Disorders of Hemoglobin: Genetics, Pathophysiology and Clinical Management. Cambridge, England: Cambridge University Press; 2001: 494-526.

5.

263

Case # 4

Hemoglobin S with hereditary persistence of fetal hemoglobin (HPFH)

African-American adult male, apparently healthy and without any previously known major clinical condition visited his family physician for his annual check-up.

Laboratory Data:
Hemoglobin 13.4 RBC 4.78 MCV 80.9 MCH 28.0 Hb A 5.7 Hb S 56.0 Hb A2 3.3 Hb F 35.0 (Hemoglobin fractions from HPLC) Peripheral Blood Smear: No abnormality was noticed. Sickle cell solubility test for Hb S: Positive. 13.5 - 18.5 g/dL 3 4.6 - 6.2 Mil/mm 80 - 100 fL 27 - 34 pg 94.3 - 98.5 1.5 - 3.7% 0.0 - 2.0%

Agarose Gel Electrophoresis (pH 8.6)

264

Case # 4 Hemoglobin S with hereditary persistence of fetal hemoglobin (HPFH)

Citrate Agar Electrophoresis (pH 6.2)

Isoelectric focusing

265

Case # 4 Hemoglobin S with hereditary persistence of fetal hemoglobin (HPFH)

Capillary zone electrophoresis

High performance liquid chromatography

266

Case # 4 Hemoglobin S with hereditary persistence of fetal hemoglobin (HPFH)

Interpretation & Discussion:

Summary of Results Method Hb F area Alk Major Agarose band (Hb F)
Acid Agar / Agarose

Hb A area

Hb S area Major band (Hb S) Major band (Hb S) Major peak (Hb S ) Zone 5 Major band (Hb S) Major peak (Hb S) RT=4.38

Hb A2/C area Faint band (Hb A2)

CZE

Major band (Hb F) Major peak (Hb F) Zone 7 Major band (Hb F) Major peak (Hb F) RT=1.16

IEF

HPLC

Minor peak (Hb A2) Zone 3 Faint band (Hb A2) Minor peak (Hb A2) RT=3.6

These laboratory results must have been somewhat of a surprise for an asymptomatic patient. The few commonly encountered hemoglobins which migrate in the position of Hb S on alkaline agarose gel electrophoresis are Hb D, Hb G and Lepore all of which are ruled out by the results of acid agar gel electrophoresis. In addition the positive Hb S solubility test assures this patient has only Hb S and High Persistence of Fetal Hemoglobin. Analysis of this specimen by CZE requires a modification of the procedure because there is no hemoglobin A present for the software to use as a home base for comparison to other hemoglobin mobilities. The CZE analysis would be repeated after mixing in
267

1:1 ratio with a normal blood specimen.

From all the five laboratory methods, three abnormalities are evident: a) b) c) Absence of Hb A Hb S (56%) Hb F (35%)

The percentage of Hb S and Hb F suggests the following diagnostic possibilities: i) ii) iii) Homozygous Hb S disease with failure to suppress Hb F production Hb S-- thalassemia, with failure to suppress Hb F production Hb S- HPFH due to a deletional mutation in the non S gene

Hb S HPFH patients are the result of a point mutation on one beta gene forming Hb S and a deletion of the delta and beta area on the other gene permitting the production of Hb F to continue. The Hb F expression will be 25 35%. This is a pancellar condition so every erythrocyte will contain Hb F as well as Hb S and the damage caused by Hb S is not seen. Generally speaking patients with Hb S-HPFH are clinically well, with a benign clinical course, little evidence of hemolysis and without severe anemia. It is prudent to make a clinical diagnosis based on all available resources. In this case other laboratory data showed a positive sickle solubility test, a normal CBC, serum iron and ferritin, and no other abnormalities except for some sickling. Consultation with the physician indicated the patient was clinically well and certainly had not been treated with hydroxyurea. This patient is presumed to be Hb S HPFH.

Approximately 1% of Homozygous S patients present with 5% or less Hb F and these patients clinically do better than those without Hb F. Therefore much
268

has been done to increase the production of Hb F in homozygous S patients in general. Degree of success of hydroxyurea treatment has been very variable for unknown but at least to some extent genetic reasons. A patient with homozygous Hb S disease may present with Hb F levels (15% - 30%) following treatment with Hydroxyurea. Among the symptom ameliorating effects of hydroxyurea is the apparent interference in the suppression of Hb F manufacture and the production of nitric oxide. Since Hb F is higher in oxygen affinity than Hb S and deoxyhemoglobin S polymerizes, its presence protects the cells from sickling and other but not all symptoms of Sickle Cell Disease. The problem with this type of fetal persistence is that it is not pancellular. Not all erythrocytes contain Hb F even though the Hb F is elevated. Those cells without the Hb F are not protected. That said as a result of several Clincal Trials including BABY HUG several agencies have recommended use of hydroxyl urea for treatment of Sickle Cell Disease [McGann PT, Ware RE. Hydroxyurea for sickle cell anemia: What have we learned and what questions still remain? Curr Opin Hematol 2011; 18(3): 158-165]. In the unlikely circumstance that it was not known if the patient had been treated with hydroxyl urea there is the possibility that he might have been a homozygous patient who at the moment his blood was drawn was not very symptomatic but his Hb F had been chemically altered. The two conditions could be separated by doing a Kleihuer Betke acid elution test or flow cytometry (monoclonal antibody agains - chains) for the study of pancellular vs
269

heterocellular distribution of the Hb F. An essentially homogeneous distribution establishes the Hb S-HPFH diagnosis. Hb S--thalassemia is also highly unlikely because of the clinical picture. Patients with Hb S- -thalassemia even in the presence of Hb F would have a thalassemic clinical picture. Hydroxyurea has been used for treatment of betathal patients with some success so the possibility exists that it might be helpful in a case of Hb S - -thalassemia. Far less data exists on this treatment even though it is known that the presence of Hb F lessens the clinical picture.

A few cases of clinical aberrations, e.g. minor joint or abdominal pains, asceptic necrosis of bone, palpable spleen were reported in persons with Hb SHPFH (Fairbanks VF. Hemoglobinopathies and Thalassemias. New York, NY: Brian C. Decker; 1980:136). Recently Whyte et al (see below reference # 5) reported massive splenic infarction in an adolescent with Hb S-HPFH. Therefore the condition is not benign.

References
1. Murray N, Serjeant BE, Serjeant GR. Sickle cell-hereditary persistence of fetal hemoglobin and its differentiation from other sickle cell syndromes. Br J Haemotol 1988; 6: 89-92. (available online since March 2008). 2. Hoyer JD, Connie SP, Fairbanks VF, Hanson CA, Katzmann JA. Flow cytometric measurement of hemoglobin F in RBCs: Diagnostic usefulness in the distinction of hereditary persistence of fetal hemoglobin (HPFH) and hemoglobin S-HPFH from other conditions with elevated levels of hemoglobin F. Am J Clin Pathol 2002; 117: 857-863. 3. Akinsheye I, Al-Sultan A, Solovieff N, Ngo D, Baldwin CT, Sebastiani P, Chui DH, Steinberg MH. Fetal hemoglobin in sickle cell anemia.Blood 2011; 118: 19-27.

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4. Ngo D, Aygun B, Akinsheye I, Hanjins JS, Bhan I, Luo HY, Steinberg MH, Chui DH. Fetal haemoglobin levels and haematological characteristics of compound hegterozygotes for haemoglobin S and deletional hereditary persistence of fetal hemoglobin. Br J Haematol 2012; 156(2): 259-64. 5. Whyte D, Forget BG, Chui DH, Luo HY, Pashankar F. Massive splenic infarction in an adolescent with hemoglobin S-HPFH. Pediatr Blood Cancer 2013; 60(7): 49-51. 6. Chapter 2.3 of this book: Bernard G. Forget, MD. Hereditary Persistence of Fetal Hemoglobin 7. Bain BJ. Hereditary persistence of fetal haemoglobin and other inherited causes of an increased proportion of haemoglobin F. In: Haemoglobinopathy Diagnosis, Blackwell Publishing, second edition, 2006, pp119-127.

271

Case # 5 Hemoglonin G-Philadelphia trait

Adult African-American male with no abnormalities.

Laboratory Data:
Hemoglobin 14.5 13.5 -18.5 g/dL 3 RBC 5.06 4.6 - 6.2 Mil/mm MCV 84.0 80 - 100 fL MCH 28.7 27 - 34 pg Hb A 77.1 94.3 - 98.5% Hb A2 0.9 1.5 - 3.7% Hb F 0.0 0.0 - 2.0% Hb G 22% (Hemoglobin fractions from HPLC) Peripheral Blood Smear: No abnormality was detected. Sickle cell solubility test: Negative.

Agarose Gel Electrophoresis (pH 8.6)

272

Case # 5 Hemoglobin G-Philadelphia trait

Citrate Agar Electrophoresis (pH 6.2)

Isoelectric focusing

273

Case # 5 Hemoglobin G-Philadelphia trait

Capillary zone electrophoresis

High performance liquid chromatography

274

Case # 5 Hemoglobin G-Philadelphia trait

Interpretation & Discussion

Summary of Results Method Hb A area Major band (Hb A) Hb S area Major band (Hb G) Hb A2/C area Minor band (Hb A2)

Alk Agarose

Minor band close to carbonic anhydrase

Acid Agar/Agarose

CZE

Major band (Hb A+ Hb A2 + Hb G+ Hb G2) Major peak (Hb A) Zone 9 Major band (Hb A)

Major peak (Hb G) Zone 6

Major band anodal to Hb S (Hb G)

IEF

Minor peak (Hb A2) Zone 3 Minor band (Hb A2) Minor peak (Hb A2) RT=3.6

Minor peak (Hb G2) Zone 1

Minor band (Hb G2) as far cathodal to A2 as G is anodal to it.

HPLC

Major peak (Hb A) RT=2.45

Major peak (Hb G) RT=4.04

Minor peak RT=4.5-4.6

* Note: HPLC retention time (RT) varies with the type of the instrument used and several other factors, e.g. temperature etc.

Electrophoretic migration bands at less than 1% may be difficult to detect on alkaline electrophoresis. If the presence of a minor band is expected the sample amount may be increased. Quantification of the results must not be done
275

on over applied samples because you will have exceeded the quantitative linearity of the system.

The sickle cell solubility test was negative, ruling out the possibility of Hb S. The separation table shows the presence of a non Hb S band migrating in the Hb S area for all methods except Acid Electrophoresis. Common variants found anodal to Hb S on alkaline electrophoresis that migrate in Hb A position on acid electrophoretic conditions are Hb D, Hb G-Philadelphia and Lepore. Of these options only Hb G- Philadelphia is an -chain variant. If -chain variant is expressed in a large enough percentage to compete with normal -chains for combination with chains a new small modified delta band is created. In individuals with Hb G-Philadelphia [68(E17)AsnLys], a combination of Hb G Philadelphia -chains with normal -chains leads to the formation of about 1% of a molecule Hb G2 (2G2). Hb G2 has no clinical significance, but plays an important role in the distinction between Hb D and Hb G-Philadelphia. Since Hb G-Philadelphia is entirely innocuous, globin chain electrophoresis and DNA studies are usually not necessary.

There is a temptation to analyze the available hemoglobin variants by percentage since Hemoglobin Lepore runs less than 15 % and Hb D runs about 40 while Hb G-Philadelphia trait runs 20-25% in the heterozygote. Differentiation between Hb D and Hb G-Philadelphia on the basis of the percentage of the variant is not advised because the percentages of either would be effected by a concurrent -thalassemia -2 trait or homozygous -thalassemia-2 (see below).
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The single alpha gene deletion resulting in -thalassemia-2 trait is found in 1/3 of African Americans therefore this silent mutation could be likely found in association with Hb G-Philadelphia in this ethnic population.

Of the four alpha genes located on chromosome 16 (two on each chromosome), alpha gene mutations lead to the following possibilities (adopted with the permission of College of American pathologists: Hoyer JD and Kroft SH, eds. Color Atlas of Hemoglobin Disorders. College of American Pathologists, Northfield, IL, 2003; 67).

1. 2.

Hb G trait with no thalassemia, Hb G 20 -25% no hematologic effect Hb G trait, One gene deleted (-thalassemia-2 trait), Hb G 25-35% usually no hematologic effect Hb G-trait; Two genes deleted (homozygous -thallasemia-2), Hb G 3545%; microcytosis. Homozygous Hb G; Two genes deleted (homozygous - thalassemia-2), Hb G 95%, microcytosis.

3.

4.

References
1. Keren DF. Clinical Evaluation of Hemoglobinopathies: Part II. Structural Changes, Ward Medical Laboratory, Archived Issues 2003; 3: 1-11. Available online (http://www.wardlab.com/14-3.html). Hoyer JD and Kroft SH, eds. Color Atlas of Hemoglobin Disorders. College of American Pathologists, Northfield, IL, 2003; 65. Bain BJ. Hemoglobin G-Philadelphia trait: In: Haemoglobinopathy Diagnosis, Blackwell Publishing, second edition, 2006, pp 212. Milner PF, Huisman TH. Studies of the proportion and synthesis of haemoglobin G-Philadelphia in red cells of heterozygotes, a homozygote, and a heterozygote for both haemoglobin G and alpha thalassemia. Br J Haematol 1976; 34: 207-220. (Available online from July 2008).
277

2. 3. 4.

5.

6.

7.

Baine BM, Rucknagel DL, Dublin DA Jr, Adams JG III. Trimodality in the proportion of hemoglobin G-Philadelphia in heterozygotes; evidence of heterogeneity in the number of human alpha chain locations. Proc Natl Acad Sci. 1976; 73: 3633-36. Reider RF, Woodbury DH, Rucknagel DL. The interaction of thalassemia and hemoglobin G-Philadelphia. Br. J Haematol. 1976; 32: 159-65. Khalil MSM, Timbs A, Hendrson S, Schuh A, Hussein MRA, Old J. Haemoglobin (Hb) G-Philadelphia, Hb Stanleyville-II, Hb G-Norfolk, Hb Matsue-Oki and Hb Mizushi can form a panel of -chain variants that overlap in their phenotype: the novel use of StyI to screen for Hb GPhiladelphia. Intl Jnl Lab Hem 2011; 33: 318-325.

278

Case # 6 Hemoglobin S-G Philadelphia

Adult African American female who was asymptomatic.

Laboratory Data:
Hemoglobin 11.7 RBC 4.29 MCV 81.6 MCH 27.4 Hb A 54.0 Hb S 19.9 Hb G 17.8 Hb A2 1.1 Hb F 0.2 Hb S-G Hybrid 7.0 (Hemoglobin fractions from HPLC) Peripheral Blood Smear: No abnormality. Sickle cell solubility test for hemoglobin S: Positive. Unstable hemoglobin (isopropanol) Test: Negative. No record of blood transfusion during the past six months. 12.0 -16.0 g/dL 3 4.0 5.5 Mil/mm 79 - 98 fL 27- 34 pg 94.3 98.5%

1.5-3.7% 0.0-2.0%

Agarose Gel Electrophoresis (pH 8.6)

279

Case # 6 Hemoglobin S-G Philadelphia

Citrate Agar Electrophoresis (pH 6.2)

Isoelectric focusing

280

Case # 6 Hemoglobin S-G Philadelphia

Capillary zone electrophoresis

High performance liquid chromatography

281

Case # 6 Hemoglobin S-G Philadelphia

Interpretation & Discussion

Summary of Results Method Alk Agarose Hb A area Major band Hb A Major band (Hb A + G) Major peak (Hb A) Zone 9 Hb S area Major band Hb S + G Major band (Hb S) Major peak Zone 6
Major peak poorly separated from Zone 6 in Zone 5

Hb A2/C area Minor band (Hb A2)

Minor band cathodal to A2

Acid Agar / Agarose

CZE

Minor Hb A2 peak Zone 3

Major Hb GS hybrid peak Zone 2 Medium band (Hb S-G hybrid + A2 )

Minor Hb G A2 peak Zone 1 Minor Hb G2 Band Medium peak (Hb S-G hybrid) RT=4.8

IEF

Major Hb G band Anodal to Hb S

*

Major band (Hb S) Minor peak (Hb A2) RT=3.58 Medium peak (Hb G) RT=4.0

HPLC

Medium peak (Hb A) RT=2.35

Medium peak (Hb S) RT=4.24

*Note:

HPLC retention time (RT) varies with the type of the instrument used and several other factors, e.g. temperature etc.

Agarose gel electrophoresis (pH 8.6) showed three major bands in the familiar positions of Hb A ( 50%), Hb S ( 38%), and Hb A2/C (>10%) and a barely visible minor band slightly cathodal to the carbonic anhydrase position. It should be emphasized here that Hb A, Hb S, and Hb C cannot all be
282

manufactured in any single person because there are only 2 beta genes and these hemoglobins represent three different beta compositions. Either this patient had a transfusion or one of the hemoglobin variants is not a beta chain variant. The transfusion could be to a patient with Hb S and C or a transfusion using blood from an Hb A-S heterozygous donor or an Hb A-C heterozygous donor to a patient who was a heterozygote of the other type. These unlikely scenarios were all ruled out as the patient received no blood transfusion.

Beta-thalassemia in conjunction with Hb A-S trait can result in an elevated Hb A2 which migrates with or near Hb C by most of these methods. In S-thalassemia the Hb A2 is rarely higher than 10% so a >10% band is unlikely Hb A2. Secondly in S--thalassemia patients Hb F concentration is often increased especially if the patient is thalassemic to the point that the Hb A2 is very elevated but in this patient the Hb F was normal ( 0.2%).

The identity of the small barely visible minor band is the key to the identification. The most common alpha chain variant is Hb G-Philadelphia which would present in the Hb S area at 30 to 35%. This alpha chain variant then competes with the unmodified alpha chains to combine with the beta and delta chains available. Since the sickle solubility test was positive we know the band in the position of Hb S is indeed at least partly due to the S beta gene combined with normal alpha chains. This Hb S beta gene when combined with a modified
283

Hb G-Philadelphia alpha gene creates a new double hemoglobin variant combination, Hb S-G Philadelphia hybrid which unfortunately migrates with Hb A2 on alkaline, acid or IEF electrophoresis. This explains the elevated Hb A 2. half of the alpha chains are modified they would be competing also with the unmodified alpha chains for delta chains. The unmodified alpha chain delta combination is Hb A2 seen normally and the modified alpha variant delta combination is new hemoglobin, Hb G2 which migrates close to the carbonic anhydrase. The number of different hemoglobin molecules created by a Hb S GPhiladelphia double mutation is 6. The Hb S-G hybdrid migrates with A2 on acid, alkaline and IEF electrophoresis. If

IEF, CZE and HPLC data support the presence of a heterozygous Hb GPhiladelphia [68(E17)AsnLys] and Hb S in that two distinct, appro ximately equal bands or peaks were seen in the position of Hb S and Hb G. IEF indicated that the Hb G band is closer to the Hb A band (more anodal) than the Hb S. Two additional bands in the position of Hb A2 and Hb G2 were also detected from IEF although the low intensity of the Hb G2 band made it difficult to see. CZE showed six distinct peaks in the following zones with alleged hemoglobins indicated in parenthesis: i) ii) iii) iv) v) vi) Zone 9 (Hb A) Zone 6 (Hb G-Philadelphia) Zone 5 (Hb S) Zone 3 (Hb A2) Zone 2 (Hb S/G hybrid) Zone 1 (Hb G2)
284

HPLC showed the following major peaks: a) b) c) d) e) f) Hb F ( 0.2%) Hb A (54%; RT = 2.35) Hb A2 (1.1%, RT= 3.58) Hb G (17.8%, RT= 4.0) Hb S (19.9%, RT = 4.24) Hb S/G hybrid (7%, RT=4.8)

All the data affirm the presence of a double heterozygous presentation of an abnormal chain (Hb S) and an abnormal chain (Hb G-Philadelphia) in conjunction with normal and chains ( and ) found in Hb A. The abnormal chains end up competing with their normal counterparts creating all the possible combinations listed below. Hb A ( 2 2) G A Hb G ( 2 2) HB A2 ( 2 2)
A A A A A

Hb S ( 2 2) G S Hb S/G ( 2 2) Hb G2 ( 2 2)
G

Had this patient been a newborn the situation would further have been complicated by the addition of 2 new gamma chain containing forms of HbF. Hb S-G Philadelphia double heterozygous hemoglobinopathies are essentially healthy and without anemia.

References
1. Kirk CM, Papadea CN, Lazarchik J. Laboratory Recognition of a Rare Hemoglobinopathy. Hemoglobin SS and SGPhiladelphia Associated with Thalassemia -2. Arch Pathol Lab Med 1999; 123: 963-966. Gu LH, Wilson JB, Molchanova TP, McKie KM, Huisman THJ. Three Sickle Cell Anemia Patients each with a Different Chain Variant. Diagnostic Complications. Hemoglobin 1993; 17(4): 295-301.
285

2.

3.

4. 5.

Kutlar F, Kutlar A, Nuguid E, Prachal J, Huisman. Usefulness of HPLC Methodology for the Characterization of Combinations of the Common Chain variants Hb S, C, and O-Arab, and the Chain variant in GPhiladelphia. Hemoglobin 1993; 17(1):, 55-66. LeCrone CN, Jones JA, Detter JC. Hemoglobin G Trait and S Trait in the Same Patient. Hemotology 1983; 49(3): 165-167. Lawrence C, Hirsch RE, Fataliev NA, Patel S, Fabry ME, Nagel RL. Molecular interactions between Hb alpha-G Philadelphia, Hb C, Hb S: phenotypic implications for SC -G Philadelphia disease. Blood 1997; 90: 2819-2825.

286

Case # 7 Hemoglobin G-Coushatta trait

A 24 year old male resident of Cheyenne River Indian Reservation, South Dakota, USA. No physical abnormality. Blood sent to a reference laboratory for hemoglobin electrophoresis.

Laboratory Data:
Hemoglobin RBC MCV RDW Platelet Hb A Hb A2 Hb F Hb variant 12.7 4.49 81 13.2 243 56.0 2 1 41.0% 13.5-18.5 g/dL 3 4.6-6.2 Mil/mm 80-100 fL 11.5-14.5% 3 150-400 Th/mm 94.3-98.5% 1.5-3.7% 0.0-2.0%

(Hemoglobin fractions from HPLC) Peripheral Blood Smear: No abnormality noticed. Sickle cell solubility test for Hb S: Negative Unstable hemoglobin (isopropanol) test: Negative.

Agarose Gel Electrophoresis (pH 8.6)

287

Case # 7 Hemoglobin G-Coushatta trait

Citrate Agar Electrophoresis (pH 6.2)

Isoelectric focusing

288

Case # 7 Hemoglobin G-Coushatta trait

Capillary zone electrophoresis

High performance liquid chromatography

289

Case # 7 Hemoglobin G-Coushatta trait

Interpretation & Discussion

Summary of Results Method Hb A area Major band (Hb A) Major band (Hb A+ Hb A2 + Hb G) Major peak (Hb A) Zone 9 Major band (Hb A)
*

Hb S area

Alk Agarose
Acid Agar / Agarose

Major band

Hb A2/C area Minor band (Hb A2)

CZE

Major peak ( Hb G ) Zone 6

Major Hb G band anodal to S

IEF

Minor peak (Hb A2) Zone 3 Minor band (Hb A2) Major peak (Hb G + Hb A2) RT=3.6

No G2 band was detected

HPLC

Minor peak (Hb F) RT=1.05

Major peak (Hb A) RT=2.5

*Note: HPLC retention time (RT) varies with the type of the instrument used and several other factors, e.g. temperature etc.

