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Diagnostic hemoglobinopathies Second Edition

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Diagnostic Hemoglobinopathies Laboratory Methods and Case Studies Zia Uddin, PhD St. John Macomb-Oakland Hospital Warren, Michigan Second Edition August 2015
Editorial Board Diane M. Maennle, MD Chairperson Kenneth F. Tucker, MD Member Rita Ellerbrook, PhD Member Piero C. Giordano, PhD Member Kimberly R. Russell, MT (ASCP), MBA Member I
Contributors and Reviewers Antonio Amato, MD Director Centro Studi Microcitemie Di Roma A.N.M.I. ONLUS Via Galla Placidia 28/30 00159 Rome, Rome Italy Erol Omer Atalay, MD Professor, Medical Faculty Pamukkale University Kinikli, Denizli Turkey Celeste Bento, PhD Laboratorio de Anemias Congenitas e Hematologia Molecular Servico de Hematologia, Hospital Pediatrico Centro Hospitalar e Universitario de Coimbra Portugal Aigars Brants, PhD Scientific Affairs Manager Sebia, Inc 400-1705 Corporate Drive NorCross, GA 30093 USA Thomas E. Burgess, PhD Technical Director, Quest Diagnostics Tucker, Georgia USA Shahina Daar, MD, PhD Associate Professor Department of Hematology Sultan Qaboos University, Muscat Sultanate of Oman II
Angie Duong, MD Assistant Professor, Hematopathology Department of Pathology and Laboratory Medicine Medical University-South Carolina Charleston, South Carolina USA 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 III
Rosline Hassan, PhD Professor of Hematology School of Medical Sciences University Sains Malaysia, Kelanran Malaysia David Hockings, PhD Formerly with Isolab, USA and PerkinElmer Corporation, USA Raleigh-Durham, North Carolina USA Prasad Rao Koduri, MD Division of Hematology-Oncology Hektoen Institute of Medical Research Chicago, Illinois 60612 USA John Lazarchick, M.D. Professor, Pathology and Laboratory Medicine Professor, Medicine Director, Hematopathology Director, Hematopathology Fellowship Program Vice Chair, Clinical Pathology Medical University-South Carolina Charleston, SC 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 IV
Herbert L. Muncie, MD Professor, Department of Family Medicine School of Medicine, Louisiana State University 1542 Tulane Ave New Orleans, LA 70112 USA Gul M. Mustafa, PhD Post-Doctorate Fellow Department of Pathology The University of Texas Medical Branch Galveston, TX 77555 USA Diane M. Maennle, MD Associate Pathologist Department of Pathology St. John Macomb-Oakland Hospital . Warren, MI 48093 USA Jayson Miedema, MD Post-Doctorate Fellow Department of Pathology and Laboratory Medicine University of North Carolina Chapel Hill, North Carolina USA Christopher R. McCudden, PhD Assistant Professor, Department of Pathology and Laboratory Medicine, University of Ottawa Ottawa, Ontario Canada Michael A. Nardi, MS Associate Professor Department of Pediatrics and Pathology New York University School of Medicine New York, NY 100016 USA V
John Petersen, PhD Professor, Department of Pathology The University of Texas Medical Branch Galveston, TX 77555 USA Joseph M. Quashnock, PhD Laboratory Director PerkinElmer Genetics, Inc 90 Emerson Lane, Suite 1403 P.O. Box 219 Bridgeville, PA 15017 USA Semyon A. Risin, MD, PhD Professor of Pathology & Laboratory Medicine Director of Laboratory Medicine Restructuring & Strategic Planning Program University of Texas Health Science Center- Houston Medical School 6431 Fannin Street, MSB, 2.290 Houston, TX 77030 USA 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 VI
Luisella Saba, PhD Professor, Dipartimnto di Scienze Biomediche e Biotecnologie Universit degli Studi di Cagliari 09121 Cagliari, Sardina Italy Dror Sayar, MD, PhD Department of Pediatrics, Hematology-Oncology Tel Hashmer Medical Center Ramat Gan Israel Upendra Srinivas, MD Department of Hematology Kokilaben Dhirubhai Ambani Hospital & Medical Research Institute Mumbai, Maharashtra India Elizabeth Sykes, MD Clinical Pathologist William Beaumont Hospital Royal Oak, Michigan USA Ali Taher, MD, PhD Professor Medicine, Hematology & Oncology American University of Beirut Medical Center Beirut Lebanon Kenneth F. Tucker, MD Director, Hematology & Oncology Services Webber Cancer Center St. John Macomb-Oakland Hospital Warren, Michigan USA VII
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 Winfred Wang, MD Professor of Pediatrics University of Tennessee College of Medicine Pediatric Hematologist & Oncologist St Jude Children’s Research Hospital Memphis, Tennessee USA Andrew N Young, MD, PhD Department of Pathology & Laboratory Medicine Emory University School of Medicine Atlanta, GA 30303 USA VIII
Financial Disclosure I neither had nor will have financial relationship with any of the manufacturers or any other organization mentioned in the book. Similarly all the contributors and reviewers of the book have worked with gratis to further the cause of education. This book and its translations into several languages are provided at no charge. August2015 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 August 2015 Zia Uddin, PhD X
Preface Higher level education is one of the blessings of God. Unfortunately, primarily due to economic and logistic reasons a vast majority of the qualified candidates are denied this opportunity. Internet has the potential of mass education at an infinitesimal cost. This is the 3rd book launched via Internet by me at no charge. All the MD/PhD degree holders are most respectfully requested to utilize the Internet as a means of communication to launch books at no charge in their areas of expertise. Love God Love People Serve The World August 2015 Zia Uddin,PhD XI
Acknowlegement 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 August2015 Zia Uddin,PhD XII
Table of Contents Chapter 1 Hemoglobin 1 Thomas E. Burgess, PhD 1.1 Hemoglobin Structure 1.2 Hemoglobin Function 1.3 Hemoglobin Synthesis 1.4 Hemoglobin Variants Chapter 2 Red Blood Cell Morphology 10 John Lazarchick, MD Angie Duong, MD Chapter 3 Diagnostic Laboratory Methods 3.1 Basic Concepts 44 Jayson Miedema, MD and Christopher R. McCudden, PhD 3.1.1 Unstable Hemoglobins 3.1.2 Altered Affinity Hemoglobins 3.1.3 Sickle Solubility Test 3.1.4 Serum Iron, TIBC, Transferrin, Ferritin 3.1.5 Soluble Transferrin Receptor 3.1.6 Hepcidin 3.2 Microcytosis 55 Diane Maennle, MD and Kimberly Russell, MT (ASCP), MBA 3.3 Hereditary Persistence ofFetalHemoglobin 62 Bernard G. Forget, MD 3.3.1 Introduction 3.3.2 Deletions Associated with the HPFH Phenotype 3.3.3 Non-Deletion Forms of HPFH 3.3.4 HPFH Unlinked to the β-Globin Gene Cluster 3.3.5 Conclusion XIII
3.3.6 Hemoglobin F Quantification 3.4 Flow CytometryMeasurements of Cellular Fetal Hemoglobin,Oxidative Stress and Free Iron in Hemoglobinopathies 75 Eitan Fibach, MD 3.4.1 Flow Cytometry of Blood Cells 3.4.2 Measurement of Fetal Hemoglobin-Containing Erythroid Cells 3.4.3 Staining Protocols for F-RBCs and F-Retics (15) 3.4.4 F-Cell Determination for Fetal-Maternal Hemorrhage (FMH) in Pregnant Patients wit β-Thalassemia- A single Case and General Conclusion (16) 3.4.5 Oxidative Stress 3.4.6 Staining Protocols for ROS and GSH 3.4.7 Intracellular Free Iron 3.4.8 Staining Protocol for LIP 3.5 Solid Phase Electrophoretic Separation 95 Rita Ellerbrook, PhD, and Zia Uddin, PhD 3.5.1 Introduction 3.5.2 Cellulose Acetate Electrophoresis (alkaline pH) 3.5.3 Agarose Gel Electrophoresis (alkaline pH) 3.5.4 Agar Electrophoresis (acid pH) 3.5.5 Interpretation of Hemoglobin Agarose Gel (pH 8.6) and Agar Gel (pH 6.2) Electrophoresis 3.5.6 Requirements for the Identification of Complex Hemoglobinopathies 3.6 Capillary Zone Electrophoresis 107 Zia Uddin, PhD 3.6.1 Introduction 3.6.2 Basic Principle 3.6.3 Application of CZE in Diagnostic Hemoglobinopathies 3.6.4 Interpretation of CZE Results XIV
3.7 IsoelectricFocusing 117 David Hockings, PhD 3.7.1 Introduction 3.7.2 IEF of Normal Adult Hemoglobin: HbA (Adult), HbF (Fetal), HbA2 3.7.3 IEF of Normal Newborn Hemoglobins: HbF (Fetal) and HbA (Adult) 3.7.4 IEF of Beta-Chain Variant Hemoglobins 3.7.5 IEF of Alpha Chain Variant Hemoglobins 3.7.6 IEF of Thalassemias 3.8 High Performance Liquid Chromatography 129 Zia Uddin, PhD 3.8.1 Introduction 3.8.2 Basic Principle 3.8.3 Illustrations Chapter 4 Globin Chain Analysis 4.1 Solid Phase Electrophoretic Separation 136 Zia Uddin, PhD 4.1.1 Cellulose Acetate Electrophoresis (Alkaline and Acid pH) 4.2 Reverse Phase High Performance Liquid 140 Chromatography Zia Uddin, PhD, and Rita Ellerbrook, PhD 4.3 Globin Chain Gene Mutations:DNA Studies 149 Joseph M. Quashnock, PhD 4.3.1 Introduction 4.3.2 Genotyping-PCR Methodology 4.3.3 Mutations
XV 4.4 Electrospray Ionization-MassSpectrometry 166 Gul M. Mustafa, PhD and John R. Petersen, PhD 4.5 PCR and SangerSequencing 181 Elaine Lyon, PhD 4.5.1 Alpha Globin 4.5.2 Beta Globin 4.5.3 Sequencing 4.5.4 Reporting Sequence variants 4.5.5 DNA Sequence Traces 4.5.6 Conclusion Chapter 5 Alpha and Beta Thalassemia 191 Herbert L. Muncie, MD. 5.1 Epidemiology 5.2 Pathophysiology 5.3 Alpha Thalassemia 5.4 Beta Thalassemia 5.5 Diagnosis 5.6 Treatment 5.7 Complications 5.8 Other Treatment Issues 5.8.1 Hypersplenism 5.8.2 Endocrinopathies 5.8.3 Pregnancy 5.8.4 Cardiac 5.8.5 Hypercoagulopathy 5.8.6 Psychosocial 5.8.7 Vitamin Deficiencies 5.8.8 Prognosis
XVI Chapter 6 Neonatal Screening for Hemoglobinopathies 212 Zia Uddin, PhD 6.1 Introduction 6.2 Methodologies 6.3 Laboratory Reports Format & Interpretation 6.4 Examples of Neonatal Screening 6.4.1. Capillary Zone Electrophoresis 6.4.2 Isoelectric focusing 6.4.3 Isoelectric focusing and High Performance Liquid Chromatography 6.4.4 Isoelectric focusing, High Performance Liquid Chromatography and DNA studies 6.5 Genetic Counseling & Screening Chapter 7 Prenatal Diagnosis of Beta-Thalassemia and Hemoglobinopathies 236 Maria Cristina Rosatelli, PhD, and Luisella Saba, PhD Chapter 8 Hemoglobin A1c 266 Zia Uddin, PhD 8.1 Introduction 8.2 HbA1c Diagnostic Role in Diabetes Mellitus, and Glycemic Control in Adults 8.3 Measurement of HbA1c 8.4 Factors Affecting the Accuracy of Hb A1c Assay XVII
Case Studies 278 Introduction Case # 1 Normal Adult 281 Case # 2 HemoglobinS trait 286 Case # 3 HemoglobinS homozygous 292 Case # 4 HemoglobinS with hereditary persistence of fetal hemoglobin(HPFH) 298 Case # 5 HemoglobinG-Philadelphia trait 306 Case # 6 HemoglobinS-G Philadelphia 313 Case # 7 HemoglobinG-Coushatta trait 321 Case # 8 HemoglobinC trait 327 Case # 9 HemoglobinC homozygous 333 Case # 10 HemoglobinC with hereditary persistence of fetal hemoglobin(HPFH) 340 Case # 11 HemoglobinS-C disease 346 Case # 12 HemoglobinD-Los Angeles (D-Punjab) trait 353 Case # 13 HemoglobinS-D disease 360 XVIII
Case # 14 HemoglobinE and AssociatedDisorders 367 Case # 14 a HemoglobinE trait 373 Case # 14 b HemoglobinE homozygous 378 Case # 14 c HemoglobinS-E disorders 384 Case # 15 HemoglobinS-Korle Bu (G-Accra) 390 Case # 16 HemoglobinO-Arab trait 396 Case # 17 β-Thalassemia trait 402 Case # 18 HemoglobinS-β+ - thalassemia 408 Case # 19 HemoglobinC-βo – thalassemia 415 Case # 20 HemoglobinHasharon trait 421 Case # 21 HemoglobinZurich trait 428 Case # 22 HemoglobinLepore trait 434 Case # 23 HemoglobinJ-Oxford trait 442 Case # 24 HemoglobinJ-Baltimore trait 449 Case # 25 HemoglobinMalmo trait 455 Case # 26 HemoglobinKoln trait 466 Case # 27 HemoglobinQ-India trait 475 Case # 28 HemoglobinDhofar trait 488 XIX
1 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 Hemoglobin1 edited by Steinberg, Forget, Higgs and Nagel should be consulted. 1.1 Hemoglobin Structure Composed of 2 distinct globin chains, the complex protein molecule known as hemoglobin (“heme” + “globin”) is arguably THE primary component of the red blood cell in human beings. In “normal” adults, the globin chains are either alpha (α), beta (β), gamma (ϒ) or delta (δ). In addition, during embryonic life in utero, zeta (ζ) and epsilon (ε) chains are present in the first several weeks of life, being rapidly converted to alpha, beta and gamma chains as development occurs.
2 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,
3 provides a hydrophobic environment in which the heme molecules reside. This pocket protects the heme from oxidation and facilitates oxygen transfer to the tissues of the body. The previously mentioned ζ and ε chain-containing hemoglobins have very high oxygen affinities, a factor very important in the early embryonic life of the fetus. The hemoglobin molecule can be looked at in four different ways; primary, secondary, tertiary and quaternary structural views. While outside of the scope of this volume, each of these structures contributes definitive unique properties to the various hemoglobin molecules from normal hemoglobins to the very rare and functionally diverse molecules. The primary structure of all hemoglobins is the order of amino acids found in the globin chains of the molecule. It is this unique sequence that is the major differentiator of hemoglobin from each other. The secondary structure of hemoglobin is the arrangement of these amino acid chains into alpha helices separated by non-helical turns2 . 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.
4 Figure 2. Tertiary structure of a β 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 α globin chains and can,
5 via proton scavenging, keep CO2 in the soluble bicarbonate form3. Nitrous oxide is handled in two different ways by hemoglobin: one as a transporter and the other as a scavenger. Blood levels of NO are therefore, by definition, a balance between NO production and NO removal by binding to oxyhemoglobin. Since NO is an extremely potent vasodilator, hypoxic patients will have lower oxyhemoglobin and therefore higher amounts of free NO. This free NO can cause significant vasodilation, a physiological effect that is very desirable in hypoxia. All hemoglobin molecules, either normal or variant, share the same functionality in the human body. Therefore, the primary structural differences mentioned above and in more complete treatises (i.e., amino acid substitutions/deletions) will be the prime reason for functional differences. It is these amino acid variances that, along with the secondary, tertiary and quaternary structural differences, will determine if the variant hemoglobin is either benign or clinically important. The bottom line is this – whether the hemoglobin is normal or variant in nature, the prime reason for determining the hemoglobin phenotype of the patient is to assess the functionality of the hemoglobin. If the variant is normally functioning in both the heterozygous and homozygous states, the clinical picture is benign. If, however, the variant has normal properties in the heterozygous state (i.e., “trait”) but clinical issues in the homozygous state (i.e., “disease”), the phenotypic analysis and subsequent interpretation becomes ultimately important to the patient.
6 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.