Agarose gel electrophoresis (pH 8.6) indicated major bands in the position of Hb A and at the position of Hb S. Besides a minor band at the position of Hb A2 and carbonic anhydrase band no other band was detected. Citrate agar electrophoresis (pH 6.2) showed one major band at the position of Hb A, and a faint band was also detected in the position of Hb F. CZE showed major peaks
290

in Zone 9 (Hb A), and Zone 6 (Hb variant) and a minor peak in Zone 3 (Hb A2).

IEF indicated that the second major band was in the position of Hb G, or possibly Hb D but not Hb S, however a Hb G2 (2 2) band was not detected. HPLC showed two major peaks at the position of Hb A and Hb A2 rather than one toward the center of the pattern as seen with all the alkaline electrophoretic separations (pH 8.6). The tentative identification of the Hb variant (41% concentration from alkaline agarose gel electrophoresis at pH 8.6) was achieved by eliminating commonly encountered hemoglobin variants (e.g. Hb S, Hb GPhiladelphia, Hb Lepore, Hb Hasharon, etc) on the basis of the laboratory results.
G

Hb S was also ruled out by a normal sickle cell solubility test. The most commonly noticed Hb G variant (Hb G Philadelphia) is noticed mostly in African Americans. The presence of this -chain variant was ruled out because the minor Hb G2 (2 2) band was not detected by IEF or by agarose gel electrophoresis (pH 8.6) and because alpha chain variants are found in a lower percentage than -chain variants. Hb Hasharon and Hb Lepore are also ruled out on the basis of low concentration. Furthermore Hb Lepore produces a thalassemic picture including microcytosis, and that was not exhibited in this case. Hemoglobin variant of 41% is extremely high for Hb Hasharon and Hb Lepore. Hemoglobins D-Los Angeles and Hb G- trait are closely migrating variants with no clinical manifestation. Generally speaking they are found in different ethnic groups.
291
G

The safest interpretation for this case is that this patient has Hb G trait ( -chain variant ) known as Hb G-Coushatta[ 22 (4) GluAla (GAAGCA)] because of the (American Indian) ethnicity. It is emphasized that Hb G-Coushatta is not limited to American Indian tribes, and this hemoglobinopathy also know as Hb G-Saskatoon, Hb G-Taegu, or Hb G- Hsin Chu, has been reported in Chinese, Korean, Japanese, Thai, Turkish, and Algerian nationals and is harmless. Homozygous Hb G-Coushatta is very rare and exhibits microcytosis. Recently a compound heterozygote for Hb E and Hb G-Coushatta was reported in a Thai family by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). It may not be worth the cost to further solidify the identity of the hemoglobin variant in a situation like this where the variant is functioning normally.

References
1. Worrawut C, Viprakasit V. Further identification of Hb G-Coushatta [22(4) GluAla (GAAGCA)] in Thailand by the polymerase chain reaction-single-strand conformation polymorphism technique and by amplification refractory mutation system-polymerase chain reaction. Hemoglobin 2007; 31(1): 93-99. Ohba Y, Miyaji T, Hirosaki T, Matsuoka M, Koresawa M, Iuchi I. Occurrence of Hemoglobin G Coushatta in Japan. Hemoglobin 1978; 2(5): 437-441. Wong SC, Tesanovic M, Poon M-C. Detection of two abnormal hemoglobins, Hb Manitoba and Hb G-Coushatta, during analysis of glycohemoglobin (A1c) by high performance liquid chromatography. Clin Chem 1991; 38(8): 14561459. Li J, Wilson D, Plonczynski M, Harrell A, Cook CB, Scheer WD, Zeng Y-T, Coleman MB, Steinberg MH. Genetic studies suggest a multicentric origin for Hb G-Coushatta [22(4)GluAla]. Hemoglobin 1999; 23(1): 57-67. Boissel JP, Wajcman H, Labie D, Dahmane M, Benabadji M. [Hemoglobin G-Coushatta (beta 22(4) glu leads to ala) in Algeria: an homozygous case]. Nouv Rev Fr Hematol 1979; 21:225-230. Dincol G, Dincol K, Erdem S. Hb G-Coushatta or alpha 2 beta 22 (4) GluAla in a Turkish male. Hemoglobin 1989; 13: 75-77.
292

2. 3.

4.

5.

6.

Case # 8 Hemoglobin C trait

A 28 year old African American male. No physical abnormalities. Participated regularly in basketball and never complained about fatigue.

Laboratory Data:
Hemoglobin RBC MCV RDW Platelet 14.8 4.91 77 15.1 248 13.5-18.5 g/dL 3 4.6-6.2 Mil/mm 80-100 fL 11.5-14.5% 3 150-400 Th/mm 94.3-98.5% 1.5-3.7% 0.0-2.0%

Hb A 58.0 Hb A2 2 Hb F 1 Hb variant 39.0% (Hemoglobin fractions from HPLC)

+

Peripheral Blood Smear: 1 microcytosis and numerous target cells. Sickle cell solubility for Hb S: Negative Unstable hemoglobin (isopropanol) test: Negative

Agarose Gel Electrophoresis (pH 8.6)

293

Case # 8 Hemoglobin C trait

Citrate Agar Electrophoresis (pH 6.2)

Isoelectric focusing

294

Case # 8 Hemoglobin C trait

Capillary zone electrophoresis

High performance liquid chromatography

295

Case # 8 Hemoglobin C trait

Interpretation & Discussion

Summary of Results Method Hb A area Alk Major Agarose band (Hb A) Acid Agar Major /Agarose band (Hb A+ Hb A2) CZE Major peak (Hb A) Zone 9 IEF Major band (Hb A)
*

Hb S area

Hb A2/C area Major band Major band

Minor peak (Hb A2) Zone 3 Minor band (Hb A2) Minor peak (Hb A2) RT=3.6

Major peak (Hb C) Zone 2 Major band cathodal to A2 (Hb C) Major Peak (Hb C) RT=5.l0

HPLC

Major peak (Hb A) RT=2.45

* Note: HPLC retention time (RT) varies with the type of the instrument used and several other factors, e.g. temperature etc.

Agarose gel electrophoresis (pH 8.6) exhibited a major band in the position of Hb A, and another intense band (40%) in the position of Hb C/Hb E/ Hb O-Arab/ Hb A2. The intense band is not due to Hb A2 only in view of the fact that the concentration of Hb A2 is never > 10%. Citrate agar electrophoresis (pH 6.2) indicated two bands. One band was in the position of Hb A and another band in the position of Hb C. Hb E, Hb O-Arab, and Hb C-Harlem
296

are ruled out on the basis of citrate agar electrophoresis (pH 6.2), as none of these migrate in the position of Hb C by this method. Combination of alkaline and acid pH electrophoresis suggested that the Hb variant is most likely Hb C. IEF also indicated two major bands in the position of Hb A and Hb C. CZE also indicated two major peaks in Zone 9 (Hb A) and Zone 2 (Hb C). HPLC results were concordant with above stated observations from IEF and CZE, i.e. one major peak eluted in the position of Hb A (retention time 2.45 minutes) and the second major peak eluted in the C-window (retention time 5.10 minutes). The peripheral blood smear examination (1 microcytosis and target cells), negative for sickle cell solubility and hemoglobin instability tests, and the five laboratory tests led towards the assignment of the Hb variant as Hb C. In order to be Hb C trait the percentage of Hb C should be less than Hb A, therefore the diagnosis of Hb C trait was made. Hb C is a -chain variant [6 (A3) GluLys], caused by the substitution of glutamic acid by lysine in the sixth position. Hb C trait is prevalent in 2-3% in African Americans, and rarely found in other ethnic groups. Clinically the Hb C trait phenotype is insignificant.
+

297

References
1. 2. 3. Bain BJ. Hemoglobin C trait: In: Haemoglobinopathy Diagnosis, Blackwell Publishing, 2nd edition, 2006, pp 192-195. Wajcman H, Moradkhani K. Abnormal haemoglobins: detection & characterization. Indian J Med Res 2011; 134: 538-546 Joutovsky A, Nardi M. Hemoglobin C and Hemoglobin O-Arab variants can be diagnosed using the Bio-Rad Variant II High Performance Liquid Chromatography System without further confirmatory tests. Arch Pathol Lab Med 2004; 128: 435-439. Joutovsky A, Hadzi-Nesic J, Nardi MA. HPLC retention time as a diagnostic tool for hemoglobin variants and hemoglobinopathies: A study of 60 000 samples in a clinical diagnostic laboratory. Clin Chem 2004; 50: 1736-1747. Keren DF, Hedstrom D, Gulbranson R, Ou Ching-Nan, Richard B. Comparison of Sebia Capillary Electrophoresis with the Primus High-Pressure Liquid Chromatography in the evaluation of hemoglobinopathies. Am J Clin Pathol 2008; 130: 824-831

4.

5.

298

Case # 9

Hemoglobin C homozygous

African-American male (22 years old) with no physical complaints.

Laboratory Data:
Hemoglobin 12.1 RBC 4.3 MCV 73 RDW 13.3 Platelet 248 Hb A Not detected Hb A2 2.5 Hb F 1.6 Hb variant 95.9% (Hemoglobin fractions from HPLC) 13.5 - 18.5 g/dL 3 4.6 - 6.2 Mil/mm 80 -100 fL 11.5 - 14.5% 3 150 - 400 Th/mm 94.3 - 98.5% 1.5 - 3.7% 0.0 - 2.0%

Peripheral Blood Smear: Target cells, spherocytes, and poikilocytosis. Sickle cell Hb S solubility test: Negative Unstable hemoglobin (isopropanol) test: Negative

Agarose Gel Electrophoresis (pH 8.6)

299

Case # 9 Hemoglobin C homozygous

Citrate Agar Electrophoresis (pH 6.2)

Isoelectric focusing

300

Case # 9 Hemoglobin C homozygous

Capillary zone electrophoresis

High performance liquid chromatography

301

Case # 9 Hemoglobin C homozygous

Interpretation & Discussion

Summary of Results Method Alk Agarose Acid Agar /Agarose CZE Hb A area Hb S area Hb A2/C area Major band Major band Minor peak (Hb A2) Zone 3 Minor band

IEF

HPLC

Minor peak (Hb A2) RT=3.6

Major peak (Hb C) Zone 2 Major band cathodal to A2 (Hb C) Major peak (Hb C) RT=5.06

*Note: HPLC retention time (RT) varies with the type of the instrument used and several
other factors, e.g. temperature etc.

Agarose gel electrophoresis (pH 8.6) showed only one intense and major band in the position of Hb C/E/O, and Hb A was not detected. Citrate agar electrophoresis (pH 6.2) also showed one intense band in the position of Hb C, therefore at the very outset the presence of Hb E and O were ruled out. It appeared that the solitary band in the Hb C position is most likely due to the substitution of amino acid lysine with glutamic acid at the sixth position of -chain [ 6(A3) GluLys]. Hb C has prevalence of 0.017% among the African-Americans in the United States, but it has been also reported in persons of Hispanic and Sicilian ancestry.
302

Other laboratory tests (CZE, HPLC, and IEF) also indicated the prominent Hb C band or peak, however contrary to alkaline and acid electrophoresis (see above) minor bands or peaks due to Hb F ( 1.6%) and Hb A2 ( 2.5%) were also detected. Absence of Hb A by all the five methods in this person suggested either homozygous Hb C or Hb C/ -thalassemia and ruled out Hb C/ -thalassemia (Case # 19). The clear distinction between homozygous Hb C and Hb C/ -thalassemia (double heterozygous state for both Hb C and -thalassemia) is problematic, because the clinical features are similar in both cases. Careful evaluation of peripheral blood smear, CBC, anemia status, quantitative values of Hb F and Hb A2, and evaluation of hemoglobinopathy in the biological parents are helpful for the exactness of the diagnosis. Fairhurst and Casella reported a diagnosis of homozygous Hb C disease in a Ghanian child [N Engl J Med 2004; 350(26): e24], with hemoglobin (9.0 g/dL),HCT (24.3), MCV (53.8), RDW (28.8), and an uncorrected reticulocyte count of 1.6%. The peripheral blood smear (Figure 1) indicated characteristic features of homozygous Hb C: target cells (arrows), microspherocytes (arrowheads), rod-shaped cells containing hemoglobin C crystals (asterisk), anisocytosis, and poikilocytosis. Schwab and Abelson [N Engl J Med 2004; 351(15): 1577] questioned the diagnosis of homozygous Hb C on the basis of
303
0 0 0

extremely low MCV and the clinical status of the child, and suggested the diagnosis of Hb C/ -thalassemia.
0

Figure 1. Peripheral blood smear of the Ghanaian child (adopted with the permission of the N Engl J Med)

The following characteristics are helpful in the differential diagnosis between the two possibilities:
0

Test Hb A2 Hb F MCV

Homozygous Hb C 3.2 3.9% 0.8 1.9% 68 - 76

304

Hb C/ -thalassemia Elevated in most cases 3 10% (generally > 5%) 55 - 70

On the basis of Hb A2 ( 2.5%), Hb F ( 1.6), MCV (73), mild anemia, a tentative diagnosis of homozygous Hb C is reasonable, however for confirmation, additional tests in the biological parents are mandatory. Persons with homozygous Hb C rarely have clinical symptoms and live a normal life. Symptoms that may develop in these persons include: Reduced red blood cell counts during infection or illness jaundice Increased risk for gallstones Enlarged spleen Episodes of pain Increased risk for infection

Hemoglobin C is known to protect individuals against clinical Plasmodium falciparum malaria.

References
1. 2. Bunn HF, Forget BG, Hemoglobin: molecular, genetic and clinical aspects. 1 st edition, Philadelphia, PA: WB Saunders Co; 1986: 421-425. Nagel RL, Steinberg MH. Hb S/C disease and Hb C disorders. In: Steinberg MH, Forget BG, Higgs DR, Nagle RL, eds. Disorders of Hemoglobin: Genetics, Pathophysiology and Clinical Management. Cambridge, England: Cambridge University Press; 2001: 756-785. Fairhurst RM, Casella JF. Homozygous hemoglobin C disease. N Engl J Med 2004; 350: e24 (Web only). (Available at www.nejm.org/cgi/content/full/350/26/e24). Schwab JG, Abelson HT. Hemoglobin C. N Engl J Med 2004; 351(15): 1577. Weatherall DJ, Clegg JB. The thalassemia syndrome, 4th edition, Oxford, England: Blackwell Science, 2001: 415-419. Modiano D, Luoni G, Sirima BS, et al. Hemoglobin C protects against clinical Plasmodium falciparum malaria. Nature 2001; 414 (6861): 305-8. [Medline]. Rihet P, Flori L, Tall F. Hemoglobin C is associated with reduced Plasmodium falciparum parasitemia and low risk of mild malaria. Hum Mol Genet 2004; 13(1): 1-6. Hoyer JD, Kroft SH. Color Atlas of Hemoglobin Disorders. College of American Pathology 2003. Case # 8 (pp 45), Case # 15 (pp 75), Case # 29 (pp 135), Case # 30 (pp 139).
305

3.

4. 5. 6.

7.

8.

Case # 10

Hemoglobin C with hereditary persistence of fetal hemoglobin (HPFH)

A 23 years old white female presented to the Emergency Department of the hospital (2011) complaining of pelvic pain. She was found to have a ruptured right hemorrhagic ovarian cyst which was suspected on CT and ultrasound and then confirmed by laparoscopy. No blood transfusion was executed.

Laboratory Data:
Hemoglobin 11.9 12.0 -16.0 g/dL 3 RBC 4.8 4.0 - 5.5 Mil/mm MCV 74 79 - 98 fL RDW 20.8 11.5 -14.5% Hb A Not detected 94.3 - 98.5% Hb A2 2.2 1.5 - 3.7% Hb F 29.4 0.0 - 2.0% Hb variant 68.4% (Hemoglobin fractions from HPLC) Peripheral Blood Smear: Abundant target cells Sickle cell solubility test for hemoglobin S: Negative Flow cytometry (monoclonal antibody for Hb F) showed a homogeneous distribution of Hb F.

Agarose Gel Electrophoresis (pH 8.6)

306

Case # 10 Hemoglobin C with hereditary persistence of fetal hemoglobin (HPFH)

Citrate Agar Electrophoresis (pH 6.2)

Isoelectric focusing

307

Case # 10 Hemoglobin C with hereditary persistence of fetal hemoglobin (HPFH)

Capillary zone electrophoresis

High performance liquid chromatography

308

Case # 10 Hemoglobin C with hereditary persistence of fetal hemoglobin (HPFH)

Interpretation & Discussion

Note: HPLC and hemoglobin electrophoresis tests were performed at three independent laboratories, and all the results were concordant. Method Alk Agarose Acid Agar /Agarose CZE
Major band in Hb F area Major Band in Hb F area Major peak Hb F Zone 7

Hb A area

Hb S area

Hb A2/C area Major band Major band Very minor peak (Hb A2) Zone 3 Minor band (Hb A2) Very minor peak (Hb A2) RT=3.6 Major peak (Hb C) Zone 2 Major band cathodal to A2 (Hb C) Major peak (Hb C) RT=5.14

IEF

HPLC

Major band in Hb F area Major peak (Hb F) RT=1.15

*Note: HPLC retention time (RT) varies with the type of the instrument and several other factors, e.g. temperature etc.

Agarose gel electrophoresis (pH 8.6) indicated the absence of Hb A and the presence of two major bands. One major band was detected in the position of Hb F ( 29%) and another major band ( 68%) was detected in the position of Hb C/E/O. Hb E and O were ruled out on the basis of citrate agar electrophoresis
309

(pH 6.2), as only two major bands were detected in the position of Hb C and Hb F. IEF, CZE, and HPLC also confirmed the presence of only two major hemoglobins (Hb C and Hb F) in this patient.

This suggested two possibilities, a) heterozygosity for Hb C or b) heterozygosity for a deletional form of hereditary persistence of fetal hemoglobin (HPFH). The presence of Hb C > 50% also suggested the presence of HPFH.

Hb C with hereditary persistence of fetal hemoglobin is the diagnosis of this patient. Generally speaking homozygous Hb C disease (Case # 9) is rare and is associated with abundant target cells, microcytosis, reticulocytosis, and minimal hemolytic disease. Contrary to this, Hb C with HPFH is clinically similar to Hb C trait (Case # 8).

References
1. Bain BJ. Hereditary persistence of fetal hemoglobin and other inherited causes of an increased proportion of hemoglobin F: In: Hemoglobinopathy Diagnosis, Blackwell Publishing, 2nd edition, 2006, pp 119-127. 2. Bollekens JA, Forget BG. thalassemia and hereditary persistence of fetal hemoglobin. Hematol Oncol Clin North Am. 1991; 5: 399-422. 3. Hoyer JD, Penz CS, Fairbanks VF, et al. Flow cytometric measurement of hemoglobin F in RBCs: diagnostic usefulness in the distinction of hereditary persistence of fetal hemoglobin (HPFH) and hemoglobin S-HPFH from other conditions with elevated levels of hemoglobin F. Am J Clin Pathol 2002; 117: 857-863. 4. Weatherall DJ, Legg JB. Hereditary persistence of fetal hemoglobin. In: The thalassemia Syndromes. 4th ed. Oxford: Blackwell Science, 2001: 450-484.
310

5. Wood WB. Hereditary persistence of fetal hemoglobin and thalassemia. In: Steinberg MH, Forget BG, Higgs DR, Nagel RL. Disorders of Hemoglobin: Genetics, Pathophysiology, and Clinical Management, Cambridge, England: Cambridge University Press; 2001: 356-388. 6. Pissard S, Mrad A, Beuzard Y, Romeo PH. A new type of hereditary persistence of fetal hemoglobin (HPFH): HPFH Tunisia beta + (+C-200) G gamma. Br J Haematol 1996; 95(1): 67-72. 7. Martin AW, Lippmann SB, Keeling MM, Lynch JA, Martinez M. Hemoglobin C in association with hereditary persistence of fetal hemoglobin. Postgrad Med 1987; 81(8): 133-37.

311

Case # 11

Hemoglobin S-C disease

A 22 year old African American male, who was working at the Chrysler Stamping plant, complained of headache and difficulty in breathing. His supervisor suspected carbon monoxide poisoning and sent him to the Emergency Department.

Laboratory Data:
Hemoglobin 10.8 RBC 3.6 MCV 90.1 MCH 30.0 Hb A2 2.4 Hb F 1.8 Hb Variant-1 49.0% Hb Variant-2 46.8% (Hemoglobin fractions from HPLC) 13.5 - 18.5 g/dL 3 4.6 - 6.2 Mil/mm 80 - 100 fL 27 - 34 pg 1.5 - 3.7% 0.0 - 2.0%

Peripheral Blood Smear: Target cells present. Rare spherocyte seen. Slight anisocytosis and polychromasia. Sickle cell solubility test for Hb S: Positive.

Agarose Gel Electrophoresis (pH 8.6)

312

Case # 11 Hemoglobin S-C disease

Citrate Agar Electrophoresis (pH 6.2)

Isoelectric focusing

313

Case # 11 Hemoglobin S-C disease

Capillary zone electrophoresis

High performance liquid chromatography

314

Case # 11 Hemoglobin S-C disease

Interpretation & Discussion

Summary of Results Method Hb A area Hb S area Major band Major band Major peak Hb S (Zone 5) Major band (Hb S) Major peak (Hb S) RT=4.37 Hb A2/C area Major band Major band Minor peak (Hb A2) Zone 3 Very minor band (Hb A2) Very minor peak (Hb A2) RT=3.6

Alk Agarose
Acid Agar/ Agarose

CZE

Major peak (Hb C) Zone 2

Major band (Hb C) slightly cathodal to A2

IEF

HPLC

Major peak (Hb C) RT=5.12

*Note: HPLC retention time (RT) varies with the type of the instrument used and several
other factors, e.g. temperature etc.

Agarose gel electrophoresis (pH 8.6) indicated the absence of Hb A, but two intense bands were detected in the position of Hb S and Hb C/E/O/A 2. Citrate agar electrophoresis (pH 6.2) also showed the absence of a band in the usual position of Hb A, here again two bands were detected in the position of Hb S and Hb C. The citrate agar electrophoresis (pH 6.2) ruled out the possibility of Hb S-E since Hb E migrates with Hb A in this system. The possibility of Hb
315

S-O Arab was also ruled out, however with less certainty since Hb O-Arab migrates between Hb S and Hb A in this system. Since two separate major bands in the position of Hb S and Hb C were detected, Hb C-Harlem (also called Hb C-Georgetown) was also ruled out, because this variant migrates with Hb S upon citrate agar electrophoresis (pH 6.2).

IEF confirmed the above results (absence of Hb A, and two major bands in the position of Hb S and Hb C). CZE has the advantage of fewer variants with mobility similar to Hb S, HbC, and Hb E. Since Hb A was absent in the patient, no zones were detected upon CZE. Therefore, the patients blood specimen was mixed (1:1) with a normal blood specimen, and two major peaks in the patient were present in Zone 2 (Hb C), and Zone 5 (Hb S). Similarly HPLC showed two major peaks (besides very minor peaks for Hb F and Hb A2) in the S window (RT= 4.37 minutes) and C window (RT= 5.12 minutes).

All the above stated tests support the diagnosis of Hb S-C disease in this patient.

Hb S-C disease is observed in approximately 0.13% of African Americans, which is approximately half of the homozygous Hb S disease. Most clinical manifestations of homozygous Hb S disease are also seen in Hb S-C disease, but in a somewhat milder form.