7 As mentioned before, the true reason for identifying the abnormal hemoglobin or hemoglobins in patients is to identify any associated functional anomalies associated with these hemoglobins. The actual hemoglobin identification in and of itself is merely of academic interest. 1.4 Hemoglobin Variants All hemoglobin variants have one thing in common – they all involve the hemoglobin molecule and its functionality. Whether alpha, beta, gamma, delta, fusion variant, etc., the variant and its effect are judged not on its migration or concentration but rather on its functionality. The amino acid variation (e.g., glutamic acid → valine at position 6 on the beta chain for hemoglobin S) is the prime effector of the variant’s functional alteration(s) and will in most cases be the causative factor in any abnormal migration that the variant may have versus the “normal” hemoglobins (A, F, 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
8 during the interpretation of the hemoglobinopathy. This data includes, but is not limited to, pregnancy, transfusion history and ethnicity. All of these pieces of information can be critical to the proper identification/interpretation of the hemoglobin variant in the patient’s specimen. For example, an elevation of hemoglobin F in a female patient with a normal hemogram may be evidence of hereditary persistence of fetal hemoglobin; whereas, if this female is pregnant, the elevation may be a normal physiological response to the fetal presence in her body. These data may not be readily available and may require contact with the ordering healthcare professional to obtain these facts. However obtained, they are necessary for the proper identification of the hemoglobin variant or variants in the patient’s bloodstream and therefore are important in the assignment of a benign or pathological assessment of the variant hemoglobin. The variants described in the following chapters all obey the aforementioned differences, i.e., amino acid substitutions, genetic deletions, sequence modifications, etc. While not critical, the exact identification of the variant in and of itself is not normally life-threatening, especially in the heterozygous state, i.e., “trait”. It is essential that the variant be properly identified as a mis-identification can lead to other issues. For example, a mis-interpretation of a hemoglobin G trait (AG) as a sickle trait (AS), while not in and of itself is clinically an issue, presents real difficulties for a couple expecting a child. If both partners are AS, there is a 1 in 4 chance that a child born to this couple could be homozygous SS or sickle cell disease. In the case of an AS mother and an AG father (or vice versa), there is a 1 in 4 chance of a child being born with a phenotype of SG. While on the surface this
9 may appear as a problem, the SG phenotype is no more of a clinical issue than a simple AS trait. Without the exact identification of the AG trait, the interpretation and action taken by attending clinicians may be very different. References 1. Steinberg, MH, Forget, BG, Higgs, DR and Nagel, RL., Disorders of Hemoglobin, Cambridge University Press, 2001. 2. Bain, Barbara J.. in Hemoglobinopathy Diagnosis, 2nd Ed., pg. 4, Blackwell Publishing, 2006. 3. Bain, Barbara J.. in Hemoglobinopathy Diagnosis, 2nd Ed., pg. 1, Blackwell Publishing, 2006.
10 Chapter 2 Red Blood Cell Morphology John Lazarchick, MD Angie Duong, MD Knowledge of red blood cell (RBC) morphology is essential for the clinical diagnosis of hemoglobinopathy. The diameter of RBC, when mature under normal circumstances is approximately 7-8 microns, and RBC is round, anuclear and biconcave disc-shaped. A study of RBC morphology includes size, shape, color, inclusions and arrangement. In this chapter we have presented with pictures of the most commonly encountered RBC morphologies with legends and few examples of the diseases with abnormal RBC morphology. In the clinical cases of this book, we have mentioned only the main features of the peripheral blood smear, therefore a review of this chapter is advised for a naïve reader for the proper diagnosis of hemoglobinopathy. The following RBC morphology cases are presented in this chapter: Size: Macrocyte – large Fig. 1 Microcyte –small Fig. 2 Normocyte – normal Fig. 3 Hemoglobin Content: Hypochromic –low Fig. 4 Normochromic – normal Fig. 5 Polychromatic – high Fig. 6 Shape and Inclusions: Anisocytosis Fig. 7 Poikiocytosis Fig. 8 Acanthocyte Fig. 9 Basophilic Stippling Fig.10 Bite Cell Fig.11 Blister Cell Fig.12 Burr Cell (Ecchinocyte) Fig.13 Heinz Body Fig.14
11 Howell-Jolly Body Fig 15 Pappenheimer Body Fig.16 Schistocyte Fig.17 Sickle Cell Fig.18 Spherocyte Fig.19 Stomatocyte Fig. 20 Target Cell Fig. 21 Teardrop Cell Fig. 22 RBC Agglutination Fig. 23 Rouleaux Formation Fig. 24 Diseases : Erythroblastosis Fetalis Fig. 25 Hemoglobin C Disease Fig. 26 Hemoglobin C/beta Thalassemia Fig. 27 Hemoglobin S/beta Thalassemia Fig. 28 Hemoglobin SC Disease Fig. 29 Sickle Cell Disease Fig. 30 Fetal-maternal Hemorrhage: Fig. 31 Kleinhauer-Betke Stain
12 Fig. 1 – Macrocyte-large The diameter of RBC >9-14 microns (1.5 to 2 times larger than normal RBC) and the MCV >100 fL is characteristic of macrocyte. Macrocytes are mostly oval in shape.
13 Fig. 2 – Microcyte-small RBC, when abnormally smaller (< 5 micron) than normacytic RBC (7-8 micron) is defined as microcyte (also called microerythrocyte). The MCV of the microcyte RBC is < 80 fL.
14 Fig. 3 – Normocyte-normal The diameter of RBC, when mature under circumstances is approximately 7-8 microns, and are round, anuclear, biconcave disc-shaped with an internal volume of 80-100 fL. The term normocyte is used when the size of the RBC is normal.
15 Fig. 4 – Hypochromasia Hypochromasia is a descriptive term for red blood cells where the central pallor is greater than one third the diameter of the red blood cell (black arrows). This is due to a decrease in the amount of hemoglobin in the cells. Diseases with prominent hypochromasia are iron deficiency anemia, anemia of chronic disease, and sideroblastic anemia. Some cases of myelodysplastic syndrome can also have hypochromatic red blood cells. Hypochromasia is reflected in the complete blood count (CBC) by a decreased mean corpuscular hemo-globin concentration (MCHC). Also present are: target cells/codocytes (red arrow), polychromatic forms (blue arrow), fragmented red blood cells/schistocytes (green arrows), and tear drop forms/dacryocytes (yellow arrows). Overall, this smear shows moderate anisopoikilocytosis.
16 Fig. 5 – Normochromic-normal This descriptive term is applied to a red blood cell with a normal concentration of hemoglobin. The above figure is a peripheral blood cell smear of a patient treated for iron deficiency anemia. Blue arrow shows normochromic-normal RBC. Black arrow shows hypochromic-microcytic RBC.
17 Fig. 6 – Polychromatic-high This smear demonstrates polychromasia. Numerous polychromatic forms (black arrows), which are young slightly larger red blood cells with a purple-tinge due to retained RNA, are present. Polychromasia is the bone marrows response to anemia, where the bone marrow releases younger red blood cells. Sometimes, nucleated red blood cells are also released into the peripheral blood. Due to their larger size, when many polychromatic forms are present, the CBC values of mean corpuscular volume (MCV) as well as RDW (red blood cell distribution width) will be increased. In a supravital stains, such as cresyl violet, the retained RNA in the polychromatic forms precipitate out and these cells are called reticulocytes. Thus, sometimes the terms polychromatic form is used interchangeably with reticulocytes.
18 Fig. 7 – Anisocytosis The term anisocytosis refers to size variation seen among red blood cells. As demonstrated above, there are small red blood cells as well as large red blood cells, some approaching the size of a neutrophil (green arrow). Ansiocytosis is a reactive process where the bone marrow is releasing younger red blood cells, therefore an increased number of polychromatic forms can also be seen (black arrow). In the complete blood count (CBC), anisocytosis is reflected by having an increased red cell distribution width (RDW).
19 Fig. 8 – Poikilocytosis Poikilocytosis refers to shape variation. In poikilocytosis, the red blood cells have lost their normal discoid appearance. The example shown here has a predominance of ovalocytes/elliptocytes, which are red blood cells that have a length twice their diameter (a few are indicated by blue arrows). Also seen are schistocytes (red arrows), which are fragmented red blood cells. Ovalocytes/Elliptocytes are seen in peripheral blood smear in some conditions, e.g., thalassemia, iron deficiency, etc. Note: When both shape and size variation is seen in the red blood cells, the term anisopoikilocytosis can be used.
20 Fig. 9 – Acanthocyte (Spur Cell) These are red blood cells with spike-like projections (arrow) of varying length. They can be seen in both hereditary and acquired hemolytic anemias including alcoholic liver disease, pyruvate kinase deficiency, vitamin E deficiency, Huntington’s disease-like situation and abetalipoproteinemia. In the latter case, malabsorption of fat, neurologic damage and developmental delay are noted.
21 Fig. 10 – Basophilic Stippling Red blood cells have multiple fine or coarse small basophilic dot- like inclusions which are due to small clumps of ribonucleic acid and mitochondria. These inclusions can be seen in a wide variety of conditions including lead poisoning, hereditary hemoglobinopathies including unstable hemoglobins, thalassemias, sideroblastic anemias, megaloblastic anemia and hereditary pyrimidine 5’ - nucleotidase deficiency.
22 Fig. 11 – Bite Cell Bite cell (arrow) has a semicircular portion of the membrane removed. This morphologic abnormality results from splenic macrophages removing denatured precipitated hemoglobin with Heinz body formation in these cells. The most common cause of this finding is glucose-6-phosphate dehydrogenase deficiency.
23 Fig. 12 – Blister Cell Red blood cells with cytoplasmic clearing (large arrows) on one side and hemoglobin on the other side in a patient with hemolytic anemia. Multiple polychromatophilic red blood cells (reticulocytes) are noted (small arrow). In addition, a single cell with a Howell-Jolly body inclusion (double arrows) is noted,
24 Fig. 13 – Burr Cell (Echinocyte) These are red blood cells with short round membrane projections with blunt ends (large arrow). Red blood cells with more spike- like projections (small arrow) can also be seen. This finding is often an artifact of slide preparation but is typically seen in patients with uremia and pyruvate kinase deficiency.
25 Fig. 14 - Heinz Body In a RBC when the hemoglobin is denatured (either by a change of an internal amino acid or glucose-6-phosphatse deficiency, etc.), the heme portion of hemoglobin molecule is dissociated from the globin chain. The globin chain after dissociation from the heme molecule becomes denatured forming a small ball like structure (black arrow) inside the RBC, and thus called Heinz body.
26 Fig. 15 – Howell-Jolly Body This red blood cell inclusion (arrow) is round basophilic DNA remnant usually noted in the outer third of circulating red blood cells. These inclusions are normally extruded in the bone marrow during normal erythroid maturation. Howell-Jolly bodies can be seen in asplenia, conditions associated with hyposplenia including sickle cell disease and severe hemolytic anemia.
27 Fig. 16 - Pappenheimer Bodies These are small dark irregular staining granules (large arrow) of non-heme iron usually noted on the periphery of red blood cells formed by phagosomes that engulf excess iron. Basophilic stippling is present in the dysplastic nucleated RBC (small arrow) These granules stain positive with Prussian blue stain in both the nucleated RBC and mature red blood cells as shown in the lower image. They can be found in a variety of conditions including sideroblastic anemias, thalassemias and myelodyplastic syndromes.
28 Fig. 17 - Schistocyte (RBC fragments, Helmet Cells) These are red blood cell fragments typically with two pointed ends formed when RBCs are sheared by fibrin strands in clotted blood vessels. Disorders include microangiopathic hemolytic anemia, disseminated intravascular consumption (DIC), thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS).
29 Fig. 18 - Sickle Cell In inherited blood cell disease (change of an amino acid residue in the globin chain) the shape of the RBC is deformed. The deformation of RBC resembles (a waxing crescent) a moon sighted on the first day of lunar month. Since this deformation looks like a sickle (an implement with a semicircular blade attached to a short handle, used for cutting grain), therefore this deformation is called sickle cell.
30 Fig. 19 – Spherocytes This peripheral blood smear is from a patient with autoimmune hemolytic anemia (AIHA) and is characterized by many spherocytes (blue arrows) and microspherocytes (black arrows). Spherocytes are red blood cells that have no central pallor. As the name implies, microspherocytes are small spherocytes. If the majority of the cells in a peripheral smear are spherocytes, the possibility of hereditary spherocytosis arises. Hereditary spherocytosis is an autosomal dominant disease where one of the genes that code for red blood cell proteins (such as spectrin and ankyrin) become mutated.
31 Fig. 20 – Stomatocyte Red blood cells with slit-like central pallor (arrow) caused by a decrease in surface area to volume ratio associated with a membrane permeability disorder. Hereditary stomatocytosis is associated with hemolysis which can be severe. Acquired stomatocytosis can be seen in acute alcohol intoxication, chronic liver disease and as drying artifact in peripheral smear preparation.
32 Fig. 21 - Target Cells Also known as codocytes, these red blood cells appear to have a bullseye in the center of the red blood cell’s central pallor. This morphologic change is due to a relative excess of cell membrane, due to decreased cell content or increase in the cell’s surface area. Target cells can be seen in liver failure, Hemoglobin C disease, thalessemias (both alpha and beta), and iron deficiency.
33 Fig. 22 - Tear drop cells Also known as dacryocytes/dacrocytes (red circles), are distorted red blood cells where one end of the cell is drawn into a sharp point. These cells are usually seen in myelophthsic anemias, which is where the normal marrow space is occupied by non- hematopoietic elements, such as fibrosis or metastatic carcinoma. It is hypothesized that the shape of the cells is due to the red blood cells squeezing between fibers or the cells extrinsic to the marrow.
34 Fig. 23 - RBC Agglutination Clumping of the red blood cells is due to coating of the RBC surface with antibodies. Disorders causing the agglutination may be primary as in cold agglutinin disease or secondary, either clonal as in lymphoproliferative disorders or polyclonal as seen in Mycoplasma pneumonia. The upper left insert is from a slide prepared at room temperature and the upper right insert is a slide after warming the sample to 370 C with clearing of the agglutination in a patient with cold agglutinin disease.
35 Fig. 24 – Rouleaux Formation Rouleaux formation is seen in peripheral blood smears in association with plasma cell neoplasms, most commonly myeloma. The red cells become stuck together in a “stack of coins” formation, due to the excess immunoglobulin proteins released by malignant plasma cells. Not all cases of plasma cell neoplasms have rouleaux formation. Rouleaux formation is one of the causes of an increased erythrocyte sedimentation rate (ESR).
36 Fig. 25 - Erythroblastosis Fetalis This is an alloimmune hemolytic anemia in the fetus secondary to placental transfer from mother to fetus during pregnancy of anti– A or B or anti-Rh blood group IgG antibodies. These blood groups are present on the fetal RBCs but not on the maternal RBCs which then causes immune hemolysis in the fetal circulation. As noted on the smear, numerous nucleated RBCs (large arrow) and polychromatophilic RBCs (small arrow) are noted. The case shown above was due to antibodies to Rh D blood group.
37 Fig. 26 - Hemoglobin C Disease In this case of homozygous hemoglobin C disease essentially all of the RBCs are target cells (large arrow). Hemoglobin C crystals are rod shaped inclusions (Washington Monuments—small arrow) in red blood cells in both heterozygous and homozygous hemoglobin C disease as well as hemoglobin SC disease. Upper image shows the crystals at a higher magnification.
38 Fig. 27 - Hemoglobin C/beta Thalassemia Although most patients with this compound heterozygotic state for hemoglobin C and beta thalassemia are asymptomatic, a mild to moderate hemolytic anemia can be seen. The red blood cells are microcytic and hypochromic. Target cells (double arrow) and C crystals (single arrows) can be seen.
39 Fig. 28 - Hemoglobin S/beta Thalassemia Hemolytic anemia due to both production of an abnormal hemoglobin (Hemoglobin S) and decreased synthesis of beta globin chains (Beta Thalassemia). Individuals have one abnormal beta chain with substitution of glutamic acid for valine and either decreased synthesis, beta+, or complete absence of the other beta chain, beta0 . The peripheral smear shows sickle cells, nucleated red blood cells, polychromasia, microcytosis, hypochromic, target cells and basophilic stippling. Note the sickle cell in the insert and the Howell-Jolly body in the other RBC.
40 Fig. 29 - Hemoglobin SC Disease This is a representative peripheral blood smear from a patient with hemoglobin SC disease. Hemoglobin SC disease is an inherited hemoglobinopathy where the two normal genes for hemoglobin A have been replaced by one hemoglobin S gene and one hemoglobin C gene. In hemoglobin S, a single nucleotide at position 6 of the gene is substituted by another nucleotide (glutamic acid is substituted by valine). A similar phenomenon occurs in hemoglobin C, where glutamic acid is substituted by lysine. When both hemoglobin S and hemoglobin C is present, the genes are codominant and lead to many interesting peripheral blood findings. Hemoglobin S produces drepanocytes/sickle cells (black arrows) which are red blood cells that appear as crescent moon shapes or continued next page
41 sickles. Due to the abnormal hemoglobin content, the deoxygenated red blood cells become stuck in this shape, thus causing vascular occlusions which in turn lead to many complications such as pain crisis. Sickle cells are seen when there is no or decreased levels of hemoglobin A (such as hemoglobin SS disease, hemoglobin SC disease, hemoglobin S with thalessemia). In sickle cell trait, where there is one normal hemoglobin A gene and one hemoglobin S gene, sickle cells are not seen and the patients usually have no clinical symptoms. Hemoglobin C manifests in peripheral smears as numerous target cells/codocytes (green arrows). Additionally, in hemoglobin CC disease and in hemoglobin SC disease, hemoglobin C crystals (blue circle) can be seen. These crystals are desicated red blood cells with squared off/blunt edges. In hemoglobin C trait, target cells are seen but hemoglobin C crystals are not.
42 Fig. 30 - Sickle Cell Disease Sickle cell disease is a hereditary hemolytic anemia caused by a single nucleotide substitution (SNP) of valine for glutamic acid in the beta globin chain of hemoglobin. This results in hemoglobin polymerizing at low oxygen tension with sickle cell formation (small arrow). There is marked polychromasia, target cells and nucleated red blood cells (inset—large arrow) on the peripheral smear.
43 Fig. 31- Fetal-maternal Hemorrhage: Kleihauer-Betke Stain This test relies on the principle that red blood cells containing fetal hemoglobin (deep red staining RBCs) are less susceptible to acid elution than adult red blood cells. Its use is a means of quantitating fetal-maternal hemorrhage in Rh-negative mothers to determine the dose of Rho (D) immune globulin needed to inhibit formation of Rh antibodies. It can also be used to detect hereditary persistence of fetal hemoglobin (HPFH).
44 Chapter 3 Diagnostic Laboratory Methods 3.1 Basic Concepts Jayson Miedema, MD, and Christopher R. McCudden, PhD 3.1.1 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,
45 basophilic stippling, polychromasia and, hypochromasia. Since not all unstable hemoglobins will give abnormal results on HPLC or electrophoresis and/or these results can be somewhat non-specific, more definitive testing is often performed. Testing for unstable hemoglobins relies on their decreased stability in heat or isopropanol alcohol. While normal hemoglobins should be relatively stable in these conditions, hemoglobins with mutations causing instability tend to be less so and will precipitate out of solution in these environments. In the context of heat stability testing, the amount of unstable hemoglobin in a sample is given by the following equation: (Hb4°C-Hb50°C)/(Hb4°C)x100 Where Hb4°C is the hemoglobin concentration at 4 degrees centigrade and Hb50°C is the concentration of hemoglobin at 50 degrees centigrade. False positives may result from samples greater than 1 week in age as well as from samples with large amounts of fetal hemoglobin. Additional technical and clinical information on hemoglobinopathies associated with unstable hemoglobin can be obtained from: http://medtextfree.wordpress.com/2011/12/30/chapter-48-hemoglobinopathies 3.1.2 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
46 Chesapeake, and beta-chain variants, e.g. Hb Olser, Hiroshima, Andrew-Minneapolis, 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. 3.1.3 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
47 aggregation of deformed blood cells in small blood vessels. Widely believed to have 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 6th 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
48 to false negatives. False positives can occur in the settings of lipemia or samples with monoclonal proteins (dysproteinemia). Both positive and negative controls should be used as results can be somewhat subjective 3.1.4 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
49 epithelial cell sloughing in the skin and GI tract or through menstrual bleeding in 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 Fe2+ 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. This method can be affected by the presence of non-transferrin iron binding proteins, particularly in cases of
50 hemochromatosis and thalassemias. Alternatively, TIBC may be calculated based on the stoichiometric relationship between transferrin and iron (2 molecules of iron are bound to each molecule of transferrin). TIBC is calculated from measured transferrin using the following equation: TIBC (µg/dL) = 1.43 × transferrin (mg/dL). Conversely, the concentration of transferrin may be calculated from measured TIBC as follows: Transferrin (mg/dL) = 0.7 × TIBC (µg/dL). TIBC is increased in iron deficiency and decreased in chronic anemia of disease and in iron overload (it may be normal or decreased in thalassemia). From TIBC and serum iron measurement, it is also possible to calculate the % transferrin saturation (also known as iron saturation) using a simple formula: % saturation = serum Fe (µg/dL) / TIBC (µg/dL) ×100. The percent saturation is usually between 20-50%, supporting an excess capacity for iron binding. In cases of iron overload, the % saturation increases dramatically. Saturation is moderately increased in thalassemia and chronic anemia and in iron deficiency the saturation is decreased. Ferritin is a large ubiquitous protein and the major iron storing protein in the body. Ferritin serves to store thousands of iron atoms/molecule in a non-toxic form acting as an iron reserve. Ferritin is found in small amounts in the blood, where it can be measured as an indication of overall iron reserves (1 ng/mL serum iron approximates 10 mg total storage iron). 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
51 to clinical and morphological manifestations of anemia (e.g. microcytosis) making it a 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 Fe3+ 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.
52 Table 1. Iron Tests in Different Disorders Disorder Serum Iron TIBC % Saturation Transferrin Ferritin Chronic Anemia of Disease ↓ ↓ ↓ ↔ or ↓ ↔ or ↑ Iron Deficiency ↓ ↑ ↓ ↑ ↓ Thalassemia ↔ or ↑ ↔ ↔ or ↑ ↔ or ↓ ↔ or ↑ Hemochromatosis ↑ ↓ ↑↑ ↔ or ↓ ↑↑ ↓decreased; ↔ within reference interval; ↑ increased 3.1.5 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.
53 3.1.6 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.
54 Rees DC, Williams TN, Gladwin MT. Sickle-cell disease. The Lancet. 2010;376:2018- 2031. Steinberg MH. Genetic disorders of hemoglobin oxygen affinity. www.uptodate.com. Accessed April 28, 2011. Steinberg MH. Unstable hemoglobin variants. www.uptodate.com. Accessed April 28, 2011. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. Edited by Burtis CA, Ashwood ER, and Bruns DE. 5th Edition. Vichinsky EP. Sickle cell trait. www.uptodate.com. Accessed April 28, 2011.
55 Chapter 3 Diagnostic Laboratory Methods 3.2 Microcytosis Diane Maennle, MD, and Kimberly Russell, MT (ASCP), MBA Smaller-than-normal size of Red Blood Cells (RBCs) is defined as microcytosis. This is quantified by calculating the mean corpuscular volume (MCV) using the following formula employing the values of hematocrit and RBC count: MCV = Hematocrit (HCT) X 10 / RBC Count (Million) In adults, a MCV value of less than 80fL is defined as microcytosis. In pediatric subjects, the MCV and hemoglobin range distinctly vary with age (Table I). Table I Age Dependent Mean Hemoglobin and MCV Values1,2,3,4 Age Mean Hemoglobin (g/dL) Mean MCV (fL) 3 to 6 months 11.5 91 6 months to 2 years 12.0 78 2 to 6 years 12.5 81 6 to 12 years 13.5 86 12 to 18 years (female) 14.0 90 12 to 18 years (male) 14.5 88 > 18 years (female) 14.0 90 > 18 years (male) 15.75 90
56 Iron deficiency anemia, α-thalassemia trait, and β-thalassemia trait are the most common causes of microcytosis. However, other clinical conditions are also associated with microcytosis (Table II).1,3,5,6 In addition to decreased MCV, the patients with β-thalassemia trait usually have increased hemoglobin 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 β-thalassemia trait in such patients. Conversely, patients with β-thalassemia trait may acquire megaloblastic anemia or liver disease, and may exhibit a normal range for MCV.7 Table II Diagnostic Reasons of Microcytosis (listed in decending order of frequency) Children and adolescents Menstruating women Men and non-menstruating women Iron deficiency anemia Iron deficiency anemia Iron deficiency anemia Thalassemia trait Thalassemia trait Anemia of chronic disease Other hemoglobinopathies Pregnancy Unexplained anemia Lead toxicity Anemia of chronic disease Thalassemia trait Chronic inflammation Sideroblastic anemia Sideroblastic anemia Several laboratory tests in addition to the CBC, e.g. serum iron, serum ferritin, total iron- binding capacity (TIBC), transferrin saturation, hemoglobin electrophoresis, and the examination of the peripheral blood smears (by a pathologist or hematologist), are employed to provide insight and etiologies of microcytosis (Table III).3,8
57 Table III Laboratory Tests in the Differential Diagnosis of Microcytosis Suggested diagnosis Test Iron deficiency anemia Thalassemia Anemia of chronic disease Sideroblastic anemia Serum ferritin Decreased Increased Normal to increased Normal to increased RBC Increased Normal to Normal Increased distribution width increased (RDW) Serum iron Decreased Normal to Normal to Normal to increased decreased increased Total iron- Increased Normal Slightly increased Normal binding capacity Transferrin Decreased Normal to Normal to slightly Normal to saturation increased decreased increased Van Vranken3 has recently suggested a protocol for diagnosing the cause of microcytosis (Figure 1). If the cause remains unclear, the diagnosis of hemoglobinopathy by methods besides electrophoresis alone is imperative. Note: There is a type-setting error in the presentation of the protocol suggested by Van Vranken.3 We have corrected this error in the figure 1, and the journal (American Family Physician) editor was also informed.
58
59 Clinical observations of Kenneth F. Tucker, MD, FACP, a practicing hematologist for the last forty years: Ordinary hemoglobin electrophoresis (cellulose acetate or agarose gel electrophoresis) was only able to detect the more common types of thalassemias. Although there were several other types, many of them did not have microcytosis. I had a large number of patients, who had β-thalassemia minor and a few with probably α-thalassemia, in which the hemoglobin and hematocrit values were relatively normal. Microcytes may or may not be present. This diagnosis was suggested by the peripheral smear, and proven by additional laboratory tests (IFE, globin chain analysis, etc.). I believe that RDW, which is the average red cell width and reflects standard deviation of red cell volumes, is a very important test. RDW normal deviation is a bell- shaped curve. When this value is 2-3% higher, it represents red cells which have varying widths. This certainly can be seen in patients who are iron deficient with microcytosis, but have normal or large cells in addition to megaloblastic or dysplastic marrows, elevated reticulocytes, vitamin B12 or folic acid deficiency, and other conditions. Despite the availability of automated cell counters, review of the peripheral film is one of the most informative and rewarding tests that should be done (by pathologist or hematologist) in any case in which the cause of anemia is not obvious, e.g., bleeding, pure iron deficiency, pure vitamin B12 deficiency, etc. It is also emphasized that the RDW test is not sensitive or specific enough to differentiate iron deficiency and β-thalassemia trait.9 A fairly low to extremely low ferritin is an excellent measure of iron deficiency anemia. In my practice, regardless of what else is going on, any ferritin level of <10 ng/mL, means there is iron deficiency. As mentioned above (Table III), elevated ferritin levels are
60 seen in refractory anemias, all types of chronic inflammatory conditions, etc. Since this test is an acute phase reactant (similar to haptoglobin), it must not be used alone, as the ferritin level may be normal in these clinical conditions. Women in the second or third trimester are always anemic. This is similar to patients who are hypervolemic because of renal or cardiac problems. Red cells in these cases are not microcytes and when the hypervolemia is corrected, the hemoglobin and hematocrit rises. Severe anemia in childhood is usually due to the lack of iron in food, since cow’s milk does not contain iron. A naïve reader is advised to also review the “Full Color pdf of Complete Blood Count in Primary Care,” Best Practice Journal, June 2008 (www.bpac.org.nz), especially the section on Hemoglobin and Red Cell Indices (page 15). References 1. Richardson M. Microcytic anemia [published correction appear in Pediatr Rev. 2007; 28(7): 275, Pediatric Rev. 2009; 30(5): 181, and Pediatr Rev. 2007; 28(4):151]. Pediatr Rev. 2007; 28(1): 5-14. 2. Beutler E, Waalen J. The definition of anemia: what is the lower limit of normal of the blood hemoglobin concentration? Blood. 2006; 107(5): 1747-1750. 3. Van Vranken ML. Evaluation of Microcytosis. Am Fam Physician. 2010; 80(9): 1117-1122. 4. RBC indices calculation and laboratory procedure (2006). St. John Health Laboratories, Warren, MI 48093. 5. Moreno Chulila JA, Romero Colas MS, Gutierrez Martin M. Classification of anemia for gastroenterologist. World J Gastroenterol. 2009: 15(37):4627-4737. 6. Guralnik JM, Eisenstaedt RS, Ferrucci L, Klein HG, Woodman, RC. Prevalence of anemia in persons 65 years and older in the United States: evidence for a high rate of unexplained anemia. Blood. 2004; 104(8): 2263-2268. 7. Bain BJ. Hemoglobinopathy Diagnosis. 2nd ed. Malden, Mass.: Blackwell Publishing; 2006: 94-106.
61 8. Hematologic diseases. In: Wallach J. Interpretation of Diagnostic Tests. 8th 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.
62 Chapter 3 Diagnostic Laboratory Methods 3.3 Hereditary Persistence of Fetal Hemoglobin Bernard G. Forget, MD 3.3.1 Introduction Hereditary persistence of fetal hemoglobin or HPFH consists of a group of genetic disorders characterized by the presence of a substantial elevation of fetal hemoglobin (Hb F) in RBCs of heterozygotes, as well as of homozygotes and compound heterozygotes for HPFH and other hemoglobinopathies. Increased levels of Hb F can ameliorate the clinical course of hemoglobinopathies such as β thalassemia and sickle cell anemia. HPFH is usually due to deletions of different sizes involving the β-globin gene cluster, but nondeletion types of disorders have also been identified, usually due to point mutations in the γ-globin gene promoters (reviewed in refs. 1-3). Figure 1 diagrammatically illustrates the spatial organization of the β-like globin genes in the β-gene cluster on chromosome 11. However, as discussed later in this chapter, certain forms of nondeletion HPFH are clearly not linked to the β-globin gene cluster.