A characteristic of Hb S-C disease (first pointed out by Professor Virgil F. Fairbanks, MD, Mayo Clinic, Rochester, MN) is that the concentration
316

of Hb S is always slightly greater than Hb C. In addition, the cellular dehydration that occurs as a consequence of the presence of Hb C promotes the distortion of the shape of the red blood cells (Professor James D. Hoyer, MD, Mayo Clinic, Rochester, MN). Hemoglobin C-Harlem (also called Hb C-Georgetown) is a rare double chain mutation hemoglobin (
6(A3) GluVal

73(E73) AspAsn

) and patients

heterozygous for only Hb C-Harlem are asymptomatic. Compound heterozygous state (e.g. Hb S-C-Harlem) exhibits sickling, and also clinical severity.

The diagnosis of Hb S-C disease and homozygous Hb S disease is usually straight forward in the appropriate clinical context (e.g. African American patient).The diagnosis of Hb S-O Arab disease, Hb S-C-Harlem disease requires the evaluation of a large number of laboratory tests in conjunction with the clinical status of the patient. Special attention is required if the patient has been recently transfused.

References:
1. 2. Lionett F, Hammoudi N, Stojanovic KS, Avellino V, Grateau G, Girot R, Haymann J-P. Hemoglobin SC disease complications: a clinical study of 179 cases. Haematologica 2012; 97(8): 1136-1141. OKeefe EK, Rhodes MM, Woodworth A. A patient with a Previous Diagnosis of Hemoglobin S/C Disease with an unusually Severe Disease Course. Clin Chem 2008; 55(6): 1228-1231. Bain BJ. Sickle cell/hemoglobin C disease: In: Hemoglobinopathy Diagnosis, Blackwell Publishing, 2nd edition, 2006, pp 164-170.

3.

317

4.

5. 6.

7.

8.

9. 10.

Joutovsky A, Nardi M. Hemoglobin C and Hemoglobin O-Arab variants can be diagnosed using the Bio-Rad Variant II High-Performance Liquid Chromatography System without further confirmatory tests. Arch Pathol Lab Med 2004; 128: 435-439. Nagel RL, Fabry ME, Steinberg MH. The paradox of hemoglobin SC disease. Blood Reviews 2003; 17: 167-178. Powars DR. Hiti A, Ramicone E, Johnson C, Chan L. Outcome in Hemoglobin SC disease: A four-decade observational study of clinical, hematologic, and genetic factors. Am J Hematol 2002; 70: 206-215. Koduri PR, Agbemadzo B, Nathan S. Hemoglobin S-C disease revisited: Clinical study of 106 adults. Am J Hematol 2001; 68: 298300. Nagel RL, Steinberg MH, Hb S/C disease and Hb C disorders. In: Steinberg MH, Forget BG, Higgs DR, Nagle RL. Disorders of Hemoglobin: Genetics, Pathophysiology and Clinical Management. Cambridge, England: Cambridge University Press; 2001; 756-785. Bunn HF, Forget BG. Hemoglobin: Molecular, Genetic and Clinical Aspects. 1st ed. Philadelphia, PA: WB Saunders Co; 1986; 533-536. Bunn HF, Noguchi CT, Hofrichter J, Schechter GP, Schechter AN, Eaton WA. Molecular and cellular pathogenesis of hemoglobin S/C disease. Proc Natl Acad Sci USA. 1982; 79: 7527-7531.

318

Case # 12 Hemoglobin D-Los Angeles (D-Punjab) trait

First year resident (male, 26 years old) in the Department of Surgery. Originally from India (State of Punjab). Healthy and physically robust.

Laboratory Data:
Hemoglobin 14.7 RBC 4.9 MCV 82 MCH 30.2 Platelet 239 Hb A 58.0 Hb A2 1.5 Hb F 0.3 Hb Variant 40.2 (Hemoglobin fractions from HPLC) Peripheral Blood Smear: No abnormality Unstable hemoglobin (isopropanol) test: Negative Sickle cell solubility test for Hb S: Negative 13.5 - 18.5 g/dL 3 4.6 - 6.2 Mil/mm 80 - 100 fL 27 - 34 pg 3 150 - 400 Th/mm 94.3 - 98.5% 1.5 - 3.7% 0.0 - 2.0%

Agarose Gel Electrophoresis (pH 8.6)

319

Case # 12 Hemoglobin D-Los Angeles (D-Punjab) trait

Citrate Agar Electrophoresis (pH 6.2)

Isoelectric focusing

320

Case # 12 Hemoglobin D-Los Angeles (D-Punjab) trait

Capillary zone electrophoresis

High performance liquid chromatography

321

Case # 12 Hemoglobin D-Los Angeles (D-Punjab) trait

Interpretation & Discussion

Summary of Results Method Hb A area Major band (Hb A) Major band Major peak (Hb A) Zone 9 Major band (Hb A) Major peak (Hb A) RT=2.42 Hb S area Major band Hb A2/C area Minor band

Alk Agarose Acid Agar /Agarose CZE

Major peak (Hb D) Zone 6

Major Hb D band slightly anodic to S

IEF

HPLC

Minor peak (Hb A2) Zone 3 Minor band (Hb A2) Minor peak (Hb A2) RT=3.6

Major peak (Hb D) RT=3.99

*Note: HPLC retention time (RT) varies with the type of the instrument and several other
factors, e.g. temperature etc.

Agarose gel electrophoresis (pH 8.6) showed two major bands in approximately equal intensity at the positions of Hb A and Hb S. Citrate agar electrophoresis (pH 6.2) showed only one major band ( 100%) and barely visible staining in the Hb F position. Several hemoglobin variants migrate in the position of Hb S upon agarose gel electrophoresis (pH 8.6), and among them, the most frequently noticed are Hb G, Hb D, and very rarely Hb Korle-Bu (GAcra).

322

Hb S was easily ruled out on the basis of the negative sickle solubility test, Hb G (-chain variant) was ruled out on the basis of the absence of Hb G 2 band (2G2) and the observation that the percentage of the abnormal variant approaches 50%. -chain variants percentages do not run this high without other genetic complications (see Case# 5).

The differentiation between Hb D-Los Angeles and other kinds of heterozygous D hemoglobins (which are also -chain variants) or heterozygous Hb Korle-Bu (G-Accra) on the basis of electrophoretic tests (alkaline, acid, IEF, CZE) was not possible with certainty due to identical mobilities. HPLC differentiated Hb D-Los Angeles from Hb Korle-Bu. We have summarized the HPLC retention times from three separate studies for Hb D-Los Angeles and Hb Korle-Bu: Nardi et al* Nardi-2013** Hoyer et al

Hb Korle-Bu Hb D- Los Angeles

3.92 + 0.050 4.18+ 0.007

3.9+ 0.034 4.11+ 0.078

3.88+ 0.08 4.08+ 0.08

* Bio-Rad Variant II (Clin Chem 2004; 50: 1736-1747) ** Bio-Rad Variant II (personal communication) Bio-Rad Variant Classic (Intl J Lab Hematol 2012; 34: 594-604) It is the observation of Professor Michael A. Nardi (personal communication) that Hb Korle-Bu rarely separates from Hb A2 (due to the closeness of their retention times), while Hb D-Los Angeles always separates from Hb A2.

323

In view of the laboratory tests, the diagnosis of Hb D-Los Angeles trait was most likely. Since the patient had a clinically silent and harmless condition, it was not advised to perform globin chain analysis and DNA studies.

Hb D-Los Angeles results from a substitution of glutamic acid by glutamine on position 121 of the -chain [121(GH4)GluGln.GAA>CAA] and is a harmless condition. Hb D-Los Angeles has been found double heterozygotes for other variants (e.g., Hb S, Hb C, Hb E). Hb D-Los Angeles in combination with Hb S causes a severe sickling disorder (Case # 13).

Homozygous Hb D-Los Angeles patients exhibit normal hematologic indices (e.g. hemoglobin, RBC), and no evidence of hemolysis. However, patients with Homozygous Hb D-Los Angeles and -Thalassemia do have a mild anemia and mild hemolysis.
o

References
1. 2. Pandey S, Mishra RM, Pandey S, Saxena R. Homozygous hemoglobin D with alpha thalassemia: case report. Open Journal of Hematology 2011; 2: 1-4. Basmanj MT, Karimpoor M, Amirian A, Jafrinejad M, Katouzian L, Valei A, Bayat F, Kordafshari A, Zeinali S. Co-inheritance of Hemoglobin D and thalassemia Traits in Three Iranian Families: Clinical Relevance. Archives of Iranian Medicine 2011;14(1): 61-63. Srinivas U, Pati HP, Saxena R. Hemoglobin D-Punjab syndromes in India: a single center experience on cation-exchange high performance liquid chromatography. Hematology 2010; 15 (3): 178-181. Yavarian M, Karimi M, Paran F, Neven C, Harteveld CL, Giordano PC. Multi Centric Origin of Hemoglobin D-Punjab [121(GH4)GluGLN, GAA>CAA]. Hemoglobin 2005; 29 (4): 307-310.

3.

4.

324

5.

6. 7.

8. 9.

10. 11.

12.

13. 14.

Atalay EO, Koyuncu H, Turgut B, Atalay A, Yildiz S, Bahadir A, Koseler A. High incidence of Hb D-Los Angeles [121(GH4)GluGln] in Denizli Province, Aegean Region of Turkey. Hemoglobin 2005; 29(4): 307-310. Owaidah TM, Al-Saleh MM, Al-Hellani AM. Hemoglobin D/-thallasemia and -thalassemia major in a Saudi family. Saudi Med J 2005; 26(4): 674-677. Thornburg CD, Zimmerman SA, Schultz WH, Ware RE. An infant with Homozygous D-Iran. Journal of Pediatric Hematology/Oncology 2001; 23(1): 67-68. El-Kalla S, Mathews AR. Hb D-Punjab in the United Arab Emirates. Hemoglobin 1997; 21(4): 369-375. Zago MA, Costa FF. Hb D-Los Angeles in Brazil: Simple Heterozygotes and Associations with -Thalassemia and with Hb S. Hemoglobin 1988; 12(4): 399-403. Harano T, Harano K, Ueda S, Nakaya K. Hb D-Los Angeles [121 GluGln] in Japan. Hemoglobin 1987; 11(2): 177-180. Li HJ, Liu DX, Li L, Liu ZG, Lo SL, Zhao J, Han XP, Yu WZ. A Note About The Incidence And Origin of Hb D-Punjab in Xinjiang, Peoples Republic of China. Hemoglobin 1986; 10(6): 667-671. Husquinet H, Parent MT, Galacteros F. Hemoglobin D-Los Angeles [121 (GH4)GluGln] in the Province of Liege, Belgium. Hemoglobin 1986; 10(6): 587-592. Baiget M, del Rio E, Gimferrer E. Hemoglobin D-Punjab (121 GluGln) in a Spanish Family. Hemoglobin 1982; 6(2):193-198. Ramot B, Rotem J, Rahbar S, Jacobs AS, Udem L, Ranney HM. Hemoglobin D-Punjab in a Bulgarian Jewish Family. Israel J. Med. Sci. 1969; 5(5):10661070.

325

Case # 13

Hemoglobin S-D disease

17 years old male patient. No other information provided due to the privacy requested by the patient. No record of blood transfusion during the past three months.

Laboratory Data:
Hemoglobin RBC MCV MCH Platelet Hb A (mostly Hb A1c) 10.2 3.2 90.7 30.9 229 6.0 1.5 - 3.7% 0.0 - 2.0% 13.5 - 18.5 g/dL 3 4.6 - 6.2 Mil/mm 80 - 100 fL 27 - 34 pg 3 150 - 400 Th/mm

Hb A2 2.7 Hb F 2.5 Hb Variant-1 49.3 Hb Variant-2 39.5 (Hemoglobin fractions from HPLC)

Peripheral Blood Smear: Moderate sickle cells. Target cells and polychromasia. Sickle cell solubility test for Hb S: Positive. Hemoglobin instability (isopropanol) test: Negative.

Agarose Gel Electrophoresis (pH 8.6)

326

Case # 13 Hemoglobin S-D disease

Citrate Agar Electrophoresis (pH 6.2)

Isoelectric focusing

327

Case # 13 Hemoglobin S-D disease

Capillary zone electrophoresis

High performance liquid chromatography

328

Case # 13 Hemoglobin S-D disease

Interpretation & Discussion

Summary of Results Method Hb A area Hb S area Major band Major band (Hb S) Major* peak (Hb D) Zone 6 IEF Major* peak (Hb S) Zone 5 Major band (Hb S) Major peak (Hb S) RT=4.3 Hb A2/C area Minor band

Alk Agarose Acid Agar /Agarose CZE

Major band (Hb D)

Minor peak (Hb A2) Zone 3

Major band anodal to S position (Hb D)

Minor band (Hb A2) Minor peak (Hb A2) RT=3.6

HPLC

Minor peak (Hb F) RT=1.05

Major peak (Hb D) RT=4.0

* Overlap of the two peaks (Zone 5-6) due to approximately equal and higher
concentration of Hb S and Hb D-Los Angeles. Note: HPLC retention time (RT) varies with the type of instrument used and several other factors, e.g. temperature etc.

Agarose gel electrophoresis (pH 8.6) showed a major and very intense band in the position of Hb S. Another band of faint intensity was detected in the Hb F position. A faint band in the position of Hb C/E/O/A2 was also noticed.
329

No band was detected in the position of Hb A. Citrate agar electrophoresis (pH 6.2) presented with two major bands in approximately equal intensity in the position of Hb A and Hb S. A faint band was also detected in the position of Hb F. Since the sickle cell solubility test was positive, therefore the band in the Hb S position upon citrate agar electrophoresis (pH 6.2) suggested the presence of a -chain variant (Hb S). The migration of a band of equal intensity in the position of Hb A upon citrate agar electrophoresis (pH 6.2) suggested the presence another hemoglobin variant (since Hb A was absent upon alkaline agarose gel electrophoresis). Several hemoglobin variants (e.g. Hb G, Hb D-Los Angeles, Hb Korle-Bu, etc) exhibit this kind of migration pattern, therefore assignment of this hemoglobin variant was deferred.

IEF confirmed the presence of Hb S, however another band in between the customary position of Hb G and Hb S was also prominent. The presence of Hb G from IEF was ruled out positively as no band in the position of Hb G2 (2G2) was detected. Since Hb D-Los Angeles and Hb Korle-Bu have similar mobilities upon IEF, therefore a distinction could not be made between these two possibilities. HPLC was helpful in differentiating between the Hb D-Los Angeles and Hb Korle-Bu variants, as Hb D-Los Angeles has a longer retention time (4.0 minutes) as compared to Hb Korle-Bu (3.75 minutes). Hb S eluted at retention time of 4.3 minutes, thus the two major bands in this case were separated nicely upon HPLC.

330

In a heterozygous situation upon CZE, Hb S migrates in Zone 5 and Hb DLos Angeles in Zone 6. In this case since the concentration of the two variants is intense ( 40-49% from HPLC), thus clearly separated peaks were not detected but the scan positively showed two overlapping peaks in the position of Zones 56. Distinct peaks for Hb F and Hb A2 from CZE were noticed in Zone 7 and and Zone 3 respectively.

The specimens of father and mother of this person were not available for additional studies. Furthermore globin chain and DNA studies were also not done on the blood of this person. On the basis of the available laboratory data a tentative diagnosis of a double heterozygosity of Hb S [6 (A3) GluVal] and Hb D-Los Angeles [121(GH4)GluGln.GAA>CAA] was advised to the physician.

Hb D-Los Angeles in both the heterozygous (Case # 12) and homozygous state is clinically silent and harmless. However patients with homozygous Hb DLos Angeles and -thalassemia do have mild anemia and also exhibit mild hemolysis. Hb D-Los Angeles is not itself a sickling hemoglobin, but compound heterozygosity (Hb S + Hb D-Los Angeles) produces a severe sickle cell anemia because Hb D-Los Angeles enhances Hb S polymerization by forming an additional contact stabilizing the Hb S polymer.
o

331

References
1. 2. 3. Adekile A, Mullah-Ali A, Akar NA. Does Elevated Hemoglobin F Modulate the Phenotype of Hb SD-Los Angeles?. Acta Haematol 2010; 123: 135-139. Isoa EM. Current Trends in the Management of Sickle Cell Disease: An Overview. Benin J Postgraduate Med 2009; 11:50-73. Mukherjee MB, Surve RR, Gangakhedkar RR, Mohanty D, Colah RB. Hemoglobin sickle D Punjab-a case report. Indian J Hum Genetics 2005; 11(3): 154-155. Jiskoot PMC, Halsey C, Rivers R, Bain BJ, Wilkins BS. Unusual splenic sinusoidal iron overlaod in sickle cell/haemoglobin D-Punjab disease. J Clin Pathol 2004; 57: 539-540. Athanasiou-Metaxa M, Economou M, Tstra I, Pratsidou P, Tsantali C. CoInheritance of Hemoglobin D-Punjab and Hemoglobin S: Case Report. J Ped Hematology/Oncology 2002; 24(5): 421. Perea FJ, Casas-Castaneda M, Villalobos-Arambula AR, Barajas H, Alverez F, Camacho A, Hermosillo RM, Ibrarra B. Hb D-Los Angeles Associated with Hb S or -Thalassemia in Four Mexican Mestizo Families. Hemoglobin 1999; 23(3): 231-237. Dash S. Haemoglobin S-D Disease in a Bahraini Child. Bahrain Med Bulletin 1995; 17(4): 154-56. Samperi P, Dibenedetto SP, Cataldo AD, Mancuso GR, Schiliro G. Unusual Sickle Cell Disease observed for the First Time in Italy. Haematologica 1990; 75: 464-66. McCurdy PR, Lorkin PA, Casey R, Lehmann H, Uddin DE, Dickson LG. Hemoglobin S-G (S-D) Syndrome. The American J of Med 1974; 57: 665-670. Barton LL, Stark AR, Zarkowsky HS,.Hemoglobin S-D disease in a Negro Child. The Journal of Pediatrics 1973; 82(1): 164-165. Ozsoylu S. Haemoglobin S-D Disease in a Turkish Family. Scand. J Haematol 1969; 6: 10-14. Cawein MJ, Lappat EJ, Brangle RW, Farley CH. Hemoglobin S-D Disease. Annals of Internal Medicine 1966; 64(1): 62-70.

4.

5.

6.

7. 8.

9. 10. 11. 12.

332

Case # 14 Hemoglobin E and Associated Disorders

The contents of this section are presented from Hoyer JD, Kroft SH, eds. Color Atlas of Hemoglobin Disorders: A Compendium Based on Proficiency Testing. Northfield, IL: College of American Pathologists; 2003 (Reproduced with Permission). In addition to Hb E, several other disorders of hemoglobin are prevalent in the Southeast Asian population. Therefore, Hb E may be encountered in conjunction with another abnormality. A description of the various Hb E-associated disorders is provided below. 1. Hb E trait. A harmless condition characterized by mild microcytosis and often by erythrocytosis. No icterus, no splenomegaly, no anemia. MCV about 75 fL (adult). Electrophoresis: Hb E 30-35%, Hb A 65-70%, Hb F <2%. 2. Homozygous Hb E. A harmless condition characterized only by mild microcytosis and erythrocytosis. No icterus, no splenomegaly, no anemia (hemoglobin concentration >11 g/dL in females, >14 g/dL in males). MCV about 67 fL (adults). Electrophoresis: Hb E about 99%, the rest Hb F. 3. Hb E trait/-thalassemia. This combination results in microcytosis, but usually no other adverse effects (no anemia, no splenomegaly, no icterus). Serum ferritin assay is required to differentiate this condition from Hb E trait/iron deficiency. Electrophoresis (1 gene deletion): Hb E 2530%; remainder Hb A; Hb F normal. Electrophoresis (2 gene deletion): Hb E 20-25%; remainder Hb A; Hb F normal. Since Hb E and Hb A2 comigrate in all electrophoresis media and co-elute from chromatography
333

columns, a common laboratory error is to ascribe the electrophoresis findings to -thalassemia trait. However, in the latter, Hb A2 is always <10%. 4. Hb E trait/Hb H disease. In this disorder, Hb E trait is inherited in conjunction with a three locus gene deletion. This is a moderately severe thalassemic disorder with features identical to Hb H disease. However, electrophoresis does not reveal Hb H. Instead, Hb E represents about 10-15% of hemoglobin; most of the remainder is Hb A. This paradox is due to reduced total synthesis of globin chains. As a result, not enough surplus chains are present to form tetramers (Hb H). Instead, Hb Bart's is present. (Thus, this condition has also been called "Hb A + E + Bart's Disease"). 5. Homozygous Hb E/Hb H disease. This disorder has the same features as Hb H disease. However, electrophoresis reveals mostly Hb E (about 95%) and a small proportion of Hb F. It is believed that in this condition, the tetramers co-migrate with Hb E in all electrophoresis media. 6. Hb E trait/-thalassemia/Hb Constant Spring. Features are the same as 4 and 5 above, except for faint additional hemoglobin bands (as many as five) between the positions of Hb E and the site of application. These additional faint bands represent Hb Constant Spring. 7. Hb E trait/iron deficiency. A benign condition characterized by microcytosis, often erythrocytosis, and anemia. The anemia is due to iron
334
E

deficiency and thus may be minimal to severe. There is no icterus and no splenomegaly. Electrophoresis shows the same pattern as Hb E trait/thalassemia. The combination should be suspected in an anemic patient with an "Hb A2" concentration of 10-20%. The diagnosis is confirmed by a serum ferritin assay. Following treatment, repeat electrophoresis will show Hb E representing 30-35% of total (unless the patient also has Hb E trait/-thalassemia). 8. Hb E/-thalassemia. This is a serious thalassemic disorder due to compound heterozygosity for both Hb E trait and -thalassemia trait. Characteristics are severe anemia, icterus, marked splenomegaly, and microcytosis. Affected children suffer all the problems of -thalassemia major. Most require frequent transfusions and should also receive iron chelation therapy. This is the most common severe thalassemia of Southeast Asians. Neurologic manifestations are often reported that are due to brain or spinal cord compression by extramedullary hematopoietic tumors, which may cause paraplegia. The tumors respond to radiotherapy. Electrophoresis: Hb E is 40-90% total; the rest is Hb F. (Note: Because these patients usually require transfusion, Hb A may be present from donor blood). It should be pointed out that it is not necessary to document elevated Hb A2 levels to establish a diagnosis of Hb E/thalassemia. The diagnosis is easily established on the basis of an Hb E

335

concentration >40% with the remainder representing Hb F (usually 3060%) and an absence of Hb A. 9. Hb E/-thalassemia, post-splenectomy. Same condition as # 8 (see above), but often confusing in laboratories. Splenectomy is a common treatment in Hb E/-thalassemia and is reputed to be beneficial for those with severe anemia. The post-splenectomy blood picture is characterized by marked normoblastemia and a positive solubility test for sickling hemoglobin. The latter is due to the large number of normoblast nuclei causing strong persistent turbidity. Pulmonary artery occlusion is a common complication in splenectomized patients with Hb E/thalassemia. Prophylactic therapy with daily doses of aspirin or dipyridamole is indicated for all patients with this disorder who have been splenectomized.

Note: It will not be out of place to mention here that another disorder Hb S-E heterozygous has been also diagnosed in persons of Southeast Asian origin (Case # 14 C).