63 Figure 1. Deletions of the β-globin gene cluster associated with fusion proteins and HPFH. The circle 3’ to the β-globin gene indicates the 3’ β-globin gene enhancer. The filled vertical boxes at the 3’ breakpoints of the HPFH-1 and HPFH-6 deletions indicate the locations of DNA sequences with homology to olfactory receptor genes (adopted from reference 2). The references for the individual mutations are cited in references 1, 3 and 6. HPFH is frequently contrasted with δβ thalassemia, which is another genetic disorder associated with elevated Hb F levels. However, one should probably not consider the two disorders as being unambiguously separate entities but rather as a group of disorders with a variety of partially overlapping phenotypes that sometimes defy classification as one syndrome or the other. The following is a working definition that is generally applied to the classification of these disorders: δβ thalassemia usually refers to a group of disorders associated with a mild but definite thalassemia phenotype of hypochromia and microcytosis together with a modest elevation of Hb F that, in heterozygotes, is heterogeneously distributed among red cells. In contrast, HPFH refers to a group of disorders with substantially higher levels of Hb F, and in which there is usually no associated phenotype of hypochromia and microcytosis. In addition, the increased Hb F in heterozygotes with the typical forms of HPFH is distributed in a relatively uniform (pancellular) fashion among all of the red cells rather than being distributed in a heterogeneous (heterocellular) fashion among a subpopulation of so- called F cells, as in δβ thalassemia. Homozygotes for both conditions totally lack Hb A and Hb A2, indicating absence of δ- and β-globin gene expression in cis to the δβ thalassemia and HPFH determinants. Although the apparent striking qualitative difference in cellular distribution of Hb F between HPFH and δβ thalassemia may be
64 due in great part to the quantitative differences in the amount of Hb F per cell and the sensitivity of the methods used to detect Hb F cytologically, nevertheless it would appear that the increased amount of Hb F in HPFH is caused by a genetically determined failure to suppress γ-globin gene activity postnatally in all erythroid cells, rather than being due to selective survival of the normally occurring sub-population of F cells such as occurs in sickle cell anemia, β+ and βo thalassemia. Nevertheless, heterocellular forms of HPFH, without a β-thalassemia phenotype, have been clearly defined and characterized. Therefore, in the final analysis, there is definitely some overlap between these two sets of syndromes at the level of their clinical and hematological phenotypes, as well as at the level of their molecular basis. 3.3.2 Deletions Associated with the HPFH Phenotype. Classic pancellular HPFH, with absence of δ-and β-globin gene expression from the affected chromosome, is associated with large deletions in the β-globin gene cluster that remove the δ-and β-globin genes together with variable amounts of their 5’ and 3’ flanking DNA. At least nine different HPFH deletions of this type have been characterized that vary in size or length from ~13 kb to ~ 85 kb (1-4), some of which are illustrated in Fig. 1. The mechanisms by which such deletions cause marked elevation of Hb F are not well understood, but a number of theories have been proposed. One theory is based on the model of the proposed mechanism for the marked elevation of Hb F associated with Hb Kenya. Hb Kenya is a structurally abnormal hemoglobin that, like Hb Lepore, contains a "hybrid" or fused β-like globin chain resulting from a non-homologous crossing-over event between two globin genes in the
65 β-gene cluster. However, whereas the Lepore crossover occurred between the δ- and β-globin genes, the Kenya gene resulted from crossover between the Aγ- and β-globin genes (Fig. 1). The crossover occurred in the second exons of the Aγ and β genes, between the codons for amino acids 80 to 87, and resulted in deletion of ~24 kb of DNA between the Aγ to the β gene. Unlike Hb Lepore, that is associated with a β- thalassemic phenotype, Hb Kenya is associated with a phenotype of pancellular Gγ 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 Gγ 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 Gγ- and Kenya gene promoters of a well characterized enhancer element located in the 3' flanking DNA of the β-globin gene, shown by the filled circle in Fig. 1, that becomes translocated into close proximity of the γ-globin gene promoters by the crossover/deletion event, resulting in enhanced activity of these promoters. Among the HPFH deletions, there is a relatively short deletion, called HPFH-5 or Italian HPFH, that extends from a point ~3 kb 5' to the δ gene to a point 0.7 kb 3' to the β gene, deleting the β gene but not its 3' enhancer. The molecular basis of the HPFH phenotype associated with this deletion is presumably the influence of the translocated 3' β-gene enhancer on the γ-gene promoters, in a manner analogous to that proposed for the basis of the HPFH phenotype of the Hb Kenya syndrome. In the case of some of the other larger HPFH deletions, the DNA preserved at or near the 3’ breakpoint of the deletions has been shown in various types of assays to have enhancer-like activity on gene expression (2, 5-7). Thus, it has been proposed that the DNA sequences at the
66 HPFH 3' deletion breakpoints, that become juxtaposed to the γ genes as a result of the deletion events, may influence γ-gene expression, in a manner analogous to the presumed influence of the 3' β-gene enhancer on γ-gene expression in Hb Kenya and HPFH-5. Mechanisms by which this could occur include the presence of enhancer-like sequences in the translocated 3' breakpoint DNA or the presence in this DNA of an active chromatin configuration that could have a spreading and activation effect on the expression of the neighboring γ-globin genes. A second theory for the mechanism of increased γ-gene expression in deletion- type HPFH is the nature and function of the DNA sequences conserved at the 5’ breakpoint of the deletions. The 5’ breakpoints of the HPFH deletions, as well as many of the δβ-thalassemia deletions, are located in the DNA between the Αγ and δ genes, the so-called Αγδ-intergene DNA. It has long been proposed that there may exist negative regulatory or silencer elements in this region of DNA, deletion of which in HPFH but not in δβ thalassemia, results in markedly impaired postnatal suppression of γ-gene activity in all erythroid cells (8). A number of subsequent observations have been made that support a role for the Aγδ-intergene region in the regulation of γ-gene expression (reviewed in ref. 9). The Corfu deletion in particular, involving the δ-gene and ~6 kb of upstream flanking DNA, is associated in homozygotes with a high HbF phenotype and removes some interesting structural elements, such as a poly-pyrimidine region that can serve as a binding site for a multi-protein chromatin remodeling complex containing the transcription factor Ikaros, and a region of DNA that serves as a promoter for the synthesis of an intergenic RNA transcript preferentially expressed in adult
67 erythroid cells (10). This region of DNA also appears to serve as a boundary region between fetal and adult domains of the β-globin gene cluster. The most conclusive evidence for a functional role of the Aγδ-intergene DNA in the regulation of γ gene expression consists of the observations by Sankaran et al. who have extensively characterized a negative regulatory transcription factor, called BCL11A, that down-regulates γ-gene expression in adult erythroid cells and that binds to the Aγδ-intergene DNA (11-13). BCL11A, originally identified as an important factor in B-lymphoid cell development, is a component of a multi-protein complex that plays a negative regulatory role in γ-gene expression. This complex has been shown to contain GATA1 as well as all components of the nucleosome and histone deacetylase ( NuRD)- repressive complex (14). Additional studies have shown that this complex physically interacts with another transcription factor called SOX6 that is thought to be a repressor of embryonic and fetal globin gene expression (15). Chromatin immunoprecipitation (ChIP) studies have shown that BCL11A binds to a number of regions in the β-cluster, including the upstream locus control region (LCR) and the γδ intergenic region, but does not bind to the γ- or β-gene promoters (4, 14, 15). Sankaran et al. (4) have characterized two important deletion mutants with nearly identical distal breakpoints but different upstream breakpoints around the δ-gene and its flanking DNA. One mutant with a more proximal breakpoint has a δβ-thalassemia phenotype, whereas the longer deletion removing 3.5 kb of additional upstream DNA is associated with a HPFH phenotype. The authors propose that this 3.5 kb region of DNA contains a silencer element, deletion of which can cause HPFH. This hypothesis is strengthened by the fact that the deleted region contains one of the prominent binding sites of BCL11A detected
68 in their ChIP experiments. These findings provide very strong evidence for a γ-gene silencer element in the β-gene cluster that associates with a BCL11A-containing repressor complex and that this interaction is an important factor in the suppression of γ-gene expression during the perinatal switch from expression of Hb F to Hb A. 3.3.3 Nondeletion Forms of HPFH In contrast to the deletional types of HPFH syndromes, where both linked Gγ and Aγ genes are over expressed, only one or the other γ gene is usually over expressed in the best characterized nondeletional types of HPFH associated with high levels of pancellular Hb F expression. However, less well characterized nondeletion forms of GγAγ HPFH have been described that are associated with relatively low levels of heterocellular expression of both γ genes. Because of the restricted pattern of γ-globin gene expression in the Gγ and Aγ forms of nondeletion HPFH, it was assumed that the mutations in these syndromes must be located near the affected gene and molecular studies focused initially on the DNA sequence analysis of the over expressed γ genes in these disorders.
69 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 γ type, and the G γ gene in cis to the -175 A γ mutation carried the -158 C→ T change. The references for the individual mutations are cited in references 1 and 3. The results of these structural analyses revealed a number of different point mutations in the promoter region of the over expressed γ gene in individuals with different types of nondeletion HPFH, as listed in Table 1 (reviewed in refs. 1-3). These point mutations have clustered primarily in three distinct regions of the 5'-flanking DNA of the affected γ genes. The first region is located approximately 200 base pairs from
70 the "cap site" or site of transcription initiation of the γ genes (at least five different point mutations involving single nucleotides between residues -195 to -202 from the cap site). This region of DNA, which had not previously been suspected of playing a role in the regulation of γ-gene expression, is very G+C rich and its sequence bears homology to that of known control elements of other genes that contain the binding site for the ubiquitous trans-acting protein factor called Sp1. Subsequent studies of the γ-gene promoters have demonstrated that the -200 region is also a binding site for Sp1 and for at least one other ubiquitous DNA binding protein. The second region containing a mutation associated with nondeletion HPFH is located at position -175. A point mutation (T->C) at this position of either the Gγ or Aγ gene is associated with a phenotype of pancellular HPFH with high levels of Hb F (15- 25%). This region of DNA is noteworthy because it contains an octanucleotide sequence that is present in the promoter region of a number of genes and is the binding site of another ubiquitous trans-acting factor called OCT-1. In addition, the octamer consensus sequence of the γ-gene promoters is flanked on either side by a consensus sequence for the hematopoietic-specific transcription factor GATA-1. The point mutation at position -175 affects the one nucleotide that is present in the partially overlapping binding sites of both OCT-1 and GATA-1. The third region affected by a point mutation in nondeletion HPFH is in the area of a well known regulatory element of globin and other genes: the CCAAT box sequence. In the γ genes, the CCAAT box is duplicated and the mutation associated with the Greek Aγ type of nondeletion HPFH is a G->A substitution at position -117, 2 bases upstream of the distal CCAATbox of the Aγ-globin gene promoter. The base
71 change disrupts a pentanucleotide sequence, YYTTGA (Y = pyrimidine), that is highly conserved immediately upstream of the CCAAT sequence in all animal fetal and embryonic genes. At least two other mutations involving the CCAAT box of one or the other γ gene have been reported in other cases of HPFH not associated with large deletions. The CCAAT box region is known to be the binding site of a number of trans- acting factors, including the ubiquitous factors CCAAT binding protein (CP1) and CCAAT displacement factor (CDP) as well as the erythroid-specific factor NF-E3. The unifying model by which these various mutations are thought to affect hemoglobin switching proposes that these base changes alter the binding of a number of different trans- acting factors to critical regions of the γ-gene promoters and thereby prevent the normal postnatal suppression of γ-gene expression (reviewed in refs. 1,2). The mutations could prevent the binding of negative regulatory factors or enhance the binding of positive regulatory factors. Either mechanism could be operative with one mutation or the other. 3.3.4 HPFH Unlinked to the β-Globin Gene Cluster A number of studies have identified families in which increased levels of Hb F are inherited due to a genetic determinant that is unlinked to the β-globin gene cluster. Genome-wide association studies (GWAS), using co-inheritance of single nucleotide polymorphisms (SNPs) with elevated levels of Hb F, have subsequently demonstrated the presence of two different quantitative trait loci (QTLs), unlinked to the β-globin gene cluster on chromosome 11, that are associated with inheritance of mildly elevated levels of Hb F, similar to the phenotype seen in Swiss-type heterocellular HPFH (see section
72 above on Nondeletion HPFH). These loci are located on chromosome 2 and 6 (16, 17). The locus on chromosome 2 corresponds to the site of the gene encoding BCL11A and its identification led to the elegant studies of Sankaran and co-workers demonstrating the role of BCL11A in the regulation of γ-gene expression. The locus on chromosome 6 is located between the genes encoding HBS1L and MYB. The mechanism by which this locus causes elevation of Hb F is thus far poorly understood. Finally, mutations in the gene on chromosome 19 encoding the erythroid-specific transcription factor EKLF1 have been shown to be associated with a form of HPFH (18, 19). The involved mechanism is probably through the regulation of BCL11A levels, because it has been demonstrated that EKLF1 binds to the promoter of the BCL11A gene and regulates the expression of the gene (20). 3.3.5 Conclusion Significant insights into the normal regulation of expression of the human β- globin gene cluster have been obtained by a detailed analysis of a group of disorders called HPFH. On the basis of this information, several important regulatory elements have been identified for the normal functioning of the individual genes in the cluster during the developmental switch from fetal to adult hemoglobin gene expression, as well as for the abnormal persistent expression of the γ-globin genes in adults with HPFH. These results provide a more sophisticated understanding of the molecular basis of these syndromes and point to certain strategies for potential future molecular and cellular therapies for globin gene disorders.
73 3.3.6 Hemoglobin F Quantification Hb F can be quantified by several methods, and the most commonly used procedures in a clinical laboratory are a) radial immunodiffusion, b) Elisa method, c) HPLC, and d) capillary zone electrophoresis. References 1. Bollekens JA, Forget BG. Delta beta thalassemia and hereditary persistence of fetal hemoglobin. Hematol Oncol Clin North Am 1991;5(3):399-422. 2. Forget BG. Molecular basis of hereditary persistence of fetal hemoglobin. Ann N Y Acad Sci 1998; 850:38-44. 3 Weatherall DJ, Clegg JB. The Thalassaemia Syndromes. 4th ed. Oxford ; Malden, MA: Blackwell Science; 2001. 4. Sankaran VG, Xu J, Byron R, et al. Functional element necessary for fetal hemoglobin silencing. N Engl J Med 2011; 365(9):807-14. 5. Feingold, EA, Forget BG. The breakpoint of a large deletion causing hereditary persistence of fetal hemoglobin occurs within an erythroid DNA domain remote from the β-globin gene cluster. Blood 1989; 74: 2178–2186. 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 gamma- globin gene in transgenic mice. J. Biol Chem 1995; 270: 10256-63. 8. Huisman TH, Schroeder WA, Efremov GD, et al. The present status of the heterogeneity of fetal hemoglobin in beta-thalassemia: an attempt to unify some observations in thalassemia and related conditions. Ann N Y Acad Sci 1974;232(0):107- 24. 9. Bank A, O'Neill D, Lopez R, et al. Role of intergenic human γ-δ -globin sequences in human hemoglobin switching and reactivation of fetal hemoglobin in adult erythroid cells. Ann N Y Acad Sci 2005;1054:48-54.
74 10. Chakalova L, Osborne CS, Dai YF, et al. The Corfu δβ thalassemia deletion disrupts γ-globin gene silencing and reveals post-transcriptional regulation of HbF expression. Blood 2005;105:2154-60. 11. Sankaran VG, Xu J, Orkin SH. Transcriptional silencing of fetal hemoglobin by BCL11A. Ann N Y Acad Sci. 2010;1202:64-8. 12. Sankaran VG, Xu J, Ragoczy T, et al. Developmental and species-divergent globin switching are driven by BCL11A. Nature 2009;460(7259):1093-7. 13. Sankaran VG, Nathan DG. Reversing the hemoglobin switch. N Engl J Med 2010; 363(23):2258-60. 14. Sankaran VG, Menne TF, Xu J, et al. Human fetal hemoglobin expression is regulated by the developmental stage-specific repressor BCL11A. Science 2008; 322(5909):1839-42. 15. Xu J, Sankaran VG, Ni M, et al. Transcriptional silencing of γ-globin by BCL11A involves long-range interactions and cooperation with SOX6. Genes Dev 2010; 24:783- 98. 16. Thein SL, Menzel S, Lathrop M, Garner C. Control of fetal hemoglobin: new insights emerging from genomics and clinical implications. Hum Mol Genet 2009;18(R2):R216- 23. 17. Galarneau G, Palmer CD, Sankaran VG, Orkin SH, Hirschhorn JN, Lettre G. Fine mapping at three loci known to affect fetal hemoglobin levels explains additional genetic variation. Nat Genet 2010;42(12):1049-51. 18. Borg J, Papadopoulos P, Georgitsi M, et al. Haploinsufficiency for the erythroid transcription factor KLF1 causes hereditary persistence of fetal hemoglobin. Nat Genet 2010;42(9):801-5. 19. Borg J, Patrinos GP, Felice AE, Philipsen S. Erythroid phenotypes associated with KLF1 mutations. Haematologica 2011; 96:635-8. 20. Zhou D, Liu K, Sun CW, Pawlik KM, Townes TM. KLF1 regulates BCL11A expression and γ- to β-globin gene switching. Nat Genet 2010; 42:742-4.
75 Chapter 3 Diagnostic Laboratory Methods 3.4 Flow CytometryMeasurements of Cellular Fetal Hemoglobin,Oxidative Stress and Free Iron in Hemoglobinopathies Eitan Fibach, MD 3.4.1 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
76 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.
77 FC is especially fitting for analysis of blood cells: (I) These cells which can be easily obtained by blood drawing are present as single cells, thus in contrast to cells of solid tissues, their use does not require harsh procedures for tissue disaggregation (e.g., trypsinization). (II) They are present as a mixture of various cell types, including numerous subtypes (e.g., lymphocytes), with very large (e.g., RBC) to very small (hematopoietic stem cells) representation. Cells of these sub-types can be identified and "gated" based on differences in their size (forward light scattering), granularity (side light scattering) and expression of surface antigens, and can be measured simultaneously. For measurements of various characteristics (HbF content, oxidative stress parameters and LIP content), the blood sample is stained with specific probes (as detailed below), and then with fluorescent reagents (usually antibodies) against surface markers which identify a specific subpopulation. Such markers are glycophorin A for RBC, CD61 for platelets, CD15 for neutrophils, CD19 for B-lymphocytes and CD3 for T- lymphocytes. CD45 is particularly useful since it is differentially expressed on various nucleated blood cells (Fig. 1).
78 Fig. 1. Flow cytometry of blood cells. A dot plot of blood cells with respect CD45 (FL3-H) and side light scatter (SSC-H). 3.4.2 Measurement of Fetal Hemoglobin-Containing Erythroid Cells Fetal hemoglobin (HbF, α2γ2) is the major hemoglobin (Hb) in the prenatal period that is largely replaced after birth by the adult Hb (HbA, α2β2) (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 RBC PMN Monocytes CD45 Lymphocytes
79 spectrofluorometric measurements following treatment with alkaline (to destroy non-fetal hemoglobins) and staining with benzidine (8), chromatography (ion-exchange HPLC for hemoglobins and reverse-phase HPLC for globin chains) (9), as well as immunological techniques, such as Elisa, based on antibodies against HbF (10). However, quantitative FC measurement of RBC, fluorescently stained with antibodies to HbF (as well as for the other hemoglobins), has several advantages. For example, in the differential diagnosis of Hereditary Persistence of Fetal Hemoglobin (11). This condition encompasses a heterogeneous group of disorders with marked increased levels of HbF. Based on the cellular distribution of HbF, they are characterized as pan-cellular, where all RBCs have increased levels of HbF, albeit not always uniformly so; and hetero- cellular, where nearly all the HbF is confined to a minor, distinct subpopulation of RBCs. This important distinction is most reliably ascertained by FC. Epidemiological studies have indicated that high levels of HbF improve the clinical symptoms of the underlying disease. In sickle cell anemia not only do HbF- containing cells have a lower concentration of sickle hemoglobin, but HbF inhibits polymerization of HbS directly, accounting for the lower propensity of such cells to undergo sickling (12). In β-thalassemia, elevated HbF should compensate partially for the deficiency of β-globin chains and reduce the excess of α-globin chains. Several pharmacological agents have been used to stimulate HbF production (13). Hydroxyurea (HU) is currently the drug of choice (14). When patients are monitored during HU treatment by measuring HbF in the hemolysate, an increase is usually observed after 2- 3 months (10). HU acts by a still unknown mechanism on the early erythroid precursors in the bone marrow. It takes several weeks for HbF to accumulate in the peripheral
80 blood to a quantity that allows differences before and after treatment to become apparent. Measuring differences in F-RBC by FC may be more sensitive, and measuring F-reticulocytes (retics) may provide early indication of treatment efficacy (15): Retics have a very short life-span (1-2 days) compared to mature RBC (120 days in normal subjects) and therefore measuring peripheral blood F-retics more closely characterizes the current status of HbF production in the bone marrow. Measuring F- retics can indicate the efficacy of the drug and/or the patient’s compliance several days after treatment initiation. Such follow up is very important since about 30% of the patients are non-responders. It is imperative that such patients be identified as early as possible and the treatment (that is not without potential risks) be discontinued and replaced by treatment with another drug (e.g., butyroids). 3.4.3 Staining Protocols for F-RBC and F-Retics (15) Heparinized blood is washed three times in phosphate buffered saline (PBS). For fixation, 50μl of the packed cells are resuspended in 10 ml of PBS containing 4% formaldehyde for 15-min at room temperature under constant agitation in polypropylene tubes. For permeabilization, the cells are centrifuged for 3 min at 1,500 g, and 2 ml methanol-acetone are added to the pellet, mixed and incubated for 1-min at room temperature. The cells are then washed three times and resuspended in PBS to a final volume of 0.5 ml (10% suspension). Anti-HbF monoclonal antibodies (the amount depends on the Manufacturer’s instructions or on a pre-performed titration) are added to 5x106 cells (5 μl of the 10% suspension) and incubated for 1-hr at 370C, after which the cells are washed in PBS. If
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Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition
Diagnostic hemoglobinopathies Second Edition

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Diagnostic hemoglobinopathies Second Edition

  • 1. Diagnostic Hemoglobinopathies Laboratory Methods and Case Studies Zia Uddin, PhD St. John Macomb-Oakland Hospital Warren, Michigan Second Edition August 2015
  • 2.
  • 3. Editorial Board Diane M. Maennle, MD Chairperson Kenneth F. Tucker, MD Member Rita Ellerbrook, PhD Member Piero C. Giordano, PhD Member Kimberly R. Russell, MT (ASCP), MBA Member I
  • 4. 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
  • 5. Angie Duong, MD Assistant Professor, Hematopathology Department of Pathology and Laboratory Medicine Medical University-South Carolina Charleston, South Carolina USA 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 III
  • 6. Rosline Hassan, PhD Professor of Hematology School of Medical Sciences University Sains Malaysia, Kelanran Malaysia David Hockings, PhD Formerly with Isolab, USA and PerkinElmer Corporation, USA Raleigh-Durham, North Carolina USA Prasad Rao Koduri, MD Division of Hematology-Oncology Hektoen Institute of Medical Research Chicago, Illinois 60612 USA John Lazarchick, M.D. Professor, Pathology and Laboratory Medicine Professor, Medicine Director, Hematopathology Director, Hematopathology Fellowship Program Vice Chair, Clinical Pathology Medical University-South Carolina Charleston, SC 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 IV
  • 7. Herbert L. Muncie, MD Professor, Department of Family Medicine School of Medicine, Louisiana State University 1542 Tulane Ave New Orleans, LA 70112 USA Gul M. Mustafa, PhD Post-Doctorate Fellow Department of Pathology The University of Texas Medical Branch Galveston, TX 77555 USA Diane M. Maennle, MD Associate Pathologist Department of Pathology St. John Macomb-Oakland Hospital . Warren, MI 48093 USA Jayson Miedema, MD Post-Doctorate Fellow Department of Pathology and Laboratory Medicine University of North Carolina Chapel Hill, North Carolina USA Christopher R. McCudden, PhD Assistant Professor, Department of Pathology and Laboratory Medicine, University of Ottawa Ottawa, Ontario Canada Michael A. Nardi, MS Associate Professor Department of Pediatrics and Pathology New York University School of Medicine New York, NY 100016 USA V
  • 8. John Petersen, PhD Professor, Department of Pathology The University of Texas Medical Branch Galveston, TX 77555 USA Joseph M. Quashnock, PhD Laboratory Director PerkinElmer Genetics, Inc 90 Emerson Lane, Suite 1403 P.O. Box 219 Bridgeville, PA 15017 USA Semyon A. Risin, MD, PhD Professor of Pathology & Laboratory Medicine Director of Laboratory Medicine Restructuring & Strategic Planning Program University of Texas Health Science Center- Houston Medical School 6431 Fannin Street, MSB, 2.290 Houston, TX 77030 USA 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 VI
  • 9. Luisella Saba, PhD Professor, Dipartimnto di Scienze Biomediche e Biotecnologie Universit degli Studi di Cagliari 09121 Cagliari, Sardina Italy Dror Sayar, MD, PhD Department of Pediatrics, Hematology-Oncology Tel Hashmer Medical Center Ramat Gan Israel Upendra Srinivas, MD Department of Hematology Kokilaben Dhirubhai Ambani Hospital & Medical Research Institute Mumbai, Maharashtra India Elizabeth Sykes, MD Clinical Pathologist William Beaumont Hospital Royal Oak, Michigan USA Ali Taher, MD, PhD Professor Medicine, Hematology & Oncology American University of Beirut Medical Center Beirut Lebanon Kenneth F. Tucker, MD Director, Hematology & Oncology Services Webber Cancer Center St. John Macomb-Oakland Hospital Warren, Michigan USA VII
  • 10. 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 Winfred Wang, MD Professor of Pediatrics University of Tennessee College of Medicine Pediatric Hematologist & Oncologist St Jude Children’s Research Hospital Memphis, Tennessee USA Andrew N Young, MD, PhD Department of Pathology & Laboratory Medicine Emory University School of Medicine Atlanta, GA 30303 USA VIII
  • 11. 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. August2015 Zia Uddin,PhD IX
  • 12. 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 August 2015 Zia Uddin, PhD X
  • 13. Preface Higher level education is one of the blessings of God. Unfortunately, primarily due to economic and logistic reasons a vast majority of the qualified candidates are denied this opportunity. Internet has the potential of mass education at an infinitesimal cost. This is the 3rd book launched via Internet by me at no charge. All the MD/PhD degree holders are most respectfully requested to utilize the Internet as a means of communication to launch books at no charge in their areas of expertise. Love God Love People Serve The World August 2015 Zia Uddin,PhD XI
  • 14. Acknowlegement 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 August2015 Zia Uddin,PhD XII
  • 15. Table of Contents Chapter 1 Hemoglobin 1 Thomas E. Burgess, PhD 1.1 Hemoglobin Structure 1.2 Hemoglobin Function 1.3 Hemoglobin Synthesis 1.4 Hemoglobin Variants Chapter 2 Red Blood Cell Morphology 10 John Lazarchick, MD Angie Duong, MD Chapter 3 Diagnostic Laboratory Methods 3.1 Basic Concepts 44 Jayson Miedema, MD and Christopher R. McCudden, PhD 3.1.1 Unstable Hemoglobins 3.1.2 Altered Affinity Hemoglobins 3.1.3 Sickle Solubility Test 3.1.4 Serum Iron, TIBC, Transferrin, Ferritin 3.1.5 Soluble Transferrin Receptor 3.1.6 Hepcidin 3.2 Microcytosis 55 Diane Maennle, MD and Kimberly Russell, MT (ASCP), MBA 3.3 Hereditary Persistence ofFetalHemoglobin 62 Bernard G. Forget, MD 3.3.1 Introduction 3.3.2 Deletions Associated with the HPFH Phenotype 3.3.3 Non-Deletion Forms of HPFH 3.3.4 HPFH Unlinked to the β-Globin Gene Cluster 3.3.5 Conclusion XIII
  • 16. 3.3.6 Hemoglobin F Quantification 3.4 Flow CytometryMeasurements of Cellular Fetal Hemoglobin,Oxidative Stress and Free Iron in Hemoglobinopathies 75 Eitan Fibach, MD 3.4.1 Flow Cytometry of Blood Cells 3.4.2 Measurement of Fetal Hemoglobin-Containing Erythroid Cells 3.4.3 Staining Protocols for F-RBCs and F-Retics (15) 3.4.4 F-Cell Determination for Fetal-Maternal Hemorrhage (FMH) in Pregnant Patients wit β-Thalassemia- A single Case and General Conclusion (16) 3.4.5 Oxidative Stress 3.4.6 Staining Protocols for ROS and GSH 3.4.7 Intracellular Free Iron 3.4.8 Staining Protocol for LIP 3.5 Solid Phase Electrophoretic Separation 95 Rita Ellerbrook, PhD, and Zia Uddin, PhD 3.5.1 Introduction 3.5.2 Cellulose Acetate Electrophoresis (alkaline pH) 3.5.3 Agarose Gel Electrophoresis (alkaline pH) 3.5.4 Agar Electrophoresis (acid pH) 3.5.5 Interpretation of Hemoglobin Agarose Gel (pH 8.6) and Agar Gel (pH 6.2) Electrophoresis 3.5.6 Requirements for the Identification of Complex Hemoglobinopathies 3.6 Capillary Zone Electrophoresis 107 Zia Uddin, PhD 3.6.1 Introduction 3.6.2 Basic Principle 3.6.3 Application of CZE in Diagnostic Hemoglobinopathies 3.6.4 Interpretation of CZE Results XIV
  • 17. 3.7 IsoelectricFocusing 117 David Hockings, PhD 3.7.1 Introduction 3.7.2 IEF of Normal Adult Hemoglobin: HbA (Adult), HbF (Fetal), HbA2 3.7.3 IEF of Normal Newborn Hemoglobins: HbF (Fetal) and HbA (Adult) 3.7.4 IEF of Beta-Chain Variant Hemoglobins 3.7.5 IEF of Alpha Chain Variant Hemoglobins 3.7.6 IEF of Thalassemias 3.8 High Performance Liquid Chromatography 129 Zia Uddin, PhD 3.8.1 Introduction 3.8.2 Basic Principle 3.8.3 Illustrations Chapter 4 Globin Chain Analysis 4.1 Solid Phase Electrophoretic Separation 136 Zia Uddin, PhD 4.1.1 Cellulose Acetate Electrophoresis (Alkaline and Acid pH) 4.2 Reverse Phase High Performance Liquid 140 Chromatography Zia Uddin, PhD, and Rita Ellerbrook, PhD 4.3 Globin Chain Gene Mutations:DNA Studies 149 Joseph M. Quashnock, PhD 4.3.1 Introduction 4.3.2 Genotyping-PCR Methodology 4.3.3 Mutations
  • 18. XV 4.4 Electrospray Ionization-MassSpectrometry 166 Gul M. Mustafa, PhD and John R. Petersen, PhD 4.5 PCR and SangerSequencing 181 Elaine Lyon, PhD 4.5.1 Alpha Globin 4.5.2 Beta Globin 4.5.3 Sequencing 4.5.4 Reporting Sequence variants 4.5.5 DNA Sequence Traces 4.5.6 Conclusion Chapter 5 Alpha and Beta Thalassemia 191 Herbert L. Muncie, MD. 5.1 Epidemiology 5.2 Pathophysiology 5.3 Alpha Thalassemia 5.4 Beta Thalassemia 5.5 Diagnosis 5.6 Treatment 5.7 Complications 5.8 Other Treatment Issues 5.8.1 Hypersplenism 5.8.2 Endocrinopathies 5.8.3 Pregnancy 5.8.4 Cardiac 5.8.5 Hypercoagulopathy 5.8.6 Psychosocial 5.8.7 Vitamin Deficiencies 5.8.8 Prognosis
  • 19. XVI Chapter 6 Neonatal Screening for Hemoglobinopathies 212 Zia Uddin, PhD 6.1 Introduction 6.2 Methodologies 6.3 Laboratory Reports Format & Interpretation 6.4 Examples of Neonatal Screening 6.4.1. Capillary Zone Electrophoresis 6.4.2 Isoelectric focusing 6.4.3 Isoelectric focusing and High Performance Liquid Chromatography 6.4.4 Isoelectric focusing, High Performance Liquid Chromatography and DNA studies 6.5 Genetic Counseling & Screening Chapter 7 Prenatal Diagnosis of Beta-Thalassemia and Hemoglobinopathies 236 Maria Cristina Rosatelli, PhD, and Luisella Saba, PhD Chapter 8 Hemoglobin A1c 266 Zia Uddin, PhD 8.1 Introduction 8.2 HbA1c Diagnostic Role in Diabetes Mellitus, and Glycemic Control in Adults 8.3 Measurement of HbA1c 8.4 Factors Affecting the Accuracy of Hb A1c Assay XVII
  • 20. Case Studies 278 Introduction Case # 1 Normal Adult 281 Case # 2 HemoglobinS trait 286 Case # 3 HemoglobinS homozygous 292 Case # 4 HemoglobinS with hereditary persistence of fetal hemoglobin(HPFH) 298 Case # 5 HemoglobinG-Philadelphia trait 306 Case # 6 HemoglobinS-G Philadelphia 313 Case # 7 HemoglobinG-Coushatta trait 321 Case # 8 HemoglobinC trait 327 Case # 9 HemoglobinC homozygous 333 Case # 10 HemoglobinC with hereditary persistence of fetal hemoglobin(HPFH) 340 Case # 11 HemoglobinS-C disease 346 Case # 12 HemoglobinD-Los Angeles (D-Punjab) trait 353 Case # 13 HemoglobinS-D disease 360 XVIII
  • 21. Case # 14 HemoglobinE and AssociatedDisorders 367 Case # 14 a HemoglobinE trait 373 Case # 14 b HemoglobinE homozygous 378 Case # 14 c HemoglobinS-E disorders 384 Case # 15 HemoglobinS-Korle Bu (G-Accra) 390 Case # 16 HemoglobinO-Arab trait 396 Case # 17 β-Thalassemia trait 402 Case # 18 HemoglobinS-β+ - thalassemia 408 Case # 19 HemoglobinC-βo – thalassemia 415 Case # 20 HemoglobinHasharon trait 421 Case # 21 HemoglobinZurich trait 428 Case # 22 HemoglobinLepore trait 434 Case # 23 HemoglobinJ-Oxford trait 442 Case # 24 HemoglobinJ-Baltimore trait 449 Case # 25 HemoglobinMalmo trait 455 Case # 26 HemoglobinKoln trait 466 Case # 27 HemoglobinQ-India trait 475 Case # 28 HemoglobinDhofar trait 488 XIX
  • 22. 1 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 Hemoglobin1 edited by Steinberg, Forget, Higgs and Nagel should be consulted. 1.1 Hemoglobin Structure Composed of 2 distinct globin chains, the complex protein molecule known as hemoglobin (“heme” + “globin”) is arguably THE primary component of the red blood cell in human beings. In “normal” adults, the globin chains are either alpha (α), beta (β), gamma (ϒ) or delta (δ). In addition, during embryonic life in utero, zeta (ζ) and epsilon (ε) chains are present in the first several weeks of life, being rapidly converted to alpha, beta and gamma chains as development occurs.