References 1. Fucharoen S, Weatherall DJ. The Hemoglobin E Thalassemias. Cold Spring Harb Perspect Med 2012; 2: a011734. 2. Sae-ung N, Srivorakun H, Fucharoen G, Yamsri S, Sanchaisuriya K, Fucharoen S. Phenotypic expression of hemoglobins A2 , E and F in various hemoglobin E related disorders. Blood Cells, Molecules, and Diseases 2012; 48: 11-16. 3. Tatu T, Kasinrerk W. A novel test tube method of screening for hemoglobin E. Int. Lab. Hem 2012; 34: 59-64. 4. Moiz B, Hashmi MR, Nasir A, Rashid A, Moatter T. Hemoglobin E syndromes in Pakistani population. NMC Blood Disorders 2012; 12: 1-6.
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5. Khan MR, Aziz MA, Shah MSU, Imam H. Hemoglobin E trait- in Rajshahi, Bangladesh. Bangladesh Med ResCounc Bull 2012; 38: 72-73. 6. Tamminga RYJ, Doombos ME. Muskiet FAJ, Koetse HA. Rhabdomyolysis in a child with Hb SE. Pediatric Hematology-Oncology 2012; 29(3): 267-269. 7. Edison ES, Shaji RV, Chandy M, Srivasta A. Interaction of Hemoglobin E with Other Abnormal Hmoglobins. Acta Haematol 2011; 126: 246-248. 8. Tay SH, Teng GG, Poon M, Lee VKM, Lim AYN. A Case of Hemoglobin SE Presenting with Sickle Cell Crisis: Case Report and Histological Correlation. Annl Acad Med 2011; 40 (12): 552-553. 9. Colah R, Gorakshakar A, Nadkarni A. Global burden, distribution and prevention of -thalassemias and hemoglobin E disorders. Expert Review of Hematology 2010; 3: 103-117. 10. Patel J, Patel A, Patel J, Kaur A, Patel V. Prevalence of Haemoglobinopathies in Gujrat, India: A Cross-Sectional Study. The Internet Journal of Hematology 2009; 5 (1): DOI: 10.5589/1764. 11. Intorasoot S, Thongpung R, Tragoolpua K, Chottayaporn M. Hemoglobin E Detection Using PCR with Confronting Two-Pair Primers. J Med Assoc Thai 2008; 91: 1677-1680. 12. Masiello D., Heeney MM, Adewoye AH, Eung SH, Luo Hong-Yuan, Dteinberg MH, Chui D HK. Hemoglobin S-E Disease- A Concise Review. Am H Hematol 2007; 82: 643-649. 13. Jetsrisuparb A, Sanchaisuriya K, Fucharoen G, Fucharoen S, Wiangnon S, Jetsrisuparb C, Sirijirachai J, Chansoong K. Development of Severe Anemia During Fever Episodes in Patients with Hemoglobin E trait and Hemoglobin H Disease Combinations. J Pediatr Hematol Oncol 2006; 28 (4): 249-253. 14. Bain BJ. Hemoglobin E. Other significant hemoglobinopathies. In: Hemoglobinopathy Diagnosis. 2nd Ed, 2006, pg 201-209, Blackwell Publishing, London. 15. Edison ES, Shaji RV, Srivastava A, Chandy M. Compound Heterozygosity for Hb E and Hb Lepore-Hillandia in India: First report and potential diagnostic pitfalls. Hemoglobin 2005; 29(3): 221-224. 16. Andino L, Risin SA. Pathologic Quiz case. A 24-Year-Old Woman With Abnormal Hemoglobin and Thrombocytopenia. Arch Pathol Lab Med 2005; 129: 257-258. 17. Mishra P, Pati HP, Chatterjee T, Dixit A, Choudhary DR, Srinivas MV, Mahapatra M, Choudhary VP. HB SE Disease: a clinico-hematological profile. Ann Hematol 2005; 84: 667-670. 18. Sirichotiyakul S. Tongprasert F, Tonsong T. Screening for hemoglobin E trait in pregnant women. Intl J Gyn & Obstet 2004; 86: 390-392. 19. Fucharoen S, Sanchaisuriya K, Fucharoen G, Panyasai S, Devenish R, Luv L. Interaction of hemoglobin E and several forms of -thalassemia in Cambodian families. Haematologica 2003; 88: 1092-1098. 20. Piplani S. Hemoglobin E Disorders in the North East India. JAPI 2000; 48(11): 1082-1084.
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21. Fucharoen S. Hemoglobin E disorders. In: Steinberg MH, Forget BG, Higgs DR, Nagel RL, eds. Disorders of Hemoglobin: Genetics, Pathophysiology and Clinical Management. Cambridge, England: Cambridge University Press; 2001: 1139-1154. 22. Gupta R, Jarvis M, Yardumian A. Compound Heterozygosity for hemoglobin S and hemoglobin E. Br J Haematol 2000, 108: 463. 23. Joseph VJ, Sunny AO, Pandit N, Yeshwanth M. Double Heterozygosity for Hemoglobin S and E. Indian Pediatrics 1992; 29: 895-897. 24. Fairbanks VF, Gilchrist GS, Brimhali B, Jereb JA, Goldston EC. Hemoglobin E Trait Rexamined: A Case of Microcytosis and Erythrocytosis. Blood 1979; 53(1): 109-115.

338

Case # 14 a

Hemoglobin E trait

A 56 year old male of Southeast Asian origin, migrated to America in 1972 with his parents. Physical examination showed no abnormalities.

Laboratory Data:
Hemoglobin RBC MCV RDW Platelet Hb A Hb A2 (CZE) Hb variant 14.8 5.7 74 13.5 240 64.0 2.0 34.0 13.5-18.5 g/dL 3 4.6-6.2 Mil/mm 80-100 fL 11.5-14.5% 3 150-400 Th/mm 94.3-98.5% 1.5-3.7%

Peripheral Blood Smear: Slight microcytosis, and occasional target cells Sickle cell solubility test for Hb S: Negative Hemoglobin instability (isopropanol) test: Positive

Agarose Gel Electrophoresis (pH 8.6)

339

Case # 14 a Hemoglobin E trait

Citrate Agar Electrophoresis (pH 6.2)

Isoelectric focusing

340

Case # 14 a Hemoglobin E trait

Capillary zone electrophoresis

High performance liquid chromatography

341

Case # 14 a Hemoglobin E trait

Interpretation & Discussion

Summary of Results Method Alk Agarose Hb A area Major band (Hb A) Major band (Hb A+ Hb E+ Hb A2) Major peak (Hb A) Zone 9 Major band (Hb A)
*

Hb S area

Hb A2/C area Major band (Hb E + Hb A2)

Acid Agar/Agarose

CZE

Major peak (Hb E) Zone 4

Major band (Hb E) Slightly anodal to Hb A2

IEF

Minor peak (Hb A2) Zone 3 Very minor band (Hb A2) Major peak (Hb E + Hb A2) RT=3.63

HPLC

Very faint peak (Hb F) RT=1.07

Major peak (Hb A) RT=2.42

* Note: HPLC retention time (RT) varies with the type of the instrument used and several other factors, e.g. temperature etc.

Alkaline agarose electrophoresis (pH 8.6) showed two major bands in the position of Hb A (65%), and Hb C/E/O/A2 (35%). Citrate agar electrophoresis showed only one band in the position of Hb A. This kind of electrophoretic migration pattern (pH 8.6 and 6.2) ruled out the possibility of Hb C
342

and Hb O, and suggested the possibility of Hb E, as Hb A2 is never > 10%. IEF also showed a major band in the position of Hb A and another band slightly anodal to Hb A2 suggesting the presence of Hb E variant. Hb E and Hb A2 coeluted upon HPLC, therefore their quantification was not feasible. However, CZE presented three distinct peaks in the zones for Hb A, Hb E and Hb A2 and also provided quantification of the peaks.

Hb E is a -chain variant (226Glu-Lys) and is the second most prevalent hemoglobin variant in the world after Hb S. It is prevalent in sixteen Southeast Asian countries, however it is also encountered in Europe and North America. A diagnosis of Hb E trait was made in view of the electrophoretic results and the following characteristics: Microcytosis, hypochromia, target cells, irregularly contracted cells, basophilic stippling or any combination of these features of the peripheral blood film. Negative sickle cell solubility test. Positive isopropanol test for unstable hemoglobin. Per se harmless condition and not associated with anemia. Hb A2 and Hb F in the normal range. Hb E in the range of 30-39%. Hb E is a -chain variant, however the chain is synthesized in Hb E trait at a reduced rate in comparison with . In view of this slower ribosomal synthesis of chain, a mild thalassemic blood picture is also witnessed.
E A E

343

Case # 14 b

Hemoglobin E homozygous

A 25 year old female from Laos-Northern Vietnam region, asymptomatic; physical examination showed no abnormalities. No icterus, no splenomegaly.

Laboratory Data:
Hemoglobin RBC MCV MCH Platelet Hb A Hb A2 (CZE) Hb F (HPLC) Hb variant Peripheral Blood Smear: 14.1 5.7 68 24.9 223 Not Detected 0.5 0.8 98.7% 12.0-16.0 g/dL 3 4.0-5.5 Mil/mm 80-100 fL 26-34 pg 3 150-400 Th/mm 94.3-98.5% 1.5-3.7% 0.0-2.0%

Significant microcytosis, hypochromia, target cells and occasional basophilic stippling of erythrocytes. Sickle cell solubility test for Hb S: Negative Hemoglobin instability (isopropanol) test: Positive

Agarose Gel Electrophoresis (pH 8.6)

344

Case # 14 b Hemoglobin E homozygous

Citrate Agar Electrophoresis (pH 6.2)

Isoelectric focusing

345

Case # 14 b Hemoglobin E homozygous

Capillary zone electrophoresis

High performance liquid chromatography

346

Case # 14 b Hemoglobin E homozygous

Interpretation & Discussion

Summary of Results Method Hb A area Hb S area Hb A2/C area Major band (Hb E + Hb A2) Major band (Hb E + Hb A2) Major peak (Hb E) Zone 4 Very minor peak (Hb A2) Zone 3 Major band (Hb E) slightly anodal to Hb A2 Major peak (Hb E + Hb A2) RT=3.65

Alk Agarose

Acid Agar / Agarose

CZE

IEF

HPLC*

In addition to Hb F peak at RT= 1.05 minutes, there are two additional minor peaks at RT= 1.75 minutes and RT= 2.1 minutes. The peak at RT=2.1 minutes (A0 window) must not be construed as Hb A. Similar peaks were detected upon HPLC (Bio-Rad Variant II), and were alleged to post-translationally modified Hb E (Other Significant Hemoglobinopathies. In: Hemoglobinopathy Diagnosis, Bain, Barbara J., 2nd edition, pg 206, Blackwell Publishing, 2006).
347

Alkaline agarose electrophoresis (pH 8.6) showed only one band in the position of Hb C/E/O/Hb A2, therefore Hb A was not present. Citrate agar electrophoresis (pH 6.2) indicated only one major band in the position of Hb A, thus the possibility of Hb C and Hb O was ruled out. IEF also indicated the absence of Hb A and only one major band slightly anodal to Hb A2 was detected. Absence of Hb A was also shown by HPLC, and only one major peak eluted at RT = 3.65. CZE clarified the ambiguity about the solitary band /peak in electrophoretic methods and HPLC, and two peaks were detected at Zone 4 (major peak presumably of Hb E) and Zone 3 (minor peak of Hb A2). A diagnosis of Hb E homozygous was made in view of the electrophoretic, and HPLC results and the following characteristics: Absence of anemia and hemolysis. Spleen was not enlarged. Negative sickle cell solubility test. Isopropanol test positive for unstable hemoglobin. Increased red cell count, normal hemoglobin, decreased MCV and MCH. Significant microcytosis, hypochromia, and variable number of target cells. Harmless condition. Hb A2 and Hb F in the normal range. Hb E concentration >95 %.

Homozygous Hb E is a clinically benign condition. Unfortunately, it is prevalent in the population areas (e.g. Cambodia and Northeastern India) that also have the higher frequency of -thalassemia minor. Therefore genetic counseling is advised to prevent the occurrence of severe thalassemic
348

(Hb E/ thalassemia) disorders in their children.

On the basis of hematological studies alone, homozygous Hb E may be confused with iron deficiency and -thalassemia. The following characteristic features can distinguish between Hb E/-thalassemia and homozygous Hb E. In Hb E/-thalassemia the concentration of Hb E varies between 40-70%, and the Hb F concentration is found in the range of 30-60%. In homozygous Hb E the Hb F concentration is in the normal range, and Hb E concentration is >95%.

349

Case # 14 c

Hemoglobin S-E heterozygous disorder

A 29 year old female nurse (Southeast Asian descent) complained of knee joint pain and weakness of the lower extremities. Hemoglobin electrophoresis was ordered after lower MCV was found from CBC. Clinical and laboratory data of her parents were not available to the physician.

Laboratory Data:
Hemoglobin RBC MCV MCH Platelet Hb A Hb A2 (CZE) Hb F (HPLC) Hb S Hb E 12.2 4.6 72 26 212 Not Detected 0.5 0.5 55% 44% 12.0-16.0 g/dL 3 4.0-5.5 Mil/mm 80-100 fL 26-34 pg 3 150-400 Th/mm 94.3-98.5% 1.5-3.7% 0.0-2.0%

Peripheral Blood Smear: Hypochromia, microcytosis, irregular contracted cells, occasional target cells, polychromatic cells. Hemoglobin instability (isopropanol) test: Positive Sickle cell solubility test for Hb S: Positive

Agarose Gel Electrophoresis (pH 8.6)

350

Case # 14 c Hemoglobin S-E heterozygous disorder

Citrate Agar Electrophoresis (pH 6.2)

Isoelectric focusing

351

Case # 14 c Hemoglobin S-E heterozygous disorder

Capillary zone electrophoresis

High performance liquid chromatography

352

Case # 14 c Hemoglobin S-E heterozygous disorder

Interpretation & Discussion

Summary of Results Method Hb A area Hb S area Hb A2/C area Major band (Hb E)

Alk Agarose

Major band (Hb S) Major band (Hb E) Major band (Hb S) Major peak (Hb S) Zone 5 Major band (Hb S) Major peak (Hb E) Zone 4

Acid Agar/ Agarose CZE

Minor peak (Hb A2) Zone 3 Major band (Hb E) slightly anodal to Hb A2 Major peak (Hb E + Hb A2) RT=3.65

IEF

HPLC*

Major peak (Hb S) RT=4.48

*Note: HPLC retention time (RT) varies with the type of the instrument used and several other factors, e.g. temperature etc.

Alkaline agarose gel electrophoresis (pH 8.6) showed two major bands in the position of Hb S (55%) and Hb C/E/O/A2 (45%). Citrate agar
353

electrophoresis (pH 6.2) confirmed the presence of a major band due to Hb S and presence of another band at the position of Hb A ruled out the possibility of Hb C and Hb O. IEF indicated a major band in the position of S and another major band in the position of Hb A2. These three electrophoretic methods suggested the presence of double heterozygous Hb S and Hb E. HPLC was not helpful as both Hb E and Hb A2 co-eluted at RT = 3.65. CZE provided a clear separation of the three hemoglobin entities, i.e. Hb S (Zone 5), Hb E (Zone 4) and Hb A2 (Zone 3), therefore a presumptive diagnosis of Hb S-E double heterozygous disorder was made. Hb S [6 (A3) GluVal) and Hb E [26 (8) GluLys] are the two most prevalent hemoglobin variants in the world. However, due to their existence in different ethnic groups and continents, compound heterozygosity for Hb S and Hb E is extremely rare. As of 2011 only thirty (30) cases were reported, therefore hematological parameters are too scant to provide a module for diagnostic purposes.

Majority of Hb S-E subjects have mild or absent anemia, microcytic indices, and some target cells. Contrary to some earlier reports, a severe sickle cell crisis was recently reported in a 66-year-old Bangladeshi woman in Singapore (Ann Acad Med 2011; 40: 552-553).

A recent review of Hb S-E double heterozygosity by Masiello et al (Am J Hematol 2007; 82: 643-649) mentioned that patients aged 18 and younger are
354

usually well. Sickling-related complications, including potentially life threatening acute chest syndrome was developed in a majority of cases. Generally these patients have Hb S concentration in the range of 60-65%, which is also similar to the patients with Hb S- -thalassemia, and therefore hematological features and clinical course of these patients appeared to parallel those of Hb S- thalassemia.
+ +

Coincidental complication per se not related to hemoglobinopathy was also reported, e.g. idiopathic thrombocytopenic purpura in a 28-year-old woman with Hb S-E heterozygosity (Arch Pathol Lab Med 2005, 129: 257-58).

355

Case # 15 Hemoglobin S-Korle Bu (G-Accra)

A 23 year old female administrative assistant of Ghanaian decent at the Embassy of Ghana in Washington, DC was found to have abnormal hemoglobinopathy. Physical examination was unremarkable.

Laboratory Data:
Hemoglobin RBC MCV MCH MCHC RDW Platelet Hb A Hb S (HPLC) Hb Korle-Bu Hb A2 (CZE) Hb F 13.1 4.4 82.1 27.3 32.1 12.6 267 Not Detected 53.4 % 45% 1.6 Not Detected 12.0 16.0 g/dL 3 4.0 5.5 Mil/mm 79-98 fL 26-34 pg 31-36% 11.5-14.5% 3 150-400 Th/mm 94.3-98.5%

1.5 -3.7% 0.0-2.0%

Peripheral Blood Smear: No abnormality was present. Sickle cell solubility test for Hb S: Positive Hemoglobin instability (isopropanol) test: Negative

Agarose Gel Electrophoresis (pH 8.6)

356

Case # 15 Hemoglobin S-Korle Bu (G-Accra)

Citrate Agar Electrophoresis (pH 6.2)

Isoelectric focusing

357

Case # 15 Hemoglobin S-Korle Bu (G-Accra)

Capillary zone electrophoresis

High performance liquid chromatography

358

Case #15 Hemoglobin SKorle Bu (G-Accra)

Interpretation & Discussion

Summary of Results Method Hb A area Hb S area
Broad Major band starting anodic to S (Hb Korle-Bu + Hb S) Major band slightly anodic to Hb A position (Hb KorleBu)
Major band in Hb A position (Hb KorleBu)

Alk Agarose

Hb A2/C area Very faint band (Hb A2)

Acid Agar

Major band (Hb S)

Acid Agarose

Major band (Hb S) Major peak Zone 5 (Hb S) Minor peak Zone 3 (Hb A2) Very faint band (Hb A2)

CZE

Small degradation peaks Zone 7

Major peak Zone 6 (Hb KorleBu)

Major band slightly anodal to Hb S band (HbKorleBu)

IEF

Major band (Hb S)

HPLC

Major peak RT=4.48 (Hb S)

359

Major peak (Hb A2 + Hb Korle Bu) RT=3.75

Alkaline agarose electrophoresis (pH 8.6) showed a major band in the area of Hb S and a very faint band in the Hb A2 area, but Hb A was not detected. Citrate agar electrophoresis (pH 6.2) showed two variants in the position of Hb S and very slightly anodic to the Hb A band position. IEF also indicated the presence of two variants, one in the position of Hb S and another band anodal to Hb S in the migration position of Hb D-Los Angeles/GPhiladelphia and few other variants. The presence of Hb S as one of the variants was also confirmed by HPLC, CZE and the positive solubility test. It is obvious that the three electrophoretic methods (i.e. alkaline agarose, acid agar, and IEF) could not identify the second major band with certainty. Hb G-Philadelphia was ruled out due to the absence of G2 variant ( 22) in both the alkaline agarose electrophoresis and IEF (Case # 5). In situations like this the patients history and clinical status may indicate the likelihood of the hemoglobin variant. Both of these hemoglobin variants (Hb D-Los Angeles and Hb Korle-Bu) are found in the population sector dominated by Hb S. HPLC is helpful in the separation of Hb DLos Angeles from Hb Korle-Bu, since Hb D-Los Angeles has a longer elution time (Case # 12) as compared to Hb Korle-Bu. The D-Los Angeles chain can be easily separated from
Korle-Bu G

chain by reverse phase chromatography, but all these

additional tests are not necessary. Hb S interacts with Hb D-Los Angeles causing sickle cell disease. Hb S also interacts with Hb Korle-Bu, but in opposite direction, i.e. inhibiting sickling. This patient does not have an abnormal blood picture so the second variant in this case is most likely Hb Korle-Bu (G-Accra).
360

Korle-Bu means valley of the Korle lagoon, and this hemoglobin was named after its discovery at Korle-Bu Hospital, Accra, Ghana. Initially it was called Hb G, and since several other hemoglobins with mobility similar to Hb G were discovered, its name was changed to Hb Accra. The reason for changing its name from Hb Accra to Hb Korle-Bu is not known to me. This mutation has been reported from the Ivory Coast, Costa Rica, and Mexico, so it is not highly prevalent but is widely spread. Hb Korle-Bu is the result of mutation GAT AAT at codon 73 of the chain [(E73)AspAsn]. Both the heterozygote and homozygote of Hb Korle -Bu are clinically normal.

References
1. AKL PS, Kutlar F, Patel N, Salisbury CL, Lane P, Young AN. Compound Heterozygosity for Hemoglobin S [6(A3)Glu6Val] and Hemoglobin Korle -Bu [73(E17)ASP73Asn]. Lab Hematol 2009; 15: 19-23. Chico A, Padros A, Novials A. The Korle-Bu Hb Variant in Caucasian Women With Type I Diabetes. A pitfall in the assessment of diabetes control. Diabetes Care 2004; 27(9): 2280-2281. Vukelja SJ. Hemoglobin Korle-Bu (G-Accra) in Combination with Hemoglobin C. Am J Hematol 1993; 42(4): 412. Nagel RL, Lin MJ, Witkowska HE, Fabry ME, Bestak M, Hirsch RE. Compound Heterozygosity for Hemoglobin and Korle-Bu: Moderate Microcytic Hemolytic Anemia and Acceleration of Crystal Formation. Blood 1993; 82(6): 1907-1912. Konotey-Ahulu FID, Gallo E, Lehman H, Ringelhann B. Hemoglobin Korle-Bu (73 aspartic acidasparagine). J Med Genet 1968; 5: 107-111.

2.

3. 4.

5.

361

Case # 16

Hemoglobin O-Arab trait

A 23 year old male student of Northern African descent at Michigan State University, East Lansing, Michigan, USA.

Laboratory Data:
Hemoglobin 14.8 RBC 4.9 MCV 86 MCH 29.3 MCHC 32.6 RDW 12.5 Platelet 273 Hb A 56.5% Hb O-Arab 41% Hb A2 2% Hb F 0.5% (Hemoglobin fractions from HPLC) Peripheral Blood Smear: No abnormality was present. Sickle cell solubility test for Hb S: Negative Hemoglobin instability (isopropanol) test: Negative 12.0 16.0 g/dL 3 4.0 5.5 Mil/mm 79-98 fL 26-34 pg 31-36% 11.5-14.5% 3 150-400 Th/mm 94.3-98.5 1.5 -3.7 0.0-2.0

Agarose Gel Electrophoresis (pH 8.6)

362

Case # 16 Hemoglobin O-Arab trait

Citrate Agar Electrophoresis (pH 6.2)

Isoelectric focusing

363

Case # 16 Hemoglobin O-Arab trait

Capillary zone electrophoresis

High performance liquid chromatography

364

Case # 16 Hemoglobin O-Arab trait

Interpretation & Discussion

Summary of Results Method Hb A area Alk Agarose Acid Agar Actually on Hb S side of Hb A Major band (Hb A) Major band May appear as a broader Hb A band Major band (Hb OArab) Actually migrates cathodal to Hb S) Hb S area Hb A2/C area Major band
(Hb OArab)

Actually on Hb S side of Hb C

Acid Agarose

Major band (Hb A) Small peak (Hb F) Zone 7 Major peak (Hb A) Zone 9 Major band
(Hb A)

Major band
(Hb OArab)

Actually on Hb C side of Hb S Minor peak

(Hb OArab)

CZE

IEF

Zone 3 Major band

(Hb OArab)

HPLC

Small peak (Hb F) RT=1.08

Major peak (Hb A) RT=2.38

Minor peak (Hb A2) RT=3.64

Major peak
(Hb OArab)

RT=4.3

Note: Separation under acidic conditions has traditionally been done with agar instead of
agarose because all the early descriptions of hemoglobin variants contained data collected in this manner. As the separation quality of agar has deteriorated many vendors have chosen to switch to use of the purified agarose in order to maintain a more constant product. Hemoglobin O-Arab migrates close to hemoglobin A historically and with well selected current agars. In the heterozygous state one broad band is seen starting with Hb A but extending on toward Hb S somewhat. When agarose is substituted Hb O-Arab migrates close but on the Hb C side of the Hb S location. The user is advised to take note of the separation media used for acid
electrophoresis in the interpretation of the results.