  • 23. 2 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,
  • 24. 3 provides a hydrophobic environment in which the heme molecules reside. This pocket protects the heme from oxidation and facilitates oxygen transfer to the tissues of the body. The previously mentioned ζ and ε chain-containing hemoglobins have very high oxygen affinities, a factor very important in the early embryonic life of the fetus. The hemoglobin molecule can be looked at in four different ways; primary, secondary, tertiary and quaternary structural views. While outside of the scope of this volume, each of these structures contributes definitive unique properties to the various hemoglobin molecules from normal hemoglobins to the very rare and functionally diverse molecules. The primary structure of all hemoglobins is the order of amino acids found in the globin chains of the molecule. It is this unique sequence that is the major differentiator of hemoglobin from each other. The secondary structure of hemoglobin is the arrangement of these amino acid chains into alpha helices separated by non-helical turns2 . 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.
  • 25. 4 Figure 2. Tertiary structure of a β 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 α globin chains and can,
  • 26. 5 via proton scavenging, keep CO2 in the soluble bicarbonate form3. Nitrous oxide is handled in two different ways by hemoglobin: one as a transporter and the other as a scavenger. Blood levels of NO are therefore, by definition, a balance between NO production and NO removal by binding to oxyhemoglobin. Since NO is an extremely potent vasodilator, hypoxic patients will have lower oxyhemoglobin and therefore higher amounts of free NO. This free NO can cause significant vasodilation, a physiological effect that is very desirable in hypoxia. All hemoglobin molecules, either normal or variant, share the same functionality in the human body. Therefore, the primary structural differences mentioned above and in more complete treatises (i.e., amino acid substitutions/deletions) will be the prime reason for functional differences. It is these amino acid variances that, along with the secondary, tertiary and quaternary structural differences, will determine if the variant hemoglobin is either benign or clinically important. The bottom line is this – whether the hemoglobin is normal or variant in nature, the prime reason for determining the hemoglobin phenotype of the patient is to assess the functionality of the hemoglobin. If the variant is normally functioning in both the heterozygous and homozygous states, the clinical picture is benign. If, however, the variant has normal properties in the heterozygous state (i.e., “trait”) but clinical issues in the homozygous state (i.e., “disease”), the phenotypic analysis and subsequent interpretation becomes ultimately important to the patient.
  • 27. 6 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.
  • 28. 7 As mentioned before, the true reason for identifying the abnormal hemoglobin or hemoglobins in patients is to identify any associated functional anomalies associated with these hemoglobins. The actual hemoglobin identification in and of itself is merely of academic interest. 1.4 Hemoglobin Variants All hemoglobin variants have one thing in common – they all involve the hemoglobin molecule and its functionality. Whether alpha, beta, gamma, delta, fusion variant, etc., the variant and its effect are judged not on its migration or concentration but rather on its functionality. The amino acid variation (e.g., glutamic acid → valine at position 6 on the beta chain for hemoglobin S) is the prime effector of the variant’s functional alteration(s) and will in most cases be the causative factor in any abnormal migration that the variant may have versus the “normal” hemoglobins (A, F, 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
  • 29. 8 during the interpretation of the hemoglobinopathy. This data includes, but is not limited to, pregnancy, transfusion history and ethnicity. All of these pieces of information can be critical to the proper identification/interpretation of the hemoglobin variant in the patient’s specimen. For example, an elevation of hemoglobin F in a female patient with a normal hemogram may be evidence of hereditary persistence of fetal hemoglobin; whereas, if this female is pregnant, the elevation may be a normal physiological response to the fetal presence in her body. These data may not be readily available and may require contact with the ordering healthcare professional to obtain these facts. However obtained, they are necessary for the proper identification of the hemoglobin variant or variants in the patient’s bloodstream and therefore are important in the assignment of a benign or pathological assessment of the variant hemoglobin. The variants described in the following chapters all obey the aforementioned differences, i.e., amino acid substitutions, genetic deletions, sequence modifications, etc. While not critical, the exact identification of the variant in and of itself is not normally life-threatening, especially in the heterozygous state, i.e., “trait”. It is essential that the variant be properly identified as a mis-identification can lead to other issues. For example, a mis-interpretation of a hemoglobin G trait (AG) as a sickle trait (AS), while not in and of itself is clinically an issue, presents real difficulties for a couple expecting a child. If both partners are AS, there is a 1 in 4 chance that a child born to this couple could be homozygous SS or sickle cell disease. In the case of an AS mother and an AG father (or vice versa), there is a 1 in 4 chance of a child being born with a phenotype of SG. While on the surface this
  • 30. 9 may appear as a problem, the SG phenotype is no more of a clinical issue than a simple AS trait. Without the exact identification of the AG trait, the interpretation and action taken by attending clinicians may be very different. References 1. Steinberg, MH, Forget, BG, Higgs, DR and Nagel, RL., Disorders of Hemoglobin, Cambridge University Press, 2001. 2. Bain, Barbara J.. in Hemoglobinopathy Diagnosis, 2nd Ed., pg. 4, Blackwell Publishing, 2006. 3. Bain, Barbara J.. in Hemoglobinopathy Diagnosis, 2nd Ed., pg. 1, Blackwell Publishing, 2006.
  • 31. 10 Chapter 2 Red Blood Cell Morphology John Lazarchick, MD Angie Duong, MD Knowledge of red blood cell (RBC) morphology is essential for the clinical diagnosis of hemoglobinopathy. The diameter of RBC, when mature under normal circumstances is approximately 7-8 microns, and RBC is round, anuclear and biconcave disc-shaped. A study of RBC morphology includes size, shape, color, inclusions and arrangement. In this chapter we have presented with pictures of the most commonly encountered RBC morphologies with legends and few examples of the diseases with abnormal RBC morphology. In the clinical cases of this book, we have mentioned only the main features of the peripheral blood smear, therefore a review of this chapter is advised for a naïve reader for the proper diagnosis of hemoglobinopathy. The following RBC morphology cases are presented in this chapter: Size: Macrocyte – large Fig. 1 Microcyte –small Fig. 2 Normocyte – normal Fig. 3 Hemoglobin Content: Hypochromic –low Fig. 4 Normochromic – normal Fig. 5 Polychromatic – high Fig. 6 Shape and Inclusions: Anisocytosis Fig. 7 Poikiocytosis Fig. 8 Acanthocyte Fig. 9 Basophilic Stippling Fig.10 Bite Cell Fig.11 Blister Cell Fig.12 Burr Cell (Ecchinocyte) Fig.13 Heinz Body Fig.14
  • 32. 11 Howell-Jolly Body Fig 15 Pappenheimer Body Fig.16 Schistocyte Fig.17 Sickle Cell Fig.18 Spherocyte Fig.19 Stomatocyte Fig. 20 Target Cell Fig. 21 Teardrop Cell Fig. 22 RBC Agglutination Fig. 23 Rouleaux Formation Fig. 24 Diseases : Erythroblastosis Fetalis Fig. 25 Hemoglobin C Disease Fig. 26 Hemoglobin C/beta Thalassemia Fig. 27 Hemoglobin S/beta Thalassemia Fig. 28 Hemoglobin SC Disease Fig. 29 Sickle Cell Disease Fig. 30 Fetal-maternal Hemorrhage: Fig. 31 Kleinhauer-Betke Stain
  • 33. 12 Fig. 1 – Macrocyte-large The diameter of RBC >9-14 microns (1.5 to 2 times larger than normal RBC) and the MCV >100 fL is characteristic of macrocyte. Macrocytes are mostly oval in shape.
  • 34. 13 Fig. 2 – Microcyte-small RBC, when abnormally smaller (< 5 micron) than normacytic RBC (7-8 micron) is defined as microcyte (also called microerythrocyte). The MCV of the microcyte RBC is < 80 fL.
  • 35. 14 Fig. 3 – Normocyte-normal The diameter of RBC, when mature under circumstances is approximately 7-8 microns, and are round, anuclear, biconcave disc-shaped with an internal volume of 80-100 fL. The term normocyte is used when the size of the RBC is normal.
  • 36. 15 Fig. 4 – Hypochromasia Hypochromasia is a descriptive term for red blood cells where the central pallor is greater than one third the diameter of the red blood cell (black arrows). This is due to a decrease in the amount of hemoglobin in the cells. Diseases with prominent hypochromasia are iron deficiency anemia, anemia of chronic disease, and sideroblastic anemia. Some cases of myelodysplastic syndrome can also have hypochromatic red blood cells. Hypochromasia is reflected in the complete blood count (CBC) by a decreased mean corpuscular hemo-globin concentration (MCHC). Also present are: target cells/codocytes (red arrow), polychromatic forms (blue arrow), fragmented red blood cells/schistocytes (green arrows), and tear drop forms/dacryocytes (yellow arrows). Overall, this smear shows moderate anisopoikilocytosis.
  • 37. 16 Fig. 5 – Normochromic-normal This descriptive term is applied to a red blood cell with a normal concentration of hemoglobin. The above figure is a peripheral blood cell smear of a patient treated for iron deficiency anemia. Blue arrow shows normochromic-normal RBC. Black arrow shows hypochromic-microcytic RBC.
  • 38. 17 Fig. 6 – Polychromatic-high This smear demonstrates polychromasia. Numerous polychromatic forms (black arrows), which are young slightly larger red blood cells with a purple-tinge due to retained RNA, are present. Polychromasia is the bone marrows response to anemia, where the bone marrow releases younger red blood cells. Sometimes, nucleated red blood cells are also released into the peripheral blood. Due to their larger size, when many polychromatic forms are present, the CBC values of mean corpuscular volume (MCV) as well as RDW (red blood cell distribution width) will be increased. In a supravital stains, such as cresyl violet, the retained RNA in the polychromatic forms precipitate out and these cells are called reticulocytes. Thus, sometimes the terms polychromatic form is used interchangeably with reticulocytes.
  • 39. 18 Fig. 7 – Anisocytosis The term anisocytosis refers to size variation seen among red blood cells. As demonstrated above, there are small red blood cells as well as large red blood cells, some approaching the size of a neutrophil (green arrow). Ansiocytosis is a reactive process where the bone marrow is releasing younger red blood cells, therefore an increased number of polychromatic forms can also be seen (black arrow). In the complete blood count (CBC), anisocytosis is reflected by having an increased red cell distribution width (RDW).
  • 40. 19 Fig. 8 – Poikilocytosis Poikilocytosis refers to shape variation. In poikilocytosis, the red blood cells have lost their normal discoid appearance. The example shown here has a predominance of ovalocytes/elliptocytes, which are red blood cells that have a length twice their diameter (a few are indicated by blue arrows). Also seen are schistocytes (red arrows), which are fragmented red blood cells. Ovalocytes/Elliptocytes are seen in peripheral blood smear in some conditions, e.g., thalassemia, iron deficiency, etc. Note: When both shape and size variation is seen in the red blood cells, the term anisopoikilocytosis can be used.
  • 41. 20 Fig. 9 – Acanthocyte (Spur Cell) These are red blood cells with spike-like projections (arrow) of varying length. They can be seen in both hereditary and acquired hemolytic anemias including alcoholic liver disease, pyruvate kinase deficiency, vitamin E deficiency, Huntington’s disease-like situation and abetalipoproteinemia. In the latter case, malabsorption of fat, neurologic damage and developmental delay are noted.
  • 42. 21 Fig. 10 – Basophilic Stippling Red blood cells have multiple fine or coarse small basophilic dot- like inclusions which are due to small clumps of ribonucleic acid and mitochondria. These inclusions can be seen in a wide variety of conditions including lead poisoning, hereditary hemoglobinopathies including unstable hemoglobins, thalassemias, sideroblastic anemias, megaloblastic anemia and hereditary pyrimidine 5’ - nucleotidase deficiency.
  • 43. 22 Fig. 11 – Bite Cell Bite cell (arrow) has a semicircular portion of the membrane removed. This morphologic abnormality results from splenic macrophages removing denatured precipitated hemoglobin with Heinz body formation in these cells. The most common cause of this finding is glucose-6-phosphate dehydrogenase deficiency.
  • 44. 23 Fig. 12 – Blister Cell Red blood cells with cytoplasmic clearing (large arrows) on one side and hemoglobin on the other side in a patient with hemolytic anemia. Multiple polychromatophilic red blood cells (reticulocytes) are noted (small arrow). In addition, a single cell with a Howell-Jolly body inclusion (double arrows) is noted,
  • 45. 24 Fig. 13 – Burr Cell (Echinocyte) These are red blood cells with short round membrane projections with blunt ends (large arrow). Red blood cells with more spike- like projections (small arrow) can also be seen. This finding is often an artifact of slide preparation but is typically seen in patients with uremia and pyruvate kinase deficiency.
  • 46. 25 Fig. 14 - Heinz Body In a RBC when the hemoglobin is denatured (either by a change of an internal amino acid or glucose-6-phosphatse deficiency, etc.), the heme portion of hemoglobin molecule is dissociated from the globin chain. The globin chain after dissociation from the heme molecule becomes denatured forming a small ball like structure (black arrow) inside the RBC, and thus called Heinz body.
  • 47. 26 Fig. 15 – Howell-Jolly Body This red blood cell inclusion (arrow) is round basophilic DNA remnant usually noted in the outer third of circulating red blood cells. These inclusions are normally extruded in the bone marrow during normal erythroid maturation. Howell-Jolly bodies can be seen in asplenia, conditions associated with hyposplenia including sickle cell disease and severe hemolytic anemia.
  • 48. 27 Fig. 16 - Pappenheimer Bodies These are small dark irregular staining granules (large arrow) of non-heme iron usually noted on the periphery of red blood cells formed by phagosomes that engulf excess iron. Basophilic stippling is present in the dysplastic nucleated RBC (small arrow) These granules stain positive with Prussian blue stain in both the nucleated RBC and mature red blood cells as shown in the lower image. They can be found in a variety of conditions including sideroblastic anemias, thalassemias and myelodyplastic syndromes.
  • 49. 28 Fig. 17 - Schistocyte (RBC fragments, Helmet Cells) These are red blood cell fragments typically with two pointed ends formed when RBCs are sheared by fibrin strands in clotted blood vessels. Disorders include microangiopathic hemolytic anemia, disseminated intravascular consumption (DIC), thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS).
  • 50. 29 Fig. 18 - Sickle Cell In inherited blood cell disease (change of an amino acid residue in the globin chain) the shape of the RBC is deformed. The deformation of RBC resembles (a waxing crescent) a moon sighted on the first day of lunar month. Since this deformation looks like a sickle (an implement with a semicircular blade attached to a short handle, used for cutting grain), therefore this deformation is called sickle cell.
  • 51. 30 Fig. 19 – Spherocytes This peripheral blood smear is from a patient with autoimmune hemolytic anemia (AIHA) and is characterized by many spherocytes (blue arrows) and microspherocytes (black arrows). Spherocytes are red blood cells that have no central pallor. As the name implies, microspherocytes are small spherocytes. If the majority of the cells in a peripheral smear are spherocytes, the possibility of hereditary spherocytosis arises. Hereditary spherocytosis is an autosomal dominant disease where one of the genes that code for red blood cell proteins (such as spectrin and ankyrin) become mutated.
  • 52. 31 Fig. 20 – Stomatocyte Red blood cells with slit-like central pallor (arrow) caused by a decrease in surface area to volume ratio associated with a membrane permeability disorder. Hereditary stomatocytosis is associated with hemolysis which can be severe. Acquired stomatocytosis can be seen in acute alcohol intoxication, chronic liver disease and as drying artifact in peripheral smear preparation.
  • 53. 32 Fig. 21 - Target Cells Also known as codocytes, these red blood cells appear to have a bullseye in the center of the red blood cell’s central pallor. This morphologic change is due to a relative excess of cell membrane, due to decreased cell content or increase in the cell’s surface area. Target cells can be seen in liver failure, Hemoglobin C disease, thalessemias (both alpha and beta), and iron deficiency.
  • 54. 33 Fig. 22 - Tear drop cells Also known as dacryocytes/dacrocytes (red circles), are distorted red blood cells where one end of the cell is drawn into a sharp point. These cells are usually seen in myelophthsic anemias, which is where the normal marrow space is occupied by non- hematopoietic elements, such as fibrosis or metastatic carcinoma. It is hypothesized that the shape of the cells is due to the red blood cells squeezing between fibers or the cells extrinsic to the marrow.
  • 55. 34 Fig. 23 - RBC Agglutination Clumping of the red blood cells is due to coating of the RBC surface with antibodies. Disorders causing the agglutination may be primary as in cold agglutinin disease or secondary, either clonal as in lymphoproliferative disorders or polyclonal as seen in Mycoplasma pneumonia. The upper left insert is from a slide prepared at room temperature and the upper right insert is a slide after warming the sample to 370 C with clearing of the agglutination in a patient with cold agglutinin disease.
  • 56. 35 Fig. 24 – Rouleaux Formation Rouleaux formation is seen in peripheral blood smears in association with plasma cell neoplasms, most commonly myeloma. The red cells become stuck together in a “stack of coins” formation, due to the excess immunoglobulin proteins released by malignant plasma cells. Not all cases of plasma cell neoplasms have rouleaux formation. Rouleaux formation is one of the causes of an increased erythrocyte sedimentation rate (ESR).
  • 57. 36 Fig. 25 - Erythroblastosis Fetalis This is an alloimmune hemolytic anemia in the fetus secondary to placental transfer from mother to fetus during pregnancy of anti– A or B or anti-Rh blood group IgG antibodies. These blood groups are present on the fetal RBCs but not on the maternal RBCs which then causes immune hemolysis in the fetal circulation. As noted on the smear, numerous nucleated RBCs (large arrow) and polychromatophilic RBCs (small arrow) are noted. The case shown above was due to antibodies to Rh D blood group.
  • 58. 37 Fig. 26 - Hemoglobin C Disease In this case of homozygous hemoglobin C disease essentially all of the RBCs are target cells (large arrow). Hemoglobin C crystals are rod shaped inclusions (Washington Monuments—small arrow) in red blood cells in both heterozygous and homozygous hemoglobin C disease as well as hemoglobin SC disease. Upper image shows the crystals at a higher magnification.
  • 59. 38 Fig. 27 - Hemoglobin C/beta Thalassemia Although most patients with this compound heterozygotic state for hemoglobin C and beta thalassemia are asymptomatic, a mild to moderate hemolytic anemia can be seen. The red blood cells are microcytic and hypochromic. Target cells (double arrow) and C crystals (single arrows) can be seen.
  • 60. 39 Fig. 28 - Hemoglobin S/beta Thalassemia Hemolytic anemia due to both production of an abnormal hemoglobin (Hemoglobin S) and decreased synthesis of beta globin chains (Beta Thalassemia). Individuals have one abnormal beta chain with substitution of glutamic acid for valine and either decreased synthesis, beta+, or complete absence of the other beta chain, beta0 . The peripheral smear shows sickle cells, nucleated red blood cells, polychromasia, microcytosis, hypochromic, target cells and basophilic stippling. Note the sickle cell in the insert and the Howell-Jolly body in the other RBC.
  • 61. 40 Fig. 29 - Hemoglobin SC Disease This is a representative peripheral blood smear from a patient with hemoglobin SC disease. Hemoglobin SC disease is an inherited hemoglobinopathy where the two normal genes for hemoglobin A have been replaced by one hemoglobin S gene and one hemoglobin C gene. In hemoglobin S, a single nucleotide at position 6 of the gene is substituted by another nucleotide (glutamic acid is substituted by valine). A similar phenomenon occurs in hemoglobin C, where glutamic acid is substituted by lysine. When both hemoglobin S and hemoglobin C is present, the genes are codominant and lead to many interesting peripheral blood findings. Hemoglobin S produces drepanocytes/sickle cells (black arrows) which are red blood cells that appear as crescent moon shapes or continued next page
  • 62. 41 sickles. Due to the abnormal hemoglobin content, the deoxygenated red blood cells become stuck in this shape, thus causing vascular occlusions which in turn lead to many complications such as pain crisis. Sickle cells are seen when there is no or decreased levels of hemoglobin A (such as hemoglobin SS disease, hemoglobin SC disease, hemoglobin S with thalessemia). In sickle cell trait, where there is one normal hemoglobin A gene and one hemoglobin S gene, sickle cells are not seen and the patients usually have no clinical symptoms. Hemoglobin C manifests in peripheral smears as numerous target cells/codocytes (green arrows). Additionally, in hemoglobin CC disease and in hemoglobin SC disease, hemoglobin C crystals (blue circle) can be seen. These crystals are desicated red blood cells with squared off/blunt edges. In hemoglobin C trait, target cells are seen but hemoglobin C crystals are not.
  • 63. 42 Fig. 30 - Sickle Cell Disease Sickle cell disease is a hereditary hemolytic anemia caused by a single nucleotide substitution (SNP) of valine for glutamic acid in the beta globin chain of hemoglobin. This results in hemoglobin polymerizing at low oxygen tension with sickle cell formation (small arrow). There is marked polychromasia, target cells and nucleated red blood cells (inset—large arrow) on the peripheral smear.
  • 64. 43 Fig. 31- Fetal-maternal Hemorrhage: Kleihauer-Betke Stain This test relies on the principle that red blood cells containing fetal hemoglobin (deep red staining RBCs) are less susceptible to acid elution than adult red blood cells. Its use is a means of quantitating fetal-maternal hemorrhage in Rh-negative mothers to determine the dose of Rho (D) immune globulin needed to inhibit formation of Rh antibodies. It can also be used to detect hereditary persistence of fetal hemoglobin (HPFH).
  • 65. 44 Chapter 3 Diagnostic Laboratory Methods 3.1 Basic Concepts Jayson Miedema, MD, and Christopher R. McCudden, PhD 3.1.1 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,
  • 66. 45 basophilic stippling, polychromasia and, hypochromasia. Since not all unstable hemoglobins will give abnormal results on HPLC or electrophoresis and/or these results can be somewhat non-specific, more definitive testing is often performed. Testing for unstable hemoglobins relies on their decreased stability in heat or isopropanol alcohol. While normal hemoglobins should be relatively stable in these conditions, hemoglobins with mutations causing instability tend to be less so and will precipitate out of solution in these environments. In the context of heat stability testing, the amount of unstable hemoglobin in a sample is given by the following equation: (Hb4°C-Hb50°C)/(Hb4°C)x100 Where Hb4°C is the hemoglobin concentration at 4 degrees centigrade and Hb50°C is the concentration of hemoglobin at 50 degrees centigrade. False positives may result from samples greater than 1 week in age as well as from samples with large amounts of fetal hemoglobin. Additional technical and clinical information on hemoglobinopathies associated with unstable hemoglobin can be obtained from: http://medtextfree.wordpress.com/2011/12/30/chapter-48-hemoglobinopathies 3.1.2 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
  • 67. 46 Chesapeake, and beta-chain variants, e.g. Hb Olser, Hiroshima, Andrew-Minneapolis, 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. 3.1.3 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
  • 68. 47 aggregation of deformed blood cells in small blood vessels. Widely believed to have 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 6th 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
  • 69. 48 to false negatives. False positives can occur in the settings of lipemia or samples with monoclonal proteins (dysproteinemia). Both positive and negative controls should be used as results can be somewhat subjective 3.1.4 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
  • 70. 49 epithelial cell sloughing in the skin and GI tract or through menstrual bleeding in 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 Fe2+ 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. This method can be affected by the presence of non-transferrin iron binding proteins, particularly in cases of
  • 71. 50 hemochromatosis and thalassemias. Alternatively, TIBC may be calculated based on the stoichiometric relationship between transferrin and iron (2 molecules of iron are bound to each molecule of transferrin). TIBC is calculated from measured transferrin using the following equation: TIBC (µg/dL) = 1.43 × transferrin (mg/dL). Conversely, the concentration of transferrin may be calculated from measured TIBC as follows: Transferrin (mg/dL) = 0.7 × TIBC (µg/dL). TIBC is increased in iron deficiency and decreased in chronic anemia of disease and in iron overload (it may be normal or decreased in thalassemia). From TIBC and serum iron measurement, it is also possible to calculate the % transferrin saturation (also known as iron saturation) using a simple formula: % saturation = serum Fe (µg/dL) / TIBC (µg/dL) ×100. The percent saturation is usually between 20-50%, supporting an excess capacity for iron binding. In cases of iron overload, the % saturation increases dramatically. Saturation is moderately increased in thalassemia and chronic anemia and in iron deficiency the saturation is decreased. Ferritin is a large ubiquitous protein and the major iron storing protein in the body. Ferritin serves to store thousands of iron atoms/molecule in a non-toxic form acting as an iron reserve. Ferritin is found in small amounts in the blood, where it can be measured as an indication of overall iron reserves (1 ng/mL serum iron approximates 10 mg total storage iron). 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
  • 72. 51 to clinical and morphological manifestations of anemia (e.g. microcytosis) making it a 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 Fe3+ 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.
  • 73. 52 Table 1. Iron Tests in Different Disorders Disorder Serum Iron TIBC % Saturation Transferrin Ferritin Chronic Anemia of Disease ↓ ↓ ↓ ↔ or ↓ ↔ or ↑ Iron Deficiency ↓ ↑ ↓ ↑ ↓ Thalassemia ↔ or ↑ ↔ ↔ or ↑ ↔ or ↓ ↔ or ↑ Hemochromatosis ↑ ↓ ↑↑ ↔ or ↓ ↑↑ ↓decreased; ↔ within reference interval; ↑ increased 3.1.5 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.
  • 74. 53 3.1.6 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.
  • 75. 54 Rees DC, Williams TN, Gladwin MT. Sickle-cell disease. The Lancet. 2010;376:2018- 2031. Steinberg MH. Genetic disorders of hemoglobin oxygen affinity. www.uptodate.com. Accessed April 28, 2011. Steinberg MH. Unstable hemoglobin variants. www.uptodate.com. Accessed April 28, 2011. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. Edited by Burtis CA, Ashwood ER, and Bruns DE. 5th Edition. Vichinsky EP. Sickle cell trait. www.uptodate.com. Accessed April 28, 2011.
  • 76. 55 Chapter 3 Diagnostic Laboratory Methods 3.2 Microcytosis Diane Maennle, MD, and Kimberly Russell, MT (ASCP), MBA Smaller-than-normal size of Red Blood Cells (RBCs) is defined as microcytosis. This is quantified by calculating the mean corpuscular volume (MCV) using the following formula employing the values of hematocrit and RBC count: MCV = Hematocrit (HCT) X 10 / RBC Count (Million) In adults, a MCV value of less than 80fL is defined as microcytosis. In pediatric subjects, the MCV and hemoglobin range distinctly vary with age (Table I). Table I Age Dependent Mean Hemoglobin and MCV Values1,2,3,4 Age Mean Hemoglobin (g/dL) Mean MCV (fL) 3 to 6 months 11.5 91 6 months to 2 years 12.0 78 2 to 6 years 12.5 81 6 to 12 years 13.5 86 12 to 18 years (female) 14.0 90 12 to 18 years (male) 14.5 88 > 18 years (female) 14.0 90 > 18 years (male) 15.75 90
  • 77. 56 Iron deficiency anemia, α-thalassemia trait, and β-thalassemia trait are the most common causes of microcytosis. However, other clinical conditions are also associated with microcytosis (Table II).1,3,5,6 In addition to decreased MCV, the patients with β-thalassemia trait usually have increased hemoglobin 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 β-thalassemia trait in such patients. Conversely, patients with β-thalassemia trait may acquire megaloblastic anemia or liver disease, and may exhibit a normal range for MCV.7 Table II Diagnostic Reasons of Microcytosis (listed in decending order of frequency) Children and adolescents Menstruating women Men and non-menstruating women Iron deficiency anemia Iron deficiency anemia Iron deficiency anemia Thalassemia trait Thalassemia trait Anemia of chronic disease Other hemoglobinopathies Pregnancy Unexplained anemia Lead toxicity Anemia of chronic disease Thalassemia trait Chronic inflammation Sideroblastic anemia Sideroblastic anemia Several laboratory tests in addition to the CBC, e.g. serum iron, serum ferritin, total iron- binding capacity (TIBC), transferrin saturation, hemoglobin electrophoresis, and the examination of the peripheral blood smears (by a pathologist or hematologist), are employed to provide insight and etiologies of microcytosis (Table III).3,8
  • 78. 57 Table III Laboratory Tests in the Differential Diagnosis of Microcytosis Suggested diagnosis Test Iron deficiency anemia Thalassemia Anemia of chronic disease Sideroblastic anemia Serum ferritin Decreased Increased Normal to increased Normal to increased RBC Increased Normal to Normal Increased distribution width increased (RDW) Serum iron Decreased Normal to Normal to Normal to increased decreased increased Total iron- Increased Normal Slightly increased Normal binding capacity Transferrin Decreased Normal to Normal to slightly Normal to saturation increased decreased increased Van Vranken3 has recently suggested a protocol for diagnosing the cause of microcytosis (Figure 1). If the cause remains unclear, the diagnosis of hemoglobinopathy by methods besides electrophoresis alone is imperative. Note: There is a type-setting error in the presentation of the protocol suggested by Van Vranken.3 We have corrected this error in the figure 1, and the journal (American Family Physician) editor was also informed.
  • 79. 58
  • 80. 59 Clinical observations of Kenneth F. Tucker, MD, FACP, a practicing hematologist for the last forty years: Ordinary hemoglobin electrophoresis (cellulose acetate or agarose gel electrophoresis) was only able to detect the more common types of thalassemias. Although there were several other types, many of them did not have microcytosis. I had a large number of patients, who had β-thalassemia minor and a few with probably α-thalassemia, in which the hemoglobin and hematocrit values were relatively normal. Microcytes may or may not be present. This diagnosis was suggested by the peripheral smear, and proven by additional laboratory tests (IFE, globin chain analysis, etc.). I believe that RDW, which is the average red cell width and reflects standard deviation of red cell volumes, is a very important test. RDW normal deviation is a bell- shaped curve. When this value is 2-3% higher, it represents red cells which have varying widths. This certainly can be seen in patients who are iron deficient with microcytosis, but have normal or large cells in addition to megaloblastic or dysplastic marrows, elevated reticulocytes, vitamin B12 or folic acid deficiency, and other conditions. Despite the availability of automated cell counters, review of the peripheral film is one of the most informative and rewarding tests that should be done (by pathologist or hematologist) in any case in which the cause of anemia is not obvious, e.g., bleeding, pure iron deficiency, pure vitamin B12 deficiency, etc. It is also emphasized that the RDW test is not sensitive or specific enough to differentiate iron deficiency and β-thalassemia trait.9 A fairly low to extremely low ferritin is an excellent measure of iron deficiency anemia. In my practice, regardless of what else is going on, any ferritin level of <10 ng/mL, means there is iron deficiency. As mentioned above (Table III), elevated ferritin levels are
  • 81. 60 seen in refractory anemias, all types of chronic inflammatory conditions, etc. Since this test is an acute phase reactant (similar to haptoglobin), it must not be used alone, as the ferritin level may be normal in these clinical conditions. Women in the second or third trimester are always anemic. This is similar to patients who are hypervolemic because of renal or cardiac problems. Red cells in these cases are not microcytes and when the hypervolemia is corrected, the hemoglobin and hematocrit rises. Severe anemia in childhood is usually due to the lack of iron in food, since cow’s milk does not contain iron. A naïve reader is advised to also review the “Full Color pdf of Complete Blood Count in Primary Care,” Best Practice Journal, June 2008 (www.bpac.org.nz), especially the section on Hemoglobin and Red Cell Indices (page 15). References 1. Richardson M. Microcytic anemia [published correction appear in Pediatr Rev. 2007; 28(7): 275, Pediatric Rev. 2009; 30(5): 181, and Pediatr Rev. 2007; 28(4):151]. Pediatr Rev. 2007; 28(1): 5-14. 2. Beutler E, Waalen J. The definition of anemia: what is the lower limit of normal of the blood hemoglobin concentration? Blood. 2006; 107(5): 1747-1750. 3. Van Vranken ML. Evaluation of Microcytosis. Am Fam Physician. 2010; 80(9): 1117-1122. 4. RBC indices calculation and laboratory procedure (2006). St. John Health Laboratories, Warren, MI 48093. 5. Moreno Chulila JA, Romero Colas MS, Gutierrez Martin M. Classification of anemia for gastroenterologist. World J Gastroenterol. 2009: 15(37):4627-4737. 6. Guralnik JM, Eisenstaedt RS, Ferrucci L, Klein HG, Woodman, RC. Prevalence of anemia in persons 65 years and older in the United States: evidence for a high rate of unexplained anemia. Blood. 2004; 104(8): 2263-2268. 7. Bain BJ. Hemoglobinopathy Diagnosis. 2nd ed. Malden, Mass.: Blackwell Publishing; 2006: 94-106.