365

Since the concentration of the band that migrated at or near Hb C/E/O/A 2 position on alkaline electrophoresis was significantly > 10%, Hb A2
was ruled out because Hb A2 virtually never has such an increase. Citrate agar

electrophoresis (pH 6.2) eliminates the possibility of Hb C or Hb C-Harlem. Incidentally the migration of Hb C-Harlem, Hb O-Arab and Hb E is virtually identical upon IEF, therefore it was not helpful in the differentiation of these variants. HPLC and CZE show characteristic elution times (RT) and migration mobilities respectively for Hb O-Arab. In view of the characteristic laboratory tests and normal peripheral blood smear a tentative diagnosis of Hb O-Arab trait was made.

Hemoglobin O-Arab was first discovered (in association with Hb S) in an Arabic-speaking Israeli village (Giser-A-Zarke), and thus got its name as Hb OArab. It is the same village Sayar reported the homozygous Hb O-Arab (reference 2). It is emphasized here that Hb O-Arab is not prevalent in Israel or the Jewish population. However, three homozygous Hb O-Arab cases from progeny of parents who originally came from South Sudan were recently reported [Sayar D. Clinical and Hematological Features of Homozygous Hemoglobin OArab (Beta 121 GluLys). Pediatr Blood Cancer 2013; 60: 506-507]. Hb O-Arab has been found in Northern Africa (Tunisia), African-American, Saudi Arabia, Bulgaria and the Mediterranean littoral. Hemoglobin O-Arab is a -chain variant (121 GluLys), and exhibits no evidence of hemolysis and anemia in the heterozygous state. Persons with Hb O-Arab trait are clinically well. Homozygous
366

Hb O-Arab exhibits a mild anemia. Hb O-Arab interacts with Hb S (double heterozygous) and produces a disorder similar to homozygous Hb S disease with all of its characteristic features. Hb O-Arab also interacts with -thalassemia, and these individuals exhibit moderately severe hemolytic anemia and splenomegaly.

References
1. Bain BJ. Hemoglobin O-Arab, other significant hemoglobins. In: Hemoglobinopathy Diagnosis, 2066, 2nd edition, Blackwell Publishing, pg 21315. Sayar D. Clinical and Hematological Features of Homozygous Hemoglobin-O Arab [Beta 121 GluLys]. Pediatr Blood Cancer 2013; 60: 506-507. Zimmerman SA, OBranski EE, Rosse WF, Ware RE. Hemoglobin S/O Arab : Thirteen New Cases and Review of the Literature. Am J Hematol 1999; 60: 279-284. Sangore A, Sanogo I, Meite M, Ambofo Y, Abe Sopie V, Segbena A, Tolo A. Hemoglobin O Arab Disease in Ivory Coast and West Africa. Medicine Tropicale 1992; 52(2): 163-167. Altay C, Gurgey A, Huisman Titus TJ. Homozygosity For Hemoglobin O-Arab 121 GluLys (22 ) Hb O-Arab Disease. The Turkish Journal of Pediatrics 1986; 28: 67-72. Rachmilewitz EA, Tamari H, Liff F, Ueda Y, Nagel RL. The interaction of + hemoglobin O Arab with Hb S and thalassemia among Israeli Arabs. Hum Genet 1985; 70: 119-125. Ballas SK, Atwater J, Burka ER. Hemoglonin S-O Arab--Thalassemia: Globin Biosynthesis and Clinical Picture. Hemoglobin 1977; 1(7): 651-662. Milner PF, Miller C, Grey R, Seakins M, DeJong WW, Went LN. Hemoglobin O Arab in four negro families and its interaction with hemoglobin S and hemoglobin G. N Eng J Med 1970; 283(26): 1417-1425.

2. 3.

4.

5.

6.

7. 8.

367

Case # 17 -Thalassemia trait

A 27 year old Caucasian female.

Laboratory Data:
Hemoglobin 12.5 RBC 5.13 MCV 63.8 MCH 20.5 RDW 12.1 Platelet 267 Serum Iron 110 Ferritin 75 Hb A 93.5 Hb A2 6.0 Hb F 0.5 (Hemoglobin fractions from HPLC) 12.0 16.0 g/dL 3 4.0 5.5 Mil/mm 79-98 fL 26-34 pg 11.5-14.5% 3 150-400 Th/mm 30-160 ug/dL 8-120 ng/mL 1.5 -3.7% 0.0-2.0%

Peripheral Blood Smear: Hypochromasia, microcytosis, target cells, basophilic stippling Sickle solubility test for hemoglobin S: Negative Unstable hemoglobin test (isopropanol): Negative

Agarose Gel Electrophoresis (pH 8.6)

368

Case # 17 -Thalassemia trait

Citrate Agar Electrophoresis (pH 6.2)

Isoelectric focusing

369

Case # 17 -Thalassemia trait

Capillary zone electrophoresis

High performance liquid chromatography

370

Case #17 -Thalassemia trait

Interpretation & Discussion

Summary of Results Method Hb A area Alk Agarose Acid Agar/ Agarose CZE Small peak (Hb F) Zone 7 Major band (Hb A) Major band (Hb A + Hb A2) Major peak (Hb A) Zone 9 Major band (Hb A)
*

Hb S area

Hb A2/C area Minor band (Hb A2)

IEF

HPLC

Small peak (Hb F) RT=1.05

Major peak (Hb A) RT=2.42

Minor peak (Hb A2) Zone 3 Minor band (Hb A2) Minor peak (Hb A2) RT=3.64

Note: HPLC retention time (RT) varies with the type of instrument used and several other factors, e.g. temperature etc.

Alkaline agarose electrophoresis (pH 8.6) showed no abnormality except that the staining of the band at the Hb A2 position was relatively denser than the normal adult. No abnormal band was detected from citrate agar electrophoresis (pH 6.2). Two bands in the migration position of Hb A (major band) and Hb A2 (minor band but more intense than a normal adult) were indicated by IEF. CZE and HPLC results were concordant suggesting
371

an increased concentration of Hb A2 and no other abnormal peaks. Hemoglobinopathies can be classified as a manufacture of a modified globin chain or a failure or decrease in the ability to manufacture a particular globin chain. This latter set of conditions is referred to as a thalassemia. A decreased ability to manufacture beta chains is called -thalassemia and results in small erythrocytes (microcytosis) and a decreased amount of hemoglobin per erythrocytes and thinness of the cell (hypochromasia). Due to insufficient beta chains there is a surplus of alpha chains which bind to the red blood cell membranes causing damage and an occasional clump of alpha chains is the center of the Target Cells. The delta chains compete with the beta chains present with the delta chains getting a larger proportion in this beta chain deprived environment and this accounts for an elevated Hb A2. The hematological and morphological parameters along with elevated Hb A2 suggested the diagnosis of -thalassemia trait. In the presence of serum iron deficiency Hb A2 can be falsely lower, therefore quantification should be done again after the correction of the iron deficiency. -thalassemia trait is clinically a benign condition most often found in persons of the Mediterranean, Chinese, African American and other Asian ethnic groups. However problems arise when a thalassemic gene is inherited from both parents, e.g. causing Cooleys anemia (thalassemia major). Incidentally this disease was first discovered in an Italian population by Dr. Denton Cooley in Detroit, Michigan, USA.
372

A thorough review of the articles mentioned in the references is strongly advised to make a correct diagnosis of various kinds of thalassemias (minor, intermedia, and major) and also its interactions with several other hemoglobin variants. Molecular characterization is necessary for genetic counseling when both parents are carriers of -thalassemia minor or other hemoglobinopathies.

References
1. 2. 3. Galanello R, Origa R. Review: Beta-thalassemia. Orphanet J Rare Diseases 2010; 5(11): 1-15. http://www.ojrd.com/content/5/1/11 Cao A, Galanello R. Review: Beta-thalassemia. Genetics in Medicine 2010;12(2): 61-76. Colah R, Gorakshakar A, Nadkarni A. Global burden, distribution and prevention of -thalassemias and Hemoglobin F disorders. Expert Review of Hematology 2010; 3(1): 103-116. El Rassi F, Cappellini D, Inati A, Taher A. Beta-thalassemia Intermedia: An Overview. Pediatric Annals 2008; 37(5): 302-328. Bain BJ. -thalassemia trait. The , , and -thalassemia and related conditions. In: Hemoglobinopathy Diagnosis, 2nd Edition, Blackwell Publishing; 2006: 95-105. Oliveri N, Weatherall DJ. Clinical Aspects of thalassemia. In: Steinberg MH, Forget BG, Higgs DR, Nagel RL, eds. Disorders of Hemoglobin: Genetics, Pathophysiology, and Clinical Management. Cambridge, England: Cambriadge University Press; 2001: 277-341. Weatherall DJ, Clegg JB. The thalassemias. In: The Thalassemia Syndromes, 4th ed. Oxford, England: Blackwell Science, Ltd; 2001: 287-356. Qari MH, Wali Y, Albagshi MH, Aishahrani M, Alzahrani A, Alhijji IA, Almomen A, Aljefri A, Al-Saeed HH, Abdullah S, Al-Rustamani A, Mahour K, Mousa SA. Regional consensus opinion for the management of beta thalassemia major in the Arab Gulf Area. Orphanet J Rare Diseases 2013; 8: 143. Available from http://www.ojrd.com/content/8/1/143

4. 5.

6.

7. 8.

373

Case # 18 Hemoglobin S- - thalassemia

A 37 year old African American female.

Laboratory Data:
Hemoglobin 10.3 RBC 4.28 MCV 74.8 MCH 23.9 RDW 16.2 Platelet 203 Hb A 22.1 Hb A2 6.4 Hb F 3.4 Hb S 68.1 (Hemoglobin fractions from HPLC) 12.0 16.0 g/dL 3 4.0 5.5 Mil/mm 79-98 fL 26-34 pg 11.5-14.5% 3 150-400 Th/mm 94.3-98.5% 1.5 -3.7% 0.0-2.0%

Peripheral Blood Smear: microcytosis, rare target cells, moderate poikilocytosis Sickle cell solubility test for Hb S: Positive Hemoglobin instability (isopropanol) test: Negative No record of blood transfusion during the past seven months.

Agarose Gel Electrophoresis (pH 8.6)

374

Case # 18 Hemoglobin S- -thalassemia

Citrate Agar Electrophoresis (pH 6.2)

Isoelectric focusing

375

Case # 18 Hemoglobin S- -thalassemia

Capillary zone electrophoresis

High performance liquid chromatography

376

Case # 18 Hemoglobin S- -thalassemia

Interpretation & Discussion

Summary of Results Method Hb A area Small band (Hb A) Hb S area Major band (Hb S) Hb A2/C area Minor band (Hb A2)

Alk Very Agarose weak band (Hb F) Acid Weak Agar/ band Agarose (Hb F)

Small band (Hb A+ Hb A2) Small peak (Hb A) Zone 9

Major band (Hb S)

CZE

Small peak (Hb F) Zone 7

Major peak (Hb S) Zone 5

Minor peak (Hb A2) Zone 3 Minor band (Hb A2) Minor peak (Hb A2) RT=3.6

IEF

Very small band (Hb F) Small peak (Hb F) RT=1.05

Very small band (Hb A) Small peak (Hb A) RT=2.40

Major band (Hb S) Major peak (Hb S) RT=4.32

HPLC

Agarose gel electrophoresis (pH 8.6) showed one major band in the position of Hb S, minor band (less in intensity than Hb S band) in the migration position of Hb A, and another band in the position of Hb C/E/O/A2 with intensity greater than a normal adult. Citrate agar electrophoresis (pH 6.2) indicated increased Hb F than a normal adult, and a major band in the position of Hb S,
377

major band in the position of Hb A but less in intensity than Hb S. IEF, CZE, and HPLC also provided concordant results and the evidence for the following four bands: Hb S Hb A Hb A2 Hb F Major band-positive sickle cell test Major band-concentration less than Hb S Minor band-concentration greater than a normal adult Minor band concentration greater than normal adult

Quantitatively increased Hb A2 (6.4% by HPLC) suggested the presence of thalassemia in conjunction with Hb S. Two types of Hb S--thalassemia are found in African Americans: Hb S- -thalassemia: Hb S- -thalassemia
+ + +

Type 1 with Hb A concentration 5-15% Type 2 with Hb A concentration 20-30%

This case represents Hb S- -thalassemia Type 2. It is emphasized here that precaution is warranted in the interpretation at any time Hb A is less than Hb S. This kind of situation can be encountered in homozygous sickle cell disease with recent blood transfusion. Hb S--thalassemia in African Americans is present in two clinically significant conditions. If the Hb A is completely absent then it is termed Hb S -thalassemia, and is clinically similar to homozygous sickle cell disease. If Hb A is present (Type 1 or Type 2) the person will have a milder clinical course, and elevation of Hb F is also characteristic feature of Hb S -thalassemia.
378
+ 0

Appendix:
I looked for a case of Hb S- -thalassemia for > three years but no luck. Just yesterday a 23 year female came to the Emergency Department with a severe sickle cell crisis. I immediately contacted the attending physician and my associates involved in this book to include the data of this patient for the benefit of readers for understanding the distinction between the two types of Hb S--thalassemias. CBC Hemoglobin Hematocrit RBC MCV MCH RDW Platelet Reticulocyte Count 5.3 15.1 2.13 70.7 24.8 24.3 407 13.5 12.0 16.0 g/dL 35.0 - 48.0% 3 4.0 - 5.5 Mil/mm 79 - 98 fL 26 - 34 pg 11.5 - 14,5 % 3 150 - 400 Th/mm 0.5 - 1.5%
0

Alkaline agarose (pH 8.6), Citrate agar (pH 6.2), IEF, HPLC and CZE indicated the absence of Hb A. The concentration of hemoglobin fractions from CZE were: Hb A Hb A2 Hb F Hb S Not Detected 4.5 8.6 86.9

On the basis of CBC and laboratory results a diagnosis of Hb S- -thalassemia is most likely.

References
1. Bain BJ. Sickle cell/ thalassemia, Sickle cell hemoglobin and its interactions with other variant hemoglobins and with thalassemia. In: Hemoglobinopathy Diagnosis, 2006, 2nd edition. Blackwell Publishing, England, pg 170-173. Steinberg MH. Compound heterozygous and other sickle hemoglobinopathies. In: Steinberg MH, Forget BG, Higgs DR, Nagle RL. Disorders of Hemoglobin: Genetics, Pathophsiology and Clinical Management. Cambridge , England: Cambridge University Press; 2001: 786-792.
379

2.

3.

4.

5.

Sunna EI, Gharaibeh NS, Knapp DD, Bashir NA. Prevalence of Hemoglobins S and -Thalassemia in Northern Jordan. J Obstet Gynecol Res 1996; 22(1): 17-20. Gonzalez-Redondo JM, Kutlar A, Kutlar F, McKie VC, Mckie KM, Baysai E, Huisman THJ. Molecular Characterization of Hb S (C) -Thalassemia in American Blacks. Am J Hematol 1991; 38: 9-14. Serjeant GR, Ashcroft Mt, Serjeant BE, Milner PF. The clinical features of sickle cell/-thalassemia in Jamaica. Br J Hematol 1973; 24: 19-30.

380

Case # 19 Hemoglobin C- -thalassemia

A 17 year old female student from Turkey (most likely of Eti Turkish descent) visiting a prestigious high school in Michigan to brush up her English. She was asymptomatic. She declined to participate in athletic activities because she felt fatigue upon physical activity. Low hemoglobin and MCV triggered hemoglobin electrophoresis by the physician.

Laboratory Data:
Hemoglobin 10.3 12.0 16.0 g/dL 3 RBC 5.4 4.0 5.5 Mil/mm MCV 66.5 79-98 fL MCH 20.9 26-34 pg Hb A (all methods) Not Detected 94.3-98.5% Hb A2 5.9 1.5 -3.7% Hb F 1.4 0.0-2.0% Hb C 87.0 Hb A1c + other fractions 5.7 Peripheral Blood Smear: Hypochomosia, microcytosis, target cells, anisocytosis and poikilocytosis Sickle cell solubility test for Hb S: Negative Hemoglobin instability (isopropanol) test: Negative

Agarose Gel Electrophoresis (pH 8.6)

381

Case # 19 Hemoglobin C- -thalassemia

Citrate Agar Electrophoresis (pH 6.2)

Isoelectric focusing

382

Case # 19 Hemoglobin C- -thalassemia

Capillary zone electrophoresis

High performance liquid chromatography

383

Case # 19 Hemoglobin C- -thalassemia

Interpretation & Discussion

Summary of Results Method Alk Agarose
Faint band (Hb F)

Hb A area

Hb S area

Hb A2/C area Major band

(Hb C + Hb A2)

Acid Agar /Agarose

Faint band (Hb F)

Major band
(Hb C + Hb A2)

CZE

Minor peak (Hb F) Zone 7

Minor peak (Hb A2) Zone 3 Minor band (Hb A2)

Major peak (Hb C) Zone 2

IEF

Major Hb C band cathodal to A2 Major peak

(Hb C)

HPLC

Minor peak (Hb F) RT=1.05

Minor peak (Hb A2) RT=3.6

RT=5.09

Note: Hb A was not detected by any of the six methods.

Alkaline agarose electrophoresis (pH 8.6) showed a single band in the position of Hb C/E/O/Hb A2, thus indicating the absence of Hb A. Citrate agar electrophoresis (pH 6.2) also indicated the absence of Hb A, and a major band (87%) was indicated in the position of Hb C.
384

IEF showed a major band in the position of Hb C (cathodal to Hb A2), minor band in the position of Hb A2 and a faint band in the position of Hb F. CZE also indicated three peaks:

Hb C Hb F Hb A2

major peak in Zone 2 minor peak in Zone 7 minor peak in Zone 3 but obscured by the larger peak in Zone 2 (Hb C)

HPLC separated the hemoglobins into three peaks, i.e. Hb C, Hb A2 and Hb F and also provided quantitative results.

Increased Hb A2 (5.9%), absence of Hb A, microcytosis, target cells and a major Hb C peak (87%) from HPLC suggested the presence of compound heterozygosity for Hb C and -thalassemia. It is emphasized here that a distinction between homozygous Hb C and Hb C- -thalassemia is not feasible from alkaline agarose gel electrophoresis (pH 8.6) alone due to lack of the correct quantitative value of Hb A2 because of the overlap of Hb C and Hb A2 bands. Absence of Hb A as in this case rules out the possibility of Hb C- thalassemia.
+ 0

Due to similarity in clinical features it is sometimes not possible to differentiate with certainty between homozygous Hb C and Hb C- -thalassemia. Similar clinical features of homozygous Hb C and Hb C- -thalassemia are: Mild to moderate chronic hemolytic anemia, with hemoglobin levels in the range of 8-12 g/dL and splenomegaly. The blood film shows large number of target
385
0 0

cells, folded cells, scattered spherocytes, hypochromia, microcytosis and polychromasia. The two parameters that lead to the putative diagnosis of Hb C- thalassemia in this case are MCV (55-70) and increased Hb A2. However, the Hb A2 fraction could be overestimated and HbC/beta syndromes are usually characterized by some hemolysis and spelenomegaly while this patient is asymptomatic. Microcytosis could be caused by alpha thalassemia and the genotype should be confirmed by direct sequencing of the beta genes.
0 0

References
1. Kumar S, Rana M, Handoo A, Saxena R, Verma IC, Bhargava M, Sood SK. 0 Case report of Hb C/ -thalassemia from India. Int Jnl Lab Hem 2007; 29: 381-385. Nagel RL, Steinberg MH, Hb S/C disease and Hb C disorders. In: Steinberg MH, Forget BG, Higgs DR, Nagel RL. Disorders of Hemoglobin: Genetics, Pathohysiology and Clinical Management. Cambridge, England: Cambridge University Press; 2001; 756-785. Fattoum S, Guemira F, Abdennebi M, Ben Abdeladhim A. [Hbc/betathalassemia association. Eleven cases observed in Tunisia]. Ann Pediatr (Paris) 1993; 40(1): 45-8. Maberrry MC, Mason RA, Cunningham G, Pritchard JA. Pregnancy Complicated by Hemoglobin CC and C--Thalassemia Disease. Obstet Gynecol 1990; 76: 324-327. Ozsoylu S, Sipahioglu H, Altay F. Hemoglobin C-beta (O) thalassemia. Isr J Med Sci 1989; 25: 410-412.

2.

3.

4.

5.

386

Case # 20

Hemoglobin Hasharon trait

A 55 year old male computer programmer with age onset diabetes mellitus, was screened for hemoglobinopathy since one of his family member was anemic, and another having a hemoglobin variant. His parents (Ashkenazi Jews) migrated from Poland to Detroit, Michigan.

Laboratory Data:
Hemoglobin RBC MCV MCH RDW Platelet Hb A Hb A2 (CZE) Hb F (CZE) Hb variant (CZE) 14.8 5.1 88.0 28.3 12.3 203 77.3 1.6 0.8 20.3 12.0 18.0 g/dL 3 4.6 6.2 Mil/mm 80 - 100 fL 27 - 34 pg 11.5 -14.5% 3 15 - 400 Th/mm 94.3 - 98.5% 1.5 - 3.7% 0.0 - 2.0%

Peripheral Blood Smear: No abnormality was detected. Sickle cell solubility test for Hb S: Negative Hemoglobin instability (isopropanol) test was positive but heat stability test was negative.

Agarose Gel Electrophoresis (pH 8.6)

387

Case # 20 Hemoglobin Hasharon trait

Citrate Agar Electrophoresis (pH 6.2)

Isoelectric focusing

388

Case # 20 Hemoglobin Hasharon trait

Capillary zone electrophoresis

High performance liquid chromatography

389

Case # 20 Hemoglobin Hasharon trait

Interpretation and Discussion

Summary of Results Method Hb A area Major band
(Hb A)

Hb S area
Major band slightly toward C

Alk Agarose

(Hb Hasharon)
Major band slightly toward A

Hb A2/C area Minor band (Hb A2)

Acid Agar

Major band
(Hb A)

(Hb Hasharon)
Major band directly in S position

Acid Agarose

Major band
(Hb A)

(Hb Hasharon)

CZE

Small peak
(Hb F)

Major peak
(Hb A)

Major peak (Hb

Hasharon)

Minor peak Zone 3

(Hb A2)

Zone 7

Zone 9

Very minor peak Zone 1

(HbHasharonA2) A very faint band cathodal to Hb C Hb A2 (2 2)
H

Zone 5 IEF Major band

(Hb A)

Major band slightly cathodal

of Hb S. (Hb Hasharon)

Minor band
(Hb A2)

HPLC

Very small peak

(Hb F)
RT=1.05

Major peak
(Hb A) RT=2.40

Major peak eluted

between S and C (Hb Hasharon) RT=4.74

Minor peak
(Hb A2) RT=3.58

390

Alkaline agarose electrophoresis (pH 8.6) showed a major band in the Hb A region, and another major band of lesser intensity cathodal to Hb S. A very faint band was present in Hb A2 position. Citrate agar electrophoresis (pH 6.2) also revealed two major bands with intensities equivocal to that described for agarose gel electrophoresis (pH 8.6) in the respective Hb A and Hb S positions. The sickle cell test was negative, ruling out the presence of Hb S.

IEF showed four bands: two intense bands and two faint bands. One intense band in the position of Hb A, and a second band slightly cathodal to Hb S. Additionally, there was a very faint band migrating in the Hb A2 position and a second faint band in the delta chain variant position (cathodal to Hb C). Hb A2 variants are due to the presence of an abnormal -chain as seen in Hb GPhiladelphia trait (Case # 5) or due to the presence of an abnormal delta chain. Since this specimen also has an abnormal Hb A, the Hb A2 variant is likely due to an alpha mutation. CZE showed a major peak in the Hb A zone (Zone 9), and a lesser intense peak than Hb A in Zone 5. Two minor peaks in the position of Hb F (Zone 7) and Hb A2 (Zone 3) were also detected as well as a very small peak in Zone 1. HPLC showed a major peak for Hb A and two minor peaks for Hb A2 and Hb F. Another major peak was detected between the Hb S and Hb C window.
391

A narrative report was communicated to the attending physician with a request for consultation with him to identify the exact hemoglobinopathy. The physician communicated that the patient is an orthodox Ashkenazic Jew of Polish origin.

Consistently typical migration patterns by the four electrophoretic methods, elution retention times upon HPLC and the Ashkenazic Jewish ethnic origin of the patient suggested the possibility of Hb Hasharon trait. Hb Hasharon was first discovered in Hasharon Hospital, Israel in an Ashkanezic Jew, whose father was from Poland and mother from Romania. It is -chain variant caused by a mutation on condom 47 that results in the substitution of aspartic acid by histidine (47 AspHis).

The presence of Hb Hasharon is typically found in Ashkanezic Jews (who have also migrated to several countries after World War II), and Italians from Ferrara district of Italy. Hb Hasharon has not been recognized in Sephardic Jews. No consistent clinical and hematological abnormalities are associated with Hb Hasharon. It is innocuous hemoglobinopathy, however some patients have indicated drug-Induced (sulfonamide, dapsone) hemolytic anemia.

The percentage of Hb Hasharon varies between Ashkenezic Jews and the subjects of the Ferrara district of Italy. The Hb Hasharon concentration in Italians of Ferrara district origin is usually in the range of 30-35%. Contrary to this the
392

Ashkanezic Jews have the Hb Hasharon concentration in the range of 15-20%. The DNA studies have determined that this difference is because of an underlying -thalassemia (-thalassemia-2) in Italians of Ferrara area. Thus, these individuals have both an alpha chain mutation and an alpha deletion. The Ashkanezic Jews have no evidence of the presence of -thalassemia-2 trait.

References
1. Unstable hemoglobin variants, Martin H. Steinberg, MD, www.uptodate.com 2013 UpTodate. http://www.uptodate.com/contents/unstable-hemoglobin-variants Zur B, Ludwig M, Stoffel-Wagner B. Hemoglobin Hasharon and Hemoglobin NYU in subjects of German origin. Biochemia Medica 2011; 21: 321-25. Chinelato-Fernandes AR, Mendiburu CF, Bonini-Domingos CR. Utilization of different methodologies for the characterization of Hb Hasharon heterozygotes. Genet Mol Res 2006; 5: 1-6. Eliakim R, Rachmilewitz EA. Hemoglobinopathise in Israel. Hemoglobin 1983; 7: 479-85. Mavilio F, Marinucci A, Fontanarosa PP, Tentori L, Cappellozza G. Hemoglobin Hasharon [2 47(CD5) AspHis2] linked to -Thalassemia in Northern Italian carriers. Acta Haemat. 1980; 63: 305-311. del Senno L, Bernardi F, Marchetti G, et al. Organization of globin genes 47 and mRNA translation in subjects carrying hemoglobin Hasharon ( Asp replaced by His) from the Ferrara region (Northern Italy). Eur J Biochem 1980; 111(1): 125-130. Alberti R, Mariuzzi GM, Marinucci M, Bruni E, Tentori L. Hemoglobin Hasharon in a north Italian community. J Med Genet 1975; 12: 294-98.

2. 3.

4. 5.

6.

7.

393

Case # 21

Hemoglobin Zurich trait

A 18 year old white female student from Grand Rapids, Michigan. Parents migrated from Europe, but no information available about their ethnicity and country of origin. Her physical examination was normal.

Laboratory Data:
Hemoglobin RBC MCV MCH RDW Platelet Hb A Hb A2 (HPLC) Hb F (HPLC) Hb variant 11.6 4.4 102.0 28.4 12.3 228 66.0 1.6 0.8 31.6% 12.0 16.0 g/dL 3 4.0 5.5 Mil/mm 79 - 98 fL 26 - 34 pg 11.5 -14.5% 3 15 - 400 Th/mm 94.3 - 98.5% 1.5 - 3.7% 0.0 - 2.0%

Peripheral Blood Smear: Macrocytic red blood cells. Serum iron and ferritin were normal; very mild anemia with slight reticulocytosis. Sickle cell solubility test for Hb S: Negative Hemoglobin instability (isopropanol) test: Positive No congenital deficiency of glucose-6-phosphate dehydrogenase.

Agarose Gel Electrophoresis (pH 8.6)

394

Case # 21 Hemoglobin Zurich trait

Citrate Agar Electrophoresis (pH 6.2)

Isoelectric focusing

395

Case # 21 Hemoglobin Zurich trait

Capillary zone electrophoresis

High performance liquid chromatography

396

Case # 21 Hemoglobin Zurich trait

Interpretation & Discussion

Summary of Results Method Hb A area Major band
(Hb A)

Hb S area Major band

Alk Agarose Acid Agar

Hb A2/C area Minor band

(Hb A2)

Major band
(Hb A + Hb A2+
Hb variant)

CZE

IEF

Major peak Zone 9 (Hb A) Major band

(Hb A)

Major peak Zone 6 Major band

Slightly cathodal to Hb S

Minor peak Zone 3

(Hb A2)

Minor band
(Hb A2)

HPLC

Very minor peak

(Hb F)

Major peak (Hb A) RT=2.34

Major peak RT=3.55

RT=1.06

*The hemoglobin variant eluted with Hb A2, and Hb A2 was in the normal range from
CZE. Note: HPLC retention time (RT) varies with the type of instrument used and several other factors, e.g. temperature etc. Please note that we do not have data of acid agarose electrophoresis separation at this time.

397

Agarose gel electrophoresis (pH 8.6) showed two major bands; one at the migration position of Hb A, and one at the Hb S position. In addition, a very weak band was noticed in the position of Hb C/E/O/Hb A2. Citrate agar electrophoreis (pH 6.2) showed only one band in the position of Hb A and a very weak, smudged band in the position of Hb F.

In view of the negative sickle cell test and the migration patterns of the alkaline and acid electrophoresis, the presence of Hb S was ruled out. Similarly, the possibility of Hb G-Philadelphia and Hb D- Los Angeles was eliminated on the basis of the positive (isopropanol) instability test, the absence of G 2 band of Hb G-Philadelphia and a lower percentage of the variant as compared to Hb DLos Angeles. Hemoglobin electrophoresis on this patient was performed about seven years ago in our laboratory. At that time, neither the HPLC nor the CZE testing instruments were available in our laboratory. After consultation with the attending physician, the specimen was sent to a reference laboratory for globin chain analysis by reverse phase HPLC and DNA studies. Note: The IEF, CZE and HPLC scans inserted here in this case are adopted from other sources for educational purposes.

The globin chain analysis and DNA studies provided the correct identification of the hemoglobin variant (31.6% from alkaline agarose electrophoresis) as Hb Zurich. Hb Zurich is an unstable hemoglobin and found only in the heterozygous state. It is caused by the substitution of histidine by
398

arginine at the 63rd position of the -chain [22

63

(E7) HisArg]. Hb Zurich

was initially found in Europeans of Swiss descent, but later this variant was reported in Japanese, and Brazilian citizens of non-Swiss ancestry.

Physicians should be advised of possible induced or exacerbated hemolysis in subjects with Hb Zurich by exposure to oxidant drugs, e.g. sulfonamides, sulfones, phenacitin-like analgesics, and most of the local anesthetics.

References
1. Unstable hemoglobin variants. Steinberg MH. www.uptodate.com 2013 UpTodate. August 2013. http://www.uptodate.com/contents/unstable-hemoglobin-variant Aguinaga MdP, Wright CJ, Roa PD, Terrel F, Turner EA, Houston M. Molecular Diagnosis and Characterization of Hb Zurich [63(E7)HisArg] Carriers in a Kentucky Family. Hemoglobin 1998; 22 (5 & 6): 509-515. Harano T, Harano K, Nagasaka I, Yamasaki S. Hb Zurich [63(E7)HisArg] Found in a Japanese Woman. Hemoglobin 1996; 20 (4): 429-434. Miranda SRP, Kimura EM, Saad STO, Costa FF. Identification of Hb Zurich [2263(E7)HisArg] by DNA Analysis in a Brazilian Family. Hemoglobin 1994; 18 (4 & 5): 337-341. Zinkham WH, Winslow RM. Unstable Hemoglobins: Influence of Environment on Phenotypic Expression of a Genetic Disorder. Medicine 1989; 68(5): 309320. Zinkham WH, Houtchens RA, Caughey WS. Relation between variations in the phenotypic expression of an unstable hemoglobin disorder (hemoglobin Zurich) and carboxyhemoglobin levels. Am J Med 1983; 74: 23-29. Murata K, Yamamoto S, Hirano Y, Omine Mitsuhiro O, Tsuchiya J, Ohba Y, Miyaji T. First Japanese Family with the Unstable Hemoglobin Zurich [63(E7)HisArg]. Jap J Med 1982; 21 (1): 40-45. Dickerman JD, Holtzman NA, Zinkman WH. Hemoglobin Zurich. A Third Family Presenting with Hemolytic Reactions to Sulfonamides. Am J Med 1973; 55: 638-642.

2.

3. 4.

5.

6.

7.

8.

399

Case # 22

Hemoglobin Lepore trait

A 41 year old male employee of General Motors, Detroit, Michigan. Parents migrated from Italy.

Laboratory Data:
Hemoglobin RBC MCV MCH RDW Platelet Hb A Hb A2 Hb F Hb variant (CZE) 14.3 5.72 69 22.4 13.2 243 80.7 2.1 5.4 11.8 13.5 -18.5 g/dL 3 4.6 - 6.2 Mil/mm 80 -100 fL 27 34 pg 11.5 -14.5% 3 150 - 400 Th/mm 94.3 - 98.5% 1.5 - 3.7% 0.0 - 2.0%

Peripheral Blood Smear: Microcytosis, hypochromasia, target cells, poikilocytosis. Sickle cell solubility test for Hb S: Negative Patient was not transfused during the past four months.

Agarose Gel Electrophoresis (pH 8.6)

400

Case # 22 Hemoglobin Lepore trait

Citrate Agar Electrophoresis (pH 6.2)

Isoelectric focusing

401

Case # 22 Hemoglobin Lepore trait

Capillary zone electrophoresis

High performance liquid chromatography

402

Case # 22 Hemoglobin Lepore trait

Interpretation & Discussion

Summary of Results Method Hb A area Major band (Hb A) Hb S area Major band (Hb Lepore) Hb A2/C area Minor band (Hb A2)

Alk Agarose

Acid Agar/Agarose

Major band (Hb A+ Hb Lepore + Hb A2) Small peak (Hb F) Zone 7 Major peak (Hb A) Zone 9 Major peak (Hb Lepore) Zone 6
Major band in Hb G position (Hb Lepore)

CZE

Minor peak (Hb A2) Zone 3 Minor band (Hb A2)

Major peak Hb A2 & Hb Lepore (RT=3.5)

IEF

Major band (Hb A) Small peak (Hb F) RT=1.05 Major peak (Hb A) RT=2.45

HPLC

. Since the sickle cell test was negative and also no band was observed in the area of Hb S upon acid electrophoresis, the presence of Hb S was ruled out. The possibility of other commonly encountered variants (e.g. Hb D, Hb G, Hb

403

Russ) that exhibit alkaline and acid electrophoresis pattern similar to this case were ruled out. Hb D has a concentration of approximately 40% in the heterozygous state, and this variant quantified at 11.8% by CZE. Hb G was ruled out due to the absence of the -chain variant (
G 2 2)

band/peak by

electrophoretic methods (alkaline agarose, IEF and CZE). This variant was associated with microcytosis, while Hb Russ is not.

With IEF the hemoglobin variant migrated exactly in the position of Hb G, even though the presence of Hb G was ruled out on the basis of the absence of the delta chain Hb G band (see above). Similarly differential diagnosis of the hemoglobin variant with CZE was not helpful due to the overlap of several hemoglobins in zone 6.

HPLC showed increased intensity of the Hb A2 peak (RT=3.5), which was inconsistent with other electrophoretic methods. This suggested that the hemoglobin variant eluted with Hb A2. Another thing the HPLC ruled out was the presence of the other common variants exhibiting electrophoretic patterns similar to this case (Hb D, Hb G, Hb Russ), because none of these variants elute with Hb A2. In view of the thalassemic peripheral blood picture, low concentration of the variant (11.8%), and the separation data a diagnosis of Hb Lepore trait was made.

404

Hb Lepore is hybrid (fused globin chains) hemoglobin consisting of two -globin chains and two - hybrid chains. In the - hybrid chain the N-terminal position of the -chain joins at the C-terminal of the -chain. There are three genetically controlled - chains fusions (hybrid formation) that are characterized by their fusion points of the amino acids in the chains. This characteristic fusion of and chains leads to the following variants of Hb Lepore: i) Hb Lepore-Boston [(1-87) (115-146)]

In this case of Hb Lepore, the hybrid - chain consists of the first 87 amino acids of the -chain and the last 32 amino acids of the -chain. This variant is also called Hb Lepore-Washington. ii) iii) Hb Lepore-Baltimore Hb Lepore-Hollandia [(1-50) (86-146)] [(1-22) (50-146)]

Among these three variants, Hb Lepore-Boston is found with some frequency in people of Mediterranean descent.

Lepore- Boston, (Lepore-Washington) migrates in the same position as Hb S in alkaline conditions. However Lepore Hollandia and Lepore Baltimore migrate slightly slower than Hb S in alkaline conditions (Bain, BJ, Wild BJ, Stephens AD and Phelan L. Variant Hemoglobins: A Guide to identification; 2010: Wiley-Blackwell Publishing).

405

To the best of our knowledge both CZE and HPLC do not differentiate among these variants.

All the Lepore traits have the same clinical symptoms. Hb Lepore trait is a stable hemoglobin but exhibits features of thalassemia minor due to the decreased production of -chains. -thalassemia intermedia or major type disorder is exhibited by homozygous Hb Lepore. Hb Lepore interacts with Hb S to give Hb S/Hb Lepore, however very few cases (<18) were reported in the literature. Hb S/Hb Lepore patients exhibited mild microcytic anemia, and clinically were either asymptomatic or with complications generally associated with Hb S disease. A case of Hb E interaction with Hb Lepore-Hollandia was described in the literature but without any significant clinical condition except microcytic anemia without the need for transfusion.

References
1. McKeown SM, Carmichael H, Markowitz RB, Kutlar A, Holley L, Kutlar F. Rare Occurrence of Hb Lepore-Baltimore in African Americans: Molecular Characteristics and Variations of Hb Lepores. Ann Hematol 2009; 88: 545548. Pasanga J, George E, Nagaratnam M. Haemoglobin Lepore in a Malay family: a case report. Malasian J Pathol 2005; 27(1): 33-37. Shaji RV, Edison ES, Krishamoorthy R, Chandy M, Srivatava A. Hb Lepore in the Indian Population. Hemoglobin 2003; 27: 7-14. Viprakasit V, Pung-Amritt P, Suwanthon L, Clark K, Tanphachitr VS. Complex interactions of [delta] [beta] hybrid haemoglobin (Hb Lepore Hollandia) Hb E([beta]26G>A) and [alpha]+ thalassemia in a Thai Family. Eu J Haematol 2002; 68-107-12.

2. 3. 4.

406

5.

6.

7.

8.

9.

10.

11.

Goncalves I, Henriques A, Raimundo A, Picanco I, Reis A, Correia Jr E, Santos E, Nogueria P, Osorio-Almedia L. Fetal Hemoglobin Elevation in Hb Lepore Heterozygotes and its correlation with Globin Cluster Linked Determinants. Am J Hematol 2002; 69: 95-102. Forget BG. Molecular mechanism of beta-thalassemia. In: Steinberg MH, Forget BG, Higgs DR, Nagel RC, eds. Disorders of Hemoglobin: Genetics, Pathophysiology and Clinical Management. Cambridge, England: Cambridge University Press; 2001: 264-265. Ropero P, Gonzalez FA, Sanchez J, Anguta E, Asenjo S, Arco AD, Murga MJ, Ramos R, Fernandez C, Villegas A. Identification of the Hb Lepore phenotype by HPLC. Haematologica 1999; 84: 1081-1084. Romana M, Diara JP, Merghoub T, Leclard L, Saint-Martin C, Berchel C, Merault G. Hemoglobin Sickle-Lepore: An Unusual Case of Sickle Cell Disease. Acta Haematologica 1997; 98: 170-71. Fairbanks VF, McCormick DJ, Kubik KS, Rezuke WN, Black D, Ochaney MS. Hb S/Hb Lepore with Mild Sickling Symptoms: A Hemoglobin Variant with Mostly Delta-Chain Sequences Ameliorates Sickle-Cell Disease. Am J Hematol 1997; 54: 164-165. Waye JS, Eng B, Patterson M, Chui DHK, Chang LS, Coglonis B, Poon AO, Oliveri NF. Hb E/Hb LeporeHollandia in a Family From Bangladesh. Am J Hematol 1994; 47: 262-265. Hemoglobin Sickle-Lepore: Report: Report of Two Siblings and Review of the Literature. Am J Hematol 1993; 44: 192-195.

407

Case # 23

Hemoglobin J-Oxford trait

A 55 year old male farmer from Saginaw, Michigan. His mother belonged to a French settlement in Newfoundland, Canada. Ancestors from father side immigrated from Norway. No abnormality was found from an annual medical examination except a slight elevation of serum cholesterol.

Laboratory Data
Hemoglobin RBC MCV MCH Platelet Hb A2 Hb F Hb A Hb Variant 14.8 5.1 90.7 29.9 279 2.2 0.8 72.0 25.0 % 13.5 - 18.5 g/dL 3 4.6 - 6.2 Mil/mm 80 - 100 fL 27 - 34 pg 3 150 - 400 Th/mm 1.5 - 3.7% 0.0 - 2.0% 94.3 98.5%

Peripheral Blood Smear: No abnormality Sickle cell solubility test for Hb S: Negative Hemoglobin instability (isopropanol) test: Negative

Agarose Gel Electrophoresis (pH 8.6)

408

Case # 23 Hemoglobin J-Oxford trait

Citrate Agar Electrophoresis (pH 6.2)

Isoelectric focusing

409

Case # 23 Hemoglobin J-Oxford trait

Capillary zone electrophoresis

High performance liquid chromatography

410

Case # 23 Hemoglobin J-Oxford trait

Interpretation & Discussion

Summary of Results Method Hb A area
Major Alk Agarose band as much anodal to Hb A as Hb S is cathodal to Hb A (Hb J)

Hb S area See note below

Major band (Hb A)

Hb A2/C area Very faint band (Hb A2)

Acid Agar/ Agarose

Major band (Hb A + Hb A2+

Hb J)

CZE

Major peak (Hb J) Zone 12

Major peak (Hb A) Zone 9

Barely visible peak (Hb "JOxfordA2") Zone 6

Very minor peak (Hb A2) Zone 3 Very minor band (Hb A2)

IEF

HPLC

Major band anodal to Hb A (Hb J) Very small peak (Hb F) RT=1.05

Major band (Hb A)

Major peak (Hb A) RT=2.44

Major peak (Hb J) RT=1.62

Barely visible peak (Hb A2) RT=3.64

Note: A faint band due to Hb "A2-J" was detected in the position of Hb S, when a concentrated hemolysate was used for heavier application in alkaline agarose electrophoresis (pH 8.6).

411

In the later stages of our investigation, when Hb J was considered in this case, the alkaline agarose gel electrophoresis (pH 8.6) was repeated with a heavier application, and a faint Hb "A2-J" band was detected in the area of Hb S. The reason for this additional test was to rule out a beta chain variant form of Hb J. Because the intensity of the fast band is far less than that of the Hb A band this variant is most likely an -chain variant.

During my discussion with the attending physician the following points were brought to his attention: i) We suspect an Hb J (-chain variant) trait, probably Hb J-Oxford trait [15(A13) GlyAsp]. ii) There are >50 Hb J variants that are known in the literature. In addition to that there are > 24 Hb variants which are not designated as Hb J variant but exhibit electrophoretic mobilities akin to Hb J. Most of these Hb J variants are entirely without any clinical or hematological manifestations. Note: As of today 57 hemoglobins are designated Hb J by electrophoretic mobility and they are roughly divided equally between and chain variants. Six of these are unstable and one has increased oxygen affinity. iii) There are three Hb J variants reported in the literature as associated with clinical disorders:
412

a) b) c)

Hb J-Altgeld (unstable hemoglobin hemolytic anemia) Hb J-Cape Town (erythrocytosis due to high oxygen affinity) Hb J-Buda (erythrocytosis resulting from interaction with Hb G-Pest in persons doubly heterozygous for Hb J-Buda and Hb G-Pest)

iv)

The exact identification of the Hb J trait variant in the patient is not necessary since the patient is clinically normal. Further testing to designate the type of Hb J (DNA sequencing, LC-Mass) may be deferred indefinitely due to exorbitantly associated cost.

At the time this patient was analyzed, both the CZE and HPLC testing facilities were not available in our laboratory. For instructional purposes, we have illustrated the CZE and HPLC scans of another established Hb J-Oxford trait patient. Both the CZE (major peak in zone 12) and HPLC (minor peak at RT= 1.62) provided concurrent evidence for Hb J trait (-chain variant).

Hb J was first discovered in 1956 (Thorup OA, Itano HH, Wheyby M, Leavll BS. Hemoglobin J. Science 1956; 123: 889-90), and the 57 variants found so far are roughly divided equally between -chain and -chain variants. Hb J variants are rarely found, but have been reported from the USA, northern European countries, China and Japan. The only Hb J variant which is encountered with any notable frequency, is Hb J-Baltimore (Case # 24).

413

Hb H, J, I, N, K, Camden and Hope are designated "fast hemoglobins" in view of their faster mobility on agarose gel electrophoresis (pH 8.6). Additional cases of other "fast hemoglobins" will be included in the 2nd edition of the book.

References
1. Caruso D, Crestani M, Riva LD, Mitro N, Giavarini F, Mozzi R, Franzini C. Mass spectrometry and DNA sequencing are complementary techniques for characterizing hemoglobin variants: the example of hemoglobin J-Oxford. Haematologica 2004; 89(5): 608-609. Joutousky A, Hadzi-Nesic J, Nardi MA. HPLC Retention Time as a Diagnostic Tool for Hemoglobin Variants and Hemoglobinopathies: A study of 60 000 Samples in a Clinical Diagnostic Laboratory. Clin Chem 2004; 50(10): 17361747. Harano K, Harano T, Shibata S, Mori H, Ueda S, Imai K, Ohba Y, Irimajiri K. Hb J-Oxford [15(A13) Gly---Asp] in Japan. Hemoglobin 1984; 8(2): 197-198.

2.

3.

414

Case # 24

Hemoglobin J-Baltimore trait

A 33 year old male, residing in Windsor, Canada, whose ancestors migrated from Europe. While donating blood, his hemoglobin was found to be low, therefore his family physician ordered hemoglobin electrophoresis.

Laboratory Data
Hemoglobin RBC MCV MCH Platelet Hb A2 Hb F Hb A Hb Variant (HPLC) 12.8 4.9 86.0 28.3 232 2.3 3.8 55.2 38.7 % 13.5 - 18.5 g/dL 3 4.6 - 6.2 Mil/mm 80 - 100 fL 27 - 34 pg 3 150 - 400 Th/mm 1.5 - 3.7% 0.0 - 2.0% 94.3 98.5%

Peripheral Blood Smear: No abnormality Sickle cell solubility test for Hb S: Negative Hemoglobin instability (isopropanol) test: Negative

Agarose Gel Electrophoresis (pH 8.6)

415

Case # 24 Hemoglobin J-Baltimore trait

Citrate Agar Electrophoresis (pH 6.2)

Isoelectric focusing

416

Case # 24 Hemoglobin J-Baltimore trait

Capillary zone electrophoresis

High performance liquid chromatography

417

Case # 24 Hemoglobin J-Baltimore trait

Interpretation & Discussion

Summary of Results Method Hb A area Major band (Hb A) Note: Both major bands of equal intensity Hb S area Hb A2/C area Very faint band (Hb A2)

Major Alk Agarose band as much anodal to Hb A as Hb S is cathodal to Hb A (Hb J)

Acid Agar/ Agarose

Major band (Hb A + Hb A2+

Hb J)

CZE

Major peak (Hb J) Zone 12

Major peak (Hb A) Zone 9 Note: Both major bands of equal intensity Major peak (Hb J) RT=1.8

IEF

Major Major band band anodal to (Hb A) Hb A (Hb J) Small peak (Hb F) RT=1.05 Major peak (Hb A) RT=2.4

Minor peak (Hb A2) Zone 3 Minor band (Hb A2)

HPLC

Very small peak (Hb A2) RT=3.6

Note:

Since Hb J-Baltimore is a -chain variant, therefore the major bands (Hb A and Hb J-Baltimore) are approximately in equal concentration.

418

Agarose gel electrophoresis (pH 8.6) showed two major bands. One band was in the position of Hb A. Another major band was approximately as much anodal to Hb A as Hb S is cathodal to Hb A. Visually, the intensity of both of these two bands was similar. No other band was detected besides a minor band for Hb A2. There are several fast moving hemoglobin variants. Among them are Hb H and Hb I, which migrate much faster towards anode than Hb J (see Case # 23). Similarly, Hb N also migrates slightly faster than Hb J. There are several chain variants that exhibit similar migration patterns as this patient. These chain variants are usually present in a 1:3 ratio relative to Hb A. Since the intensity of the two major bands on alkaline agarose electrophoresis was similar, it was suggestive of a -chain variant in this case.

Citrate agar electrophoresis (pH 6.2) showed only one major band in the position of Hb A. IEF also showed two major bands, i.e. one in the position of Hb A and another more anodal to it in the position of Hb J. The attending physician was consulted about the clinical condition of the patient. Since no abnormality was noted by the physician except a slightly lower hemoglobin, a report was submitted advising the presence of a harmless Hb J- trait without any hematological or clinical consequences..

For instructional purposes, we have included the CZE and HPLC scans of Hb J-Baltimore (-chain variant), which is the most prevalent -chain variant among the category of Hb J variants (both and chains).
419

CZE showed a major peak (approximately 40%) in zone 12 (Hb JBaltimore), and HPLC also showed a major peak at a retention time of 1.8 minutes (Hb J-Baltimore). We suspect that this case was most likely a representative of Hb J-Baltimore. Confirmation by DNA studies, globin chain analysis, and LC-Mass spectrometry would be necessary for definitive diagnosis. However, for financial reasons, all these additional tests are not required in view of the benign status of the hemoglobinopathy in the patient. Hb J-Baltimore (also called J-Trinidad, J-Ireland, J-Georgia) is a -chain variant [ 16(A13) GlyAsp] and is encountered rarely in Afro Americans, and very rarely in Europeans.

References
1. Arribalzaga K, Ricard MP, Carreno DL, Sanchez J, Gonzalez A, Ropero P, + Villegas A. Hb J-Baltimore [16(A13)GlyAsp] Associated with Thalassemia in a Spanish Family. Hemoglobin 1996; 20(1): 79-84. Landin B, Jeppsson J-O. Rare -Chain Hemoglobin Variants Found in Swedish Patients During Hb A1c Analysis. Hemoglobin 1993; 17(4): 303-318. Vandenesch F, Baklouti F, Francina A, Vianey-Liaud C, Bertrand A, Le Devehat C, Delaunay J. Hemoglobin J-Baltimore [16(A13)GlyAsp]: Interference with the assay of Hb A1c. Clin Chim Acta 1987; 168(2): 121-28. Musumeci S, Schiliro G, Fisher A, Musco A, Marinucci M, Mavilio F, Fontanarosa PP, Tentori L. Hb J-Baltimore [16(A13)GlyAsp] in Association with -Thalassemia in a Sicilian Family. Hemoglobin 1979; 3(6): 459-464.

2. 3.

4.

420

Case # 25 Hemoglobin Malmo trait

An 18 year old high school student from Warren, Michigan was hurt during football practice and brought to the Emergency Department of the hospital. He had a ruddy face and complained of pain in the lower extremities.

Laboratory Data:
Hemoglobin RBC MCV MCH Platelets Hb A (HPLC) Hb A2 Hb F Hb Variant (HPLC) 20.1 6.8 88.0 29.7 270.0 56.0 2.0 4.0 38% 13.5 -18.5 g/dL 3 4.6 - 6.2 Mil/mm 80 - 100 fL 27 - 34 pg 3 150 400 Th/mm 94.3 - 98.5% 1.5 - 3.7% 0.0 - 2.0%

Peripheral Blood Smear: Crowding of erythrocytes. Sickle cell solubility test for Hb S: Negative Hemoglobin instability (isopropanol) test: Negative

Agarose Gel Electrophoresis (pH 8.6)

421

Case # 25 Hemoglobin Malmo trait

Citrate Agar Electrophoresis (pH 6.2)

Isoelectric focusing

422

Case # 25 Hemoglobin Malmo trait

Capillary zone electrophoresis

High performance liquid chromatography

423

Case # 25 Hemoglobin Malmo trait

Interpretation & Discussion:

Note: Acid agarose data was not available for Hb Malmo. Method Hb A area Major band (Hb A + Hb Variant) Major band (Hb A + Hb Variant) Major peak (Hb A + Hb Malmo) Zone 9 Major band in Hb A position Hb S area Hb A2/C area Very faint band (Hb A2)

Alk Agarose

Acid Agar Minor Hb F band detected CZE

Minor peak (Hb A2) Zone 3 Faint band in Hb A2 area Minor peak (Hb A2) RT=3.6

IEF

Faint band in Hb F area

*

HPLC

Major band slightly anodal to Hb A band Minor Major peak peak (Hb F) (Hb RT=1.03 Malmo) RT=1.66

Major peak (Hb A) RT=2.44

*Note:

HPLC retention times varies with the type of the instrument used and several other factors, e.g. temperature, etc.

424

CZE was not available in our laboratory when we encountered this case, therefore the CZE scan provided here is of another Hb Malmo patient. However the CZE scan of a Hb Malmo trait was not helpful in the identification of the variant since both the Hb A and Hb Malmo migrated together in zone 9.

IEF did separate Hb Malmo from Hb A in a pattern identical with a proven case of Malmo hemoglobinopathy reported in the literature. Hb Malmo migrated slightly anodal to Hb A, and the migration mobility was much less than some fast hemoglobins (e.g. Hb J-Oxford, Hb J-Baltimore, Hb N, Hb I, etc).

HPLC provided two clearly separated major peaks and three small peaks of Hb F, Hb A1c and Hb A2. One major peak was due to Hb A (RT= 2.43 minutes), and another with a faster elution time (RT=1.66), was due to the hemoglobin variant of this case. In summary, Hb Malmo elutes with Hb A or before, depending on the chromatographic system used. In our system it eluted before Hb A.

We were aware that occasionally erythrocytosis has been found to be associated with high-oxygen affinity hemoglobins, but we did not have the capability to determine a hemoglobin-oxygen dissociation curve and its p50 ( the point on the curve where the hemoglobin molecule is half-saturated with oxygen).

425

Normally, the hemoglobin-oxygen dissociation curve is sigmoid-shaped. High affinity hemoglobins, e.g. Hb Malmo, show a markedly leftward shifted curve (p50 of about 13 torr compared to normal values of 26-30 torr) resulting in a hyperbolic shape. The oxygen delivery to the tissues is impaired whenever the oxygen affinity is high (low p50). Erythropoietin production is stimulated, which in turn increases the red cell mass, resulting in erythrocytosis.

After consultation with the attending physician, a narrative report was submitted stating that a hemoglobin variant is present and in view of marked erythrocytosis a possibility of a high affinity hemoglobin cannot be ruled out.

Fortunately, the parents of the patient agreed to provide their blood for analysis. The mother was found to have a normal CBC and hemoglobin pattern. The father, who had immigrated from Sweden to USA belonged to a family with known erythrocytosis. Some years ago, when he complained of fatigue, headaches and lethargy a diagnosis of Hb Malmo was made in Sweden. In order to relieve his symptoms, phelebotomy was performed. The electrophoretic (alkaline, acid, IEF) results and HPLC curve were identical for both the father and son.

426

In view of the ancestral background and the laboratory results on both the patient and the parents, a putative diagnosis of Hb Malmo was made. The attending physician and the family were advised that the hemoglobin disorder was essentially benign. However, the patient should refrain from smoking and should be followed periodically for any signs of fatigue, headaches or lightheadedness.

More than 100 high oxygen affinity hemoglobin variants are reported in the literature. Hb Malmo is a member of this class and is the result of the substitution of glutamine for a histidine amino acid at the 97th amino acid of the chain [97(FG4)HisGln]. This mutation is in the area of the peptide chain that moves during the oxygenation deoxygenation process. The substitution inhibits movement in such a manner that deoxygenation becomes more difficult and deters transfer to the tissue to the point that the patient would become anemic if the body did not compensate by making excess erythrocytes. The amino acids from position 94 through 103 constitute a nonhelical section of the beta chain and mutations effecting ionicity of those positions effects the spacing between the alpha and beta chains near the point of oxygenation. This area of the globin chain is called the FG segment or FG corner and a list of these variants is found in Table 1 (courtesy of Hoyer & Kraft, College of American Pathologists).

427

The fit between the alpha and beta chains is critical because the gap becomes narrower when oxygen is attached to the ferrous iron and expands as oxygen is released. A second region of the beta chain (amino acids 143 through 146 on the Carboxy end of the molecule) also effects this spatial control. Several hemoglobin variants have been identified as possessing mutations in this area and thus assisting in understanding the synchronous action involved in the oxygenation / deoxygenation process. Table 2 is a list of these variants (also courtesy of Hoyer & Kraft, College of American Pathologists). In all the cases on this list except for Heathrow and Brigham the mutation effects the shape of the globin chains such that the electrophoretic mobility is altered so the mutations are not silent (personal communication, Rita Ellerbrook, PhD, Helena Laboratories, USA).

Hb Malmo only exists in the heterozygous state. The homozygous state has not been reported and is thus most probably incompatible with life.

Reference
1. 2. 3. Bain BJ. High-affinity Hemoglobins. In: Hemoglobinopathies Diagnosis, Blackwell Publishing, Oxford, United Kingdom. 2006, 224-226. Steinberg MH. Genetic disorders of hemoglobin oxygen affinity. www.uotodate.com 2013 UpToDate Fernandez FAG, Villegas A, Ropero P, Carreno MD, Anguita E, Polo M, Pascual A, Henandez A. Hemoglobinopathies with high oxygen affinity. Experience of Erythropathology Cooperative Spanish Group. Ann Hematol 2009; 88: 235-238.
428

4.

5. 6.

7.

Giordano PC, Harteveld, Brand A, Willems LNA, Kluin-Nelemans HC, Plug RJ, Batelaan DN, Bernini LF. Hb Malmo[-97(FG-4) HisGln] leading to polycythema in a Dutch family. Ann Hematol 1996; 73: 183-188. Landin B, Berglund S, Wallman K. Two Different Mutations in Codon 97 of the -Globin Gene Cause Hb Malmo in Sweden. Am J Hematol 1996; 51: 32-36. Girino M, Riccardi A, Mosca A, Paleari R, Bonomo P. Double Heterozygosity for Hemoglobin Malmo [97 (FG4) HisGln] and -Thalassemia Traits. Haematologica 1989; 74: 187-90. Boyer SH, Charache S, Fairbanks VF, Maldonado JE, Noyes A, Gayle EE. Hemoglobin Malmo -97 (FG-4) HistidineGlutamine: A Cause of Polycythemia. J Clin Invest 1972; 51: 666-676.

429

Table 1. Chain Variants in FG Corner and G Helix Position 94 Helical # FG1 Substitution AspHis AspAsn LysAsn LysGlu HisGln HisLeu ValMet ValGly ValAla 99 G1 AspAsn AspHis AspAla AspTyr AspGly AspVal ProLeu GluGly GluGln GluAsp GluLys AsnLys AsnThr AsnSer AsnTyr PheLeu
430

Name Barcelona Bunbury

Effect polycythemia normal

95

FG2

Detroit normal N-Baltimore normal Malmo Wood Koln polycythemia polycythemia

97

FG4

98

FG5

hemolysis, O2 Affinity Nottingham hemolysis Djelfa (?) Kempsey Yakima Radcliffe Ypsilanti Hotel Dieu Chemilly Brigham Alberta Rush Potomac British Columbia Richmond Kansas Beth Israel Saint Mande Heathrow polycythemia polycythemia polycythemia polycythemia polycythemia polycythemia polycythemia polycythemia hemolysis polycythemia polycythemia

100 101

G2 G3

102

G4

normal cyanosis cyanosis cyanosis polycythemia

103

G5

Table 2. Chain Variants Near the C-Terminus

Position 143

Helical # H21

Substitution HisArg HisGln HisPro HisAsp HisTyr

Name Abbruzzo Little Rock Syracuse Rancho Mirage Old Dominion

Effect polycythemia polycythemia polycythemia (?)

normal CBC O2 affinity

144

HC1

LysAsn LysGlu

Andrewpolycythemia Minneapolis Mito Bethesda Rainier Osler McKees Rocks Hiroshima York Cochin-Port Royal Cowtown Kodaira polycythemia polycythemia polycythemia polycythemia polycythemia

145

HC2

TyrHis TyrCys TyrAsn TyrStop HisAsp HisPro HisArg HisLeu HisGln

146

HC3

polycythemia polycythemia (?) polycythemia polycythemia

The contents of these tables are presented from Hoyer JD, Kroft SH, eds. Color Atlas of Hemoglobin Disorders: A Compendium Based on Proficiency Testing. Northfield, IL: College of American Pathologists: 2003 (Reproduced with Permission)

431

Case # 26

Hemoglobin Koln trait

A 18 year old female student. No ancestral information was available. Physical examination revealed scleral icterus and spleen palpable 4 cm below left costal margin.

Laboratory Data:
Hemoglobin RBC MCV RDW Platelet WBC Reticulocyte Serum Iron Total Bilirubin Indirect Bilirubin Hb A (HPLC) Hb A2 (HPLC) 10.7 3.9 106 13.6 235 7.1 2.9 39 2.8 2.4 74.8% 2.2 12.0 -16.0 g/dL 3 4.0 - 5.5 Mil/mm 79 - 98 fL 11.5 -14.5% 3 150 400 Th/mm 4.0 11.0 Th/mm 0.7 - 1.8 % 30 160 ug/dL 0.0 1.5 mg/dL 0.0 0.4 mg/dL 1.5 - 3.7%
3

Peripheral Blood Smear: Mild macrocytic anemia with slight hypochromasia, polychromasia, and occasional target cells. Sickle cell solubility test for Hb S: Negative Hemoglobin instability (isopropanol) test: Positive

Agarose Gel Electrophoresis (pH 8.6)

432

Case # 26 Hemoglobin Koln trait

Citrate Agar Electrophoresis (pH 6.2)

Isoelectric focusing

433

Case # 26 Hemoglobin Koln trait

Capillary zone electrophoresis

High performance liquid chromatography

434

Case # 26 Hemoglobin Koln trait

Interpretation & Discussion

Summary of Results Method Hb A area Major band Hb A Major band (All Hbs) Major peak (Hb A +
Hb Kln)

Hb S area
Broad smudge from Hb S through Hb C

Hb A2/C area
No discrete band detected

Alk Agarose

Acid Agar / Agarose CZE

Zone 9 IEF Major band (Hb A)

Minor peak (Hb Koln) Zone 6

Broad smudge of Hb Koln from Hb A-Hb A2

Minor peak (Hb Koln) Zone 4

HPLC

Major peak (Hb A) RT=2.39

Two barely visible minor peaks (Hb Koln) RT=4.54.8

Minor peak (Hb A2 + Hb Koln) Zone 3 Barely visible minor band Hb A2 Minor peak Hb A2 RT=3.6

Minor peak (Hb Koln) RT=4.95

# Due to the instability of Hb Koln, several minor bands were noticed in HPLC. A similar phenomenon was observed in other electrophoretic methods except in the acid agar/agarose electrophoresis, where only one major band was detected.

Alkaline agarose gel electrophoresis (pH 8.6) showed a major band in the position of Hb A and a smudged band migrating on both the anodal and cathodal
435

sides of Hb S extending further towards the area of Hb C. Citrate agar electrophoresis (pH 6.2) showed only one band in the position of Hb A. IEF showed an intense band in the position of Hb A, and a broad smear between Hb A and Hb A2 (similar to a fresh painting with a brush). CZE showed a major peak in zone 9 (Hb A area) and three minor peaks in zones 3, 4 and 6. HPLC showed a major peak at a retention time (RT) of 2.39 minutes (Hb A), and minor peaks from a RT of 3.5 5.0 minutes.

The laboratory tests indicated that either the hemoglobin variant did not separate from Hb A, or it formed discrete peaks, or it formed blurred bands. Since the hemoglobin instability test was positive, the attending physician was advised of the possibility of an unstable hemoglobin variant.

In August 2013, Professor Steinberg reviewed (reference # 1) the current clinical and hematological characteristics of unstable hemoglobins. According to this recent review, approximately 140 of the1028 known mutations of hemoglobin were found to be unstable. To date, it is not feasible to identify an unstable hemoglobin variant (either alpha-, beta-, gamma, and delta-globin chains abnormalities) by the commonly used laboratory methods.

In most worldwide laboratories, Hb E, Hb H, Hb Hasharon and Hb Koln are the most frequently reported unstable hemoglobin variants. Three of these variants (Hb E, Hb H, and Hb Hasharon) were excluded in our patient on the basis of their electrophoretic mobilities and retention times on HPLC. Hb E
436

(Case # 14a) migrates in the position of Hb A2/E/O/C. Hb H is a fast migrating hemoglobin variant (more anodal to Hb A) on alkaline agarose gel electrophoresis (pH 8.6). Hb Hasharon (Case # 20) migrates in the Hb S area on both the alkaline and acid electrophoresis.

Hemoglobin Koln, a prevalent unstable hemoglobin, has a rather atypical electrophoretic migration pattern, which is helpful in its identification in conjunction with the associated clinical and hematological manifestations of the patient. In Hb Koln, a valine amino acid at the 98th position of the -chain (98ValMet) is substituted by the amino acid methionine. Since both the valine and methionine amino acids are neutral amino acids, there would be no net change in the charge. However Hb Koln does separate from Hb A on agarose gel electrophoresis (pH 8.6). This anomaly is explained by the modification at the site of -chain contact with the heme molecule, which causes a quaternary structure change of the hemoglobin molecule. As a consequence of this, especially during alkaline electrophoresis, Hb Koln loses heme groups from the abnormal -chain and thus loses negative charge. The hemoglobin migrating in the Hb S position on alkaline electrophoresis is essentially des-heme hemoglobin Koln.

The diagnosis of Hb Koln was substantiated by the following observations: i) ii) Unstable hemoglobin Negative sickle cell solubility test for Hb S
437

iii) iv) v) vi) vii)

Minimal or no anemia Splenomegaly and regenerative erythrocyte changes even in the absence of anemia Hypochromasia and macrocytosis are usually evident Increased oxygen affinity Atypical migration pattern on alkaline agarose gel electrophoresis (pH 8.6), IEF, CZE and multiple peaks on HPLC.

In view of the above considerations, the physician was advised of the possibility of Hb Koln. Due to the autosomal dominant mode of familial transmission, the parents of the patient should also be analyzed for the confirmation of Hb Koln.

Generally, patients with Hb Koln are asymptomatic unless complications develop, e.g. blow to the left side of the abdomen by contact sports, exacerbation of hemolysis during infection (e.g. upper respiratory tract infection), or after use of some medications, e.g. sulfonamides. Hb Koln exists only in the heterozygous state; life is not compatible with homozygous Hb Koln.

Hb Koln and Hb Hasharon (Case # 20) are two of the many unstable hemoglobins that have been identified. Unstable hemoglobins may be suspected in patients presenting with the symptoms of congenital non-sherocytic anemia with splenomegaly and pigmented gallstones, or with hemolytic anemia in which the red cells contain Heinz bodies and are sensitive to oxidant drugs as sulfonamides, or with mild anemia in which the reticulocyte count is elevated compared to the amount of hemoglobin, or with a peripheral blood smear containing target cells, basophilic stippling, a few anisocytes and
438

hypochromic red cells. If the condition was present in a newborn, the cause might be a gamma chain variant whose effect would be eliminated as beta chain production increased. Likewise, an asymptomatic newborn, in which clinical condition began to develop at 4 to 6 months of age might be a beta chain variant. A suspicious peripheral blood smear would lead to a Heinz body test and hemoglobin instability (isopropanol) test, but the later two tests should not be run in the presence of Hb F levels >5%.

References
1. 2. Steinberg MH. Unstable hemoglobin variants. www.uptodate.com UpToDate. Literature review current through August 2013. Chang YH, Hur M, Lee DS, Park SS, Kim BK, Park S, Ohba Y, Hattori Y, Cho HI. The first case of Hb Koln [98(FG5)ValMet] in Korea. Hemoglobin 1999; 23(3): 287-289. Chang J-G, Yang T-Y, Perng L-I, Wang J-C, Tsan K-W. Hb Koln [98(FG5)ValMet] : The first case found in a Chinese family. Hemoglobin 1998; 22 (5&6): 535-536. Landin B, Frostad B, Brune M, Ljung R. Haemoglobin Koln as de novo mutations in Sweden: Diagnosis by PCR and specific enzymatic cleavage. Eur J Haematol 1994; 52:156-61. Indrak K, Brabec V, Wilson JB, Webber BB, Huisman THJ. Hb Koln or 2298(FG5)VALMet in a Czechoslovakian family. Hemoglobin 1991; 15(1& 2): 133-135. Ohba Y. Unstable hemoglobins. Hemoglobin 1990; 14: 353-388. Bird AR, Karabus CD, Hartley PS, Lehman H. Haemoglobin Koln in Cape Town. A case reprt. S Afr Med J 1987; 72: 154-156. Gurgey A, Altay C. Hemoglobin Koln [ 98(FG5) ValMet] in a Turkish child. The Turkish Journal of Pediatrics 1982; 24: 271-73. Ricco G, Ravazzolo R, Rege-Cambrin G, Capaldi A, Trento M, Leechi M, Sartori ML, Furlani C, Rietto GB, Rabino-Massa E. Koln haemoglobinopathy in Italy. Pan. Med 1981; 23:227-233. Stirling M. Koln Haemoglobinopathy in a Second Scotish Family. Scott Med J 1980; 25: 121-125. Egan EL, Fairbanks VF. Postsplenectomy Erythrocytosis in Hemoglobin Koln Disease. N Eng J Med 1973; 288: 929-931.
439

3.

4.

5.

6. 7. 8. 9.

10. 11.

12. 13.

Hallen J, Charlesworth D, Lehmann H. Haemoglobin Koln in a Jewish Family. Acta Med. Scand. 1972; 191: 177-180. Luan Eng L-I, Lopez CG, Eapen JS, Eravelly J, Wiltshire BG, Lehmann H. Unstable Haemoglobin Koln Disease in Members of a Malay Family. J Med Genetics 1972; 9: 340-43.

440

Case # 27 Hemoglobin Q-India trait

27 year old male from India. No abnormality detected. Physical examination was unremarkable.

Laboratory Data
Hemoglobin RBC MCV MCH Platelet Hb A2 Hb F Hb A (HPLC) Hb Variant (HPLC) 14.8 5.4 86.0 28.3 232 1.1 0.8 78.8 19.3 % 13.5 - 18.5 g/dL 3 4.6 - 6.2 Mil/mm 80 - 100 fL 27 - 34 pg 3 150 - 400 Th/mm 1.5 - 3.7% 0.0 - 2.0% 94.3 - 98.5%

Peripheral Blood Smear: No abnormality Sickle cell solubility test for Hb S: Negative Hemoglobin instability (isopropanol) test: Negative

Agarose Gel Electrophoresis (pH 8.6)

441

Case # 27 Hemoglobin-Q India trait

Citrate Agar Electrophoresis (pH 6.2)

Isoelectric focusing

442

Case # 27 Hemoglobin-Q India trait

Capillary zone electrophoresis

High performance liquid chromatography

443

Case # 27 Hemoglobin Q-India trait

Interpretation & Discussion

Summary of Results Method Hb A area Major band (Hb A) Hb S area
Major band slightly towards Hb A side of Hb S (Hb QIndia)

Alk Agarose

Hb A2/C area Faint band (Hb A2)

Acid Agar

Major band between Hb A and Hb S (Hb A + Hb Q-India)

Hb A and Hb QIndia combine as a broader band

Slightly anodal towards Hb S

Acid Agarose

Major band (Hb A + Hb QIndia) Major peak (Hb A) Zone 9

Exactly in the position of Hb A

CZE

Major peak (QIndia) Zone 6 Major band (Hb QIndia) Major peak (Hb QIndia) RT=4.7
444 Slightly anodal towards Hb A

Minor peak (Hb A2) Zone 3

Minor peak (Hb QIndia + Hb A2 ) Zone 1

IEF

Major band (Hb A)

Faint band (Hb A2) Faint peak (Hb A2) RT=3.6

HPLC

Major peak (Hb A) RT=2.39

The diagnosis of Hb Q-India trait is cumbersome on alkaline electrophoresis and may be mistaken for Hb S/D/ Lepore because the band in the Hb S area is close to Hb S. Sickling or sickle solubility tests should be employed to rule out Hb S. These and other variants are characterized through additional laboratory tests (HPLC and IEF). Molecular analysis is required for establishing a definitive diagnosis. This can be achieved through ARMS-PCR, RFLP-PCR and mass spectrometry. However, first two of these are fraught with technical pitfalls and should be done with inclusion of appropriate controls to acquire correct results. LC/ESI-MS offers a rapid and unambiguous characterization of individual chains. DNA sequence is currently the most accurate way of identifying Hb Q-India in a blood sample.

Review of Hemoglobin Q
Bushra Moiz, PhD

Introduction
Hb Q was first reported in 1958 in a Chinese patient (1). Since then a number of cases have been described in Asians. It is a rare hemoglobinopathy resulting from a single point mutation (GACCAC) of -1 globin gene present on chromosome 16. The resulting hemoglobin is modified structurally at polypeptide chain replacing aspartic acid by histidine. Depending on the implicated codon, three variants have been described namely Hb Q-India ( 64 AspHis), Hb Q-Thailand ( 74 AspHis), and Hb
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Q-Iran ( 75 AspHis). Using computerized models for protein structure, it was observed that there is no difference between the predicted secondary structures of normal -globin and that of Hb Q-India (2). In contrast, Hb Q-Iran carries an extra helix while Hb Q-Thailand carries two extra helices. The predicted results of tertiary structure also support these findings (2). Since the residue and hence charge changes involve the surface of the hemoglobin tetramer, the properties of the hemoglobin molecule are not affected (3). Hb Q-Thailand [74(EF3) AspHis] is often found in Thailand, China, and other Southeast Asian countries (4). It has several synonyms including Hb Mahidol, QChinese, G-Taichung, Kurashiki, and Asabara (5). The alpha-Q-Thailand gene is strongly linked to gene deletion and has important implications in the identification and diagnosis of hemoglobinopathies and thalassemias. Subjects with Hb Q-Thailand invariably show microcytosis as the variant is invariably linked to (-
o 4.2

). However,

individuals who are doubly mutated for Hb Q-Thailand and thalassemia may be more severely anemic (6,7). More complex interaction of Hb Q-Thailand with Hb E, Constant Spring and hereditary persistence of fetal hemoglobin has been described in the literature (8-10). Hb Q-Iran was first described in 1970 by Lorkin et al (11) and later by Rahimi in an Iranian individual (12). Hb Q-India was first reported in 1972 by Sukumaran in a Sindhi family (13). Later reports were published by Dash (14), Abraham (3) and Desai (15); their observations came from Sindhi and Punjabi families. Hb Q-India usually occurs in the heterozygous
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state (

Q-India

/ and /), however double heterozygotes with both (-

Q-India o

Q-India

and /) and -thalassemia (

/ and / ) were reported (3, 14). A novel

Hb D-Punjab / Hb Q-India was recently reported in an Indian diabetic (16). No report of a homozygous state has ever been published.

Clinical manifestation
The presence of Hb Q does not impart any functional deficit since the hemoglobin is not altered structurally at its tertiary level (8). Hb Q is a stable molecule and has normal oxygen affinity. Therefore, Hb Q is clinically silent in a heterozygous individual. In contrast, subjects who are compound heterozygotes with other hemoglobinopathies exhibit a thalassemic phenotype. For example, co-inheritance of Hb Q-India and -thalassemia results in a mild anemia (17). Similarly, Hb Q-H disease caused by the co-inheritance of Hb QThailand and -thalassemia [--/- ] presents with a chronic, hemolytic anemia with associated jaundice and splenomegaly (18). The severity of anemia may warrant blood transfusions and or splenectomy (18). Subjects with a single copy of Hb Q-Iran do not show any distinctive clinical manifestation (19). Interestingly, a report from Turkey described a subject with homozygous Hb Q-Iran who was clinically asymptomatic (20).
o o

Diagnostic laboratory tests and interpretation

CBC and peripheral blood smear An individual heterozygote with Hb Q-Thailand usually shows a
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slight microcytosis. A thalassemic blood picture similar to Hb H disease is observed in Hb Q-H disease (18). The peripheral blood smear in this disorder showed anisocytosis, poikilocytosis, nucleated red cells and target cells (6). Intracellular crystals were also observed in red cells in brilliant cresyl blue preparations (6) similar to Hb H disease.

Hb Q-India usually demonstrates normochromic normocytic red cell indices with normal or near normal hemoglobin (3). However, a few cases with mild anemia and microcytosis have been reported in the literature (10). Hb Q-India with co-inherited -thalassemic trait usually shows hypochromic microcytic red cell indices with mild anemia (14,17). Hb Q-Iran shows normal red cell indices with normal hemoglobin irrespective of zygosity (12,20).

Alkaline agarose gel, citrate agar and acid agarose electrophoresis Hb Q migrates in the position of Hb S/D/Lepore on agarose gel and cellulose acetate electrophoresis (pH 8.6). Presence of chains lead to the appearance of accessory bands corresponding to abnormal hemoglobin and hence double bands of Hb A2 can be observed (14). Thus, Hb Q can easily be misinterpreted as Hb S if confirmatory testing such as sickling or sickle solubility test are not performed. On citrate agar (pH 6.2), it migrates between Hb A and Hb S. Upon electrophoretic migration in agarose medium at pH 6.2, Hb Q migrates exactly in the
448
o

position of Hb A.

HPLC .The separation of hemoglobin variants depends on their retention times (21). Hb Q-India, Iran and Thailand exhibited retention times similar to that of other -chain variants in the range of 4.76 to 4.78 minutes (3). In a heterozygote, Hb Q-India and Iran usually eluted as 17-19% of the total hemoglobin. In contrast, Hb Q-Thailand represented 30-35% of the total hemoglobin in the heterozygote state, because of the gene deletion accompanying it (5). Hb Q % may be further decreased with concomitant iron deficiency or with co-inheritance of -thalassemia trait (14,17).

IEF Hb Q-India focused in the position of Hb S. However, both the Hb Q-Iran and Hb Q-Thailand migrated slightly anodal to Hb S, i.e. towards Hb A.

Mass spectrometry Liquid chromatography electrospray ionization mass spectrometry (LC/ESI-MS) has the advantage of providing molecular information in individual polypeptide chains and of the hemoglobin molecule (23). LC/ESI-MS has been described in recent years to evaluate unknown Hb variants including Hb Q-India (24). It detects the presence of a mutant 449

chain differing in mass from a normal -chain by 22 DA. The later is assigned to a mutation of an aspartic acid residue to a histidine residue thus identifying Hb Q. The site can be identified by tandem-mass analysis of a tryptic digested fragment encompassing residues V62-K90 of hemoglobin -chain. Sequencing these fragments can establish the diagnosis of Hb Q-India.

ARMS-PCR This technique can be used for the successful detection of various hemoglobin variants including Hb Q-India (3). This technique is based on the amplification of allele specific primers because of 3-terminal matches and mismatches. The methodology is simple, rapid and inexpensive; however, it is non-specific since either sub-optimal amplification or deteriorating primers can lead to false positive results (25).

RFLP-PCR Recently, a restriction enzyme digestion assay was employed for the diagnosis of Hb Q-India (22). Restriction enzyme EaeI was utilized in RFLP-PCR since Hb Q-India abolishes the recognition site of this enzyme. It can be used as a simple, robust and alternative method to ARMS-PCR for DNA diagnosis of Hb Q-India. However, any other rare variant that abolishes the same EaeI restriction site would also be detected. Hence, RFLP-PCR can be used as an adjuvant test after HPLC and or IEF for
450

primary diagnosis of Hb Q-India.

Gene sequencing This is the most definitive technique for identifying a hemoglobin variant. Recently, Bhat described DNA sequencing in a patient with Hb QIndia (26). This methodology of sequence electrophoretogram clearly demonstrates the specific location of the mutation of Hb Q-India. Not only did it show that the codon of GAC encoding aspartic acid was mutated to the codon CAC encoding for histidine, but it also depicted the zygosity of the patient (26).

References
1. 2. 3. Vella F, Wells RH, Ager JA, Lehmann H. A haemoglobinopathy involving haemoglobin H and a new (Q) haemoglobin. Br Med J 1958; 1: 752-755. Yadav AK. Comparative analysis of protein structure of common Hb Q variants. Indian J Pathol Microbiol 2010; 53: 696-698. Abraham R, Thomas M, Britt R, Fischer C, Old J. Hb Q-India: an uncommon variant diagnosed in three Punjabi patients with diabetes is identified by a novel DNA analysis test. J Clin Pathol 2003; 56: 296-299. Higgs DR, Hunt DM, Drysdale HC, Clegg JB, Pressley L, Weatherall DJ. The genetic basis of Hb Q-H disease. Br J Haematol 1980; 46: 387-400. Hoyer JD, Kroft HS, editors. Color Atlas of Hemoglobin Disorders. A Compendium Based on Proficiency Testing, 159 pp, Northfield, Illinois, College of American Pathologists, 2003. Lieinjo LE, Pillay RP, Thuraisingham V, Further Cases of Hb Q-H disease (Hb Q-alpha thalassemia). Blood 1966, 28: 830-839. Beris P, Huber P, Miescher PA, Wilson JB, Kutlar A, Chen SS, Huisman TH. Hb Q-Thailand Hb H disease in a Chinese living in Geneva, Switzerland: Characterization of the variant and identification of the two alpha-thalassemic chromosomes. Am J Hematol 1987; 24: 395-400. Sanchaisuriya K, Chunpanich S, Fucharoen S, Fucharoen G, Sanchaisuriya P, Changtrakun Y. Association of Hb Q-Thailand with homozygous Hb E and heterozygous Hb Constant Spring in pregnancy. Eur J Haematol 2005; 74: 221-227.
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4. 5.

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9. 10.

11.

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Li D, Liao C, Li J, Xie X, Zhong H. Association of Hb Q-Thailand with heterozygous Hb E in a Chinese patient. Hemoglobin 2008; 32: 319-321. Zheng W, Liu Y, Chen D, Rong K, Ge Y, Gong C, Chen H. Complex interaction of Hb Q-Thailand and Hb E with alpha (0)-thalassemia and hereditary persistence of fetal hemoglobin in a Chinese family. Ann Hematol 2010; 89: 883-888. Lorkin PA, Charlesworth D, Lehmann H, Rahbar S, Tuchinda S, Eng Li. Two haemoglobins Q, alpha-74 (EF3) and alpha-75(EF4) aspartic acid to histidine. Br J Haematol 1970; 19: 117-125. Rahimi Z, Aktamipour R, Vaisi-Raygani A, Nagel RL, Muniz A. An Iranian child with Hb Q-Iran [alpha75(EF4)AspHis]/-alpha3.7 kb/IVSII.1 GA]: first report. J Pediatr Hematol Oncol 2007; 29: 649-651. Sukumaran PK, Merchant SM, Desai MP, Wiltshire BG, Lehmann H. Haemoglobin Q India [alpha64(E13) aspartic acid histidine] associated with beta-thalassemia observed in three Sindhi families. J Med Genet 1972; 9: 436-442. Dash S, Huisman TH. Hemoglobin Q-India [64(E13) AspHis] and beta thalassemia: a case report from Punjab (North India). Eur J Haematol 1988; 40: 281. Desai DV, Dhanani H, Kapoor AK, Yeluri SV. Hb Q-India in a Sindhi family: an uncommon hemoglobin variant. Lab Hematol 2004; 10: 212-214. Higgins T, Schnabl K, Savoy M, Rowe P, Flamini M, Bananda S. A novel double heterozygous Hb D-Pubjab / Hb Q-India hemoglobinopathy. Clin Biochem 2012; 45: 264-266. Moiz B, Moatter T, Hashmi MR, Hashmi N, Kauser T, Nasir A, Khurshid M. Identification of Hemoglobin Q India (alpha 1-64 AspHis) through ARMSPCR. First report from Pakistan. Ann Hematol 2008; 87: 385-389. Leung KF, ma ES, Chan AY, Chan LC. Clinical phenotype of haemoglobin QH disease. J Clin Pathol 2004; 57: 81-82. Rahimi Z, Rezei M, Nagel RL, Muniz A. Molecular and hematological analysis of hemoglobin Q-Iran and hemoglobin Setif in Iranian families. Arch Iran Med 2008; 11: 382-386. Ozdag H YI, Akar N. First observation of homozygote Hb Q-Iran [alpha 75(EF4) AspHis]. Turk J Hematol 2008; 25: 48-50. Joutovsky A, Hadzi-Nesic, Nardi MA. HPLC retention time as a diagnostic tool for hemoglobin variants and hemoglobinopathies: a study of 60 000 samples in a clinical diagnostic laboratory. Clin Chem 2004; 50: 1736-1747. Khalil MS, Henderson S, Schuh A, Hussein MR, Old J. The first use of Eael restriction enzyme in DNA diagnosis of Hb Q-India. Intl J Lab Hematol 2011; 33: 492-497. Wild BJ, Green BN,, Cooper EK, Lalloz MR, Erten S, Stephens AD, Layton DM. Rapid identification of hemoglobin variants by electrospray ionization mass spectrometry. Blood Cells Mol Dis 2001; 27: 691-704. Mandal AK, Bisht S, Bhat VS, Krishnaswamy PR, Balaram P. Electrospray mass spectrometric characterization of hemoglobin Q (Hb Q-India) and a
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25.

26.

double mutant hemoglobin S/D in clinical sampes. Clin Biochem 2008; 41: 7581. Old JM, Khan SN, Verma I, Fucharoen S, Kleanthous M, Ioannou P, Kotea N, Fisher C, Riazuddin S, Saxena R, Winichagoon P, Kyriacou K, Al-Qudbaili F, Khan B. A multi-center study in order to further define the molecular basis of beta-thalassemia in Thailand, Pakistan, Sri Lanka, Mauritius, Syria, and India, and to develop a simple molecular diagnostic strategy by amplification refractory mutation system-polymerase chain reaction. Hemoglobin 2001; 25: 397-407. Bhat VS, Dewan KK, Krishnaswamy PR, Mandal AK, Balaram P. Characterization of a hemoglobin variant: Hb Q-India / IVS 1-1 [G>T]-beta thalassemia. Indian J Clin Biochem 2010; 25: 99-104.

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Case # 28

Hemoglobin Dhofar trait

A 24 year old male, belonging to Qara tribes from the Dhofar region of the Sultanate of Oman. The patient had not been transfused during the past six months.

Laboratory Data:
Hemoglobin RBC MCV MCH RDW Platelet Hb A Hb A2 (HPLC) Hb F Hb Dhofar (HPLC) 13.0 5.1 68 22.4 13.2 243 81.5 4.1 0.8 13.6 13.5 -18.5 g/dL 3 4.6 - 6.2 Mil/mm 80 -100 fL 27 34 pg 11.5 -14.5% 3 150 - 400 Th/mm 94.3 - 98.5% 1.5 - 3.7% 0.0 - 2.0%

Peripheral Blood Smear: Microcytosis, hypochromasia, target cells. Hemoglobin instability (isopropanol) test: Negative Sickle cell solubility test for Hb S: Negative Note: In HPLC, Hb A2 is slightly under estimated due to overlap with Hb Dhofar peak, but in heterozygotes , Hb A2 is found to be raised if quantified by capillary zone electrophoresis or elution after cellulose acetate electrophoresis.

Agarose Gel Electrophoresis (pH 8.6)

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Case # 28 Hemoglobin Dhofar trait

Citrate Agar Electrophoresis (pH 6.2)

Isoelectric focusing

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Case # 28 Hemoglobin Dhofar trait

Capillary zone electrophoresis

High performance liquid chromatography

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Case # 28 Hemoglobin Dhofar trait

Interpretation & Discussion

Summary of Results Method Hb A area Major band (Hb A) Hb S area Medium size band (Hb Dhofar)
Hb A and Hb Dhofar combine as a broader band

Alk Agarose

Hb A2/C area Minor band (Hb A2)

Acid Agar

Major band ( Hb A + Hb Dhofar)

Acid Agarose

Major band (Hb A + Hb Dhofar)

Major band broadened by Hb Dhofar

CZE

Major peak (Hb A) Zone 9

Medium size peak (Hb Dhofar) Zone 5 Medium size band (Hb Dhofar) Medium size peak (Hb Dhofar) RT=4.04

Minor peak (Hb A2) Zone 3

IEF

Major band (Hb A)

Minor band (Hb A2)

HPLC

Major peak (Hb A) RT=2.35

Minor peak (Hb A2) RT=3.6

*Note:

HPLC retention time (RT) varies with the type of the instrument used and several other factors, e.g. temperature etc.
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Alkaline agarose gel electrophoresis (pH 8.6) showed a major band in the position of Hb A and a medium size band in the area of Hb S/G/D/Lepore/Korle Bu, and a few other variants. Citrate agar electrophoresis (pH 6.2) showed one major band in the area of Hb A and no other major band was detected. Hb S was ruled out due to the negative sickle cell solubility test and absence of a band in the Hb S area on acid electrophoresis. Hb Korle Bu was also ruled out due to the absence of a band in the Hb G region on IEF. Hb G-Philadelphia was ruled out due to the absence of a G2 band on alkaline agarose electrophoresis and IEF. Both Hb D and Hb Lepore migrate in the area of Hb G on IEF, therefore the possibility of these two variants was also ruled out; Hb Dhofar migrates in the area of Hb S on IEF.

It is emphasized here that the identification of Hb Dhofar by alkaline and acid electrophoretic methods alone is not prudent. Secondly, the range of Hb Dhofar (26 59% in homozygous and compound heterozygous and 8.8 21.5.% in heterozygous) can be a confounding factor in its differentiation with Hb D-Los Angeles trait (Case # 12).

HPLC was informative since Hb Dhofar eluted at a retention time slightly longer than Hb A2 and not in the Hb S window. Here again, the retention time (4.04 minutes) of Hb Dhofar was in the D window, thus not providing conclusive evidence for its differentiation.

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CZE scan of the patient indicated that Hb Dhofar peak migrated in zone 5 (Hb S zone), thus other possibilities (migration peak assigned in zone 6 for Hb D-Los Angeles, Hb G-Philadelphia, Hb Lepore, etc) were ruled out.
29 (GGC-GGT) Gly-Gly 58 (CCT-CGT) ProArg

Hb Dhofar [

] exists

predominantly in the Sultanate of Oman and with a thalassemic phenotype.

References
1. Daar S, Gravell D, Hussein HM, Pathare AV, Wali Y, Krishnamoorthy R. Haematological and clinical features of -thalassemia associated with Hb Dhofar. Eur J Haematol 2008; 80: 67-70. Williamson D, Brown KP, Langdown JV, Baglin TP. Haemoglobin Dhofar is + linked to the codon 29 C-T(IVSI nt-3) splice mutation which causes beta thalassemia. Br J Haematol 1995; 90: 229-31. Marengo-Rowe AJ, Lorkin PA, Gallo E, Lehmann H. Haemoglobin Dhofar- a new variant from Southern Arabia. Biochim Biophys Acta 1968; 168: 58-63. Haemoglobin Dhofar-58 (ProArg) heterozygote. In: Variant Haemoglobins: A Guide to Identification. Bain BJ, Wild BJ, Stephens AD, Phelan L. pp 188. Wiley-Blackwell, United Kingdom, 2010. Tony S, Daar S, Zachariah N, Wali Y. Prepubertal Hypertransfusion in Thalassemia Intermedia: Sustained Positive Effects on Growth, Splenic Function and Endocrine Parameters. Oman Med J 2012; 27(6). Available from http://wwwomjournal.org/fultext_pdf.aspx?DetailsID=321&type=fultext Qari MH, Wali Y, Albagshi MH, Aishahrani M, Alzahrani A, Alhijji IA, Almomen A, Aljefri A, Al-Saeed HH, Abdullah S, Al-Rustamani A, Mahour K, Mousa SA. Regional consensus opinion for the management of beta thalassemia major in the Arab Gulf Area. Orphanet J Rare Diseases 2013; 8: 143. Available from http://www.ojrd.com/content/8/1/143

2.

3. 4.

5.

6.

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