  • 82. 61 8. Hematologic diseases. In: Wallach J. Interpretation of Diagnostic Tests. 8th 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.
  • 83. 62 Chapter 3 Diagnostic Laboratory Methods 3.3 Hereditary Persistence of Fetal Hemoglobin Bernard G. Forget, MD 3.3.1 Introduction Hereditary persistence of fetal hemoglobin or HPFH consists of a group of genetic disorders characterized by the presence of a substantial elevation of fetal hemoglobin (Hb F) in RBCs of heterozygotes, as well as of homozygotes and compound heterozygotes for HPFH and other hemoglobinopathies. Increased levels of Hb F can ameliorate the clinical course of hemoglobinopathies such as β thalassemia and sickle cell anemia. HPFH is usually due to deletions of different sizes involving the β-globin gene cluster, but nondeletion types of disorders have also been identified, usually due to point mutations in the γ-globin gene promoters (reviewed in refs. 1-3). Figure 1 diagrammatically illustrates the spatial organization of the β-like globin genes in the β-gene cluster on chromosome 11. However, as discussed later in this chapter, certain forms of nondeletion HPFH are clearly not linked to the β-globin gene cluster.
  • 84. 63 Figure 1. Deletions of the β-globin gene cluster associated with fusion proteins and HPFH. The circle 3’ to the β-globin gene indicates the 3’ β-globin gene enhancer. The filled vertical boxes at the 3’ breakpoints of the HPFH-1 and HPFH-6 deletions indicate the locations of DNA sequences with homology to olfactory receptor genes (adopted from reference 2). The references for the individual mutations are cited in references 1, 3 and 6. HPFH is frequently contrasted with δβ thalassemia, which is another genetic disorder associated with elevated Hb F levels. However, one should probably not consider the two disorders as being unambiguously separate entities but rather as a group of disorders with a variety of partially overlapping phenotypes that sometimes defy classification as one syndrome or the other. The following is a working definition that is generally applied to the classification of these disorders: δβ thalassemia usually refers to a group of disorders associated with a mild but definite thalassemia phenotype of hypochromia and microcytosis together with a modest elevation of Hb F that, in heterozygotes, is heterogeneously distributed among red cells. In contrast, HPFH refers to a group of disorders with substantially higher levels of Hb F, and in which there is usually no associated phenotype of hypochromia and microcytosis. In addition, the increased Hb F in heterozygotes with the typical forms of HPFH is distributed in a relatively uniform (pancellular) fashion among all of the red cells rather than being distributed in a heterogeneous (heterocellular) fashion among a subpopulation of so- called F cells, as in δβ thalassemia. Homozygotes for both conditions totally lack Hb A and Hb A2, indicating absence of δ- and β-globin gene expression in cis to the δβ thalassemia and HPFH determinants. Although the apparent striking qualitative difference in cellular distribution of Hb F between HPFH and δβ thalassemia may be
  • 85. 64 due in great part to the quantitative differences in the amount of Hb F per cell and the sensitivity of the methods used to detect Hb F cytologically, nevertheless it would appear that the increased amount of Hb F in HPFH is caused by a genetically determined failure to suppress γ-globin gene activity postnatally in all erythroid cells, rather than being due to selective survival of the normally occurring sub-population of F cells such as occurs in sickle cell anemia, β+ and βo thalassemia. Nevertheless, heterocellular forms of HPFH, without a β-thalassemia phenotype, have been clearly defined and characterized. Therefore, in the final analysis, there is definitely some overlap between these two sets of syndromes at the level of their clinical and hematological phenotypes, as well as at the level of their molecular basis. 3.3.2 Deletions Associated with the HPFH Phenotype. Classic pancellular HPFH, with absence of δ-and β-globin gene expression from the affected chromosome, is associated with large deletions in the β-globin gene cluster that remove the δ-and β-globin genes together with variable amounts of their 5’ and 3’ flanking DNA. At least nine different HPFH deletions of this type have been characterized that vary in size or length from ~13 kb to ~ 85 kb (1-4), some of which are illustrated in Fig. 1. The mechanisms by which such deletions cause marked elevation of Hb F are not well understood, but a number of theories have been proposed. One theory is based on the model of the proposed mechanism for the marked elevation of Hb F associated with Hb Kenya. Hb Kenya is a structurally abnormal hemoglobin that, like Hb Lepore, contains a "hybrid" or fused β-like globin chain resulting from a non-homologous crossing-over event between two globin genes in the
  • 86. 65 β-gene cluster. However, whereas the Lepore crossover occurred between the δ- and β-globin genes, the Kenya gene resulted from crossover between the Aγ- and β-globin genes (Fig. 1). The crossover occurred in the second exons of the Aγ and β genes, between the codons for amino acids 80 to 87, and resulted in deletion of ~24 kb of DNA between the Aγ to the β gene. Unlike Hb Lepore, that is associated with a β- thalassemic phenotype, Hb Kenya is associated with a phenotype of pancellular Gγ 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 Gγ 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 Gγ- and Kenya gene promoters of a well characterized enhancer element located in the 3' flanking DNA of the β-globin gene, shown by the filled circle in Fig. 1, that becomes translocated into close proximity of the γ-globin gene promoters by the crossover/deletion event, resulting in enhanced activity of these promoters. Among the HPFH deletions, there is a relatively short deletion, called HPFH-5 or Italian HPFH, that extends from a point ~3 kb 5' to the δ gene to a point 0.7 kb 3' to the β gene, deleting the β gene but not its 3' enhancer. The molecular basis of the HPFH phenotype associated with this deletion is presumably the influence of the translocated 3' β-gene enhancer on the γ-gene promoters, in a manner analogous to that proposed for the basis of the HPFH phenotype of the Hb Kenya syndrome. In the case of some of the other larger HPFH deletions, the DNA preserved at or near the 3’ breakpoint of the deletions has been shown in various types of assays to have enhancer-like activity on gene expression (2, 5-7). Thus, it has been proposed that the DNA sequences at the
  • 87. 66 HPFH 3' deletion breakpoints, that become juxtaposed to the γ genes as a result of the deletion events, may influence γ-gene expression, in a manner analogous to the presumed influence of the 3' β-gene enhancer on γ-gene expression in Hb Kenya and HPFH-5. Mechanisms by which this could occur include the presence of enhancer-like sequences in the translocated 3' breakpoint DNA or the presence in this DNA of an active chromatin configuration that could have a spreading and activation effect on the expression of the neighboring γ-globin genes. A second theory for the mechanism of increased γ-gene expression in deletion- type HPFH is the nature and function of the DNA sequences conserved at the 5’ breakpoint of the deletions. The 5’ breakpoints of the HPFH deletions, as well as many of the δβ-thalassemia deletions, are located in the DNA between the Αγ and δ genes, the so-called Αγδ-intergene DNA. It has long been proposed that there may exist negative regulatory or silencer elements in this region of DNA, deletion of which in HPFH but not in δβ thalassemia, results in markedly impaired postnatal suppression of γ-gene activity in all erythroid cells (8). A number of subsequent observations have been made that support a role for the Aγδ-intergene region in the regulation of γ-gene expression (reviewed in ref. 9). The Corfu deletion in particular, involving the δ-gene and ~6 kb of upstream flanking DNA, is associated in homozygotes with a high HbF phenotype and removes some interesting structural elements, such as a poly-pyrimidine region that can serve as a binding site for a multi-protein chromatin remodeling complex containing the transcription factor Ikaros, and a region of DNA that serves as a promoter for the synthesis of an intergenic RNA transcript preferentially expressed in adult
  • 88. 67 erythroid cells (10). This region of DNA also appears to serve as a boundary region between fetal and adult domains of the β-globin gene cluster. The most conclusive evidence for a functional role of the Aγδ-intergene DNA in the regulation of γ gene expression consists of the observations by Sankaran et al. who have extensively characterized a negative regulatory transcription factor, called BCL11A, that down-regulates γ-gene expression in adult erythroid cells and that binds to the Aγδ-intergene DNA (11-13). BCL11A, originally identified as an important factor in B-lymphoid cell development, is a component of a multi-protein complex that plays a negative regulatory role in γ-gene expression. This complex has been shown to contain GATA1 as well as all components of the nucleosome and histone deacetylase ( NuRD)- repressive complex (14). Additional studies have shown that this complex physically interacts with another transcription factor called SOX6 that is thought to be a repressor of embryonic and fetal globin gene expression (15). Chromatin immunoprecipitation (ChIP) studies have shown that BCL11A binds to a number of regions in the β-cluster, including the upstream locus control region (LCR) and the γδ intergenic region, but does not bind to the γ- or β-gene promoters (4, 14, 15). Sankaran et al. (4) have characterized two important deletion mutants with nearly identical distal breakpoints but different upstream breakpoints around the δ-gene and its flanking DNA. One mutant with a more proximal breakpoint has a δβ-thalassemia phenotype, whereas the longer deletion removing 3.5 kb of additional upstream DNA is associated with a HPFH phenotype. The authors propose that this 3.5 kb region of DNA contains a silencer element, deletion of which can cause HPFH. This hypothesis is strengthened by the fact that the deleted region contains one of the prominent binding sites of BCL11A detected
  • 89. 68 in their ChIP experiments. These findings provide very strong evidence for a γ-gene silencer element in the β-gene cluster that associates with a BCL11A-containing repressor complex and that this interaction is an important factor in the suppression of γ-gene expression during the perinatal switch from expression of Hb F to Hb A. 3.3.3 Nondeletion Forms of HPFH In contrast to the deletional types of HPFH syndromes, where both linked Gγ and Aγ genes are over expressed, only one or the other γ gene is usually over expressed in the best characterized nondeletional types of HPFH associated with high levels of pancellular Hb F expression. However, less well characterized nondeletion forms of GγAγ HPFH have been described that are associated with relatively low levels of heterocellular expression of both γ genes. Because of the restricted pattern of γ-globin gene expression in the Gγ and Aγ forms of nondeletion HPFH, it was assumed that the mutations in these syndromes must be located near the affected gene and molecular studies focused initially on the DNA sequence analysis of the over expressed γ genes in these disorders.
  • 90. 69 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 γ type, and the G γ gene in cis to the -175 A γ mutation carried the -158 C→ T change. The references for the individual mutations are cited in references 1 and 3. The results of these structural analyses revealed a number of different point mutations in the promoter region of the over expressed γ gene in individuals with different types of nondeletion HPFH, as listed in Table 1 (reviewed in refs. 1-3). These point mutations have clustered primarily in three distinct regions of the 5'-flanking DNA of the affected γ genes. The first region is located approximately 200 base pairs from
  • 91. 70 the "cap site" or site of transcription initiation of the γ genes (at least five different point mutations involving single nucleotides between residues -195 to -202 from the cap site). This region of DNA, which had not previously been suspected of playing a role in the regulation of γ-gene expression, is very G+C rich and its sequence bears homology to that of known control elements of other genes that contain the binding site for the ubiquitous trans-acting protein factor called Sp1. Subsequent studies of the γ-gene promoters have demonstrated that the -200 region is also a binding site for Sp1 and for at least one other ubiquitous DNA binding protein. The second region containing a mutation associated with nondeletion HPFH is located at position -175. A point mutation (T->C) at this position of either the Gγ or Aγ gene is associated with a phenotype of pancellular HPFH with high levels of Hb F (15- 25%). This region of DNA is noteworthy because it contains an octanucleotide sequence that is present in the promoter region of a number of genes and is the binding site of another ubiquitous trans-acting factor called OCT-1. In addition, the octamer consensus sequence of the γ-gene promoters is flanked on either side by a consensus sequence for the hematopoietic-specific transcription factor GATA-1. The point mutation at position -175 affects the one nucleotide that is present in the partially overlapping binding sites of both OCT-1 and GATA-1. The third region affected by a point mutation in nondeletion HPFH is in the area of a well known regulatory element of globin and other genes: the CCAAT box sequence. In the γ genes, the CCAAT box is duplicated and the mutation associated with the Greek Aγ type of nondeletion HPFH is a G->A substitution at position -117, 2 bases upstream of the distal CCAATbox of the Aγ-globin gene promoter. The base
  • 92. 71 change disrupts a pentanucleotide sequence, YYTTGA (Y = pyrimidine), that is highly conserved immediately upstream of the CCAAT sequence in all animal fetal and embryonic genes. At least two other mutations involving the CCAAT box of one or the other γ gene have been reported in other cases of HPFH not associated with large deletions. The CCAAT box region is known to be the binding site of a number of trans- acting factors, including the ubiquitous factors CCAAT binding protein (CP1) and CCAAT displacement factor (CDP) as well as the erythroid-specific factor NF-E3. The unifying model by which these various mutations are thought to affect hemoglobin switching proposes that these base changes alter the binding of a number of different trans- acting factors to critical regions of the γ-gene promoters and thereby prevent the normal postnatal suppression of γ-gene expression (reviewed in refs. 1,2). The mutations could prevent the binding of negative regulatory factors or enhance the binding of positive regulatory factors. Either mechanism could be operative with one mutation or the other. 3.3.4 HPFH Unlinked to the β-Globin Gene Cluster A number of studies have identified families in which increased levels of Hb F are inherited due to a genetic determinant that is unlinked to the β-globin gene cluster. Genome-wide association studies (GWAS), using co-inheritance of single nucleotide polymorphisms (SNPs) with elevated levels of Hb F, have subsequently demonstrated the presence of two different quantitative trait loci (QTLs), unlinked to the β-globin gene cluster on chromosome 11, that are associated with inheritance of mildly elevated levels of Hb F, similar to the phenotype seen in Swiss-type heterocellular HPFH (see section
  • 93. 72 above on Nondeletion HPFH). These loci are located on chromosome 2 and 6 (16, 17). The locus on chromosome 2 corresponds to the site of the gene encoding BCL11A and its identification led to the elegant studies of Sankaran and co-workers demonstrating the role of BCL11A in the regulation of γ-gene expression. The locus on chromosome 6 is located between the genes encoding HBS1L and MYB. The mechanism by which this locus causes elevation of Hb F is thus far poorly understood. Finally, mutations in the gene on chromosome 19 encoding the erythroid-specific transcription factor EKLF1 have been shown to be associated with a form of HPFH (18, 19). The involved mechanism is probably through the regulation of BCL11A levels, because it has been demonstrated that EKLF1 binds to the promoter of the BCL11A gene and regulates the expression of the gene (20). 3.3.5 Conclusion Significant insights into the normal regulation of expression of the human β- globin gene cluster have been obtained by a detailed analysis of a group of disorders called HPFH. On the basis of this information, several important regulatory elements have been identified for the normal functioning of the individual genes in the cluster during the developmental switch from fetal to adult hemoglobin gene expression, as well as for the abnormal persistent expression of the γ-globin genes in adults with HPFH. These results provide a more sophisticated understanding of the molecular basis of these syndromes and point to certain strategies for potential future molecular and cellular therapies for globin gene disorders.
  • 94. 73 3.3.6 Hemoglobin F Quantification Hb F can be quantified by several methods, and the most commonly used procedures in a clinical laboratory are a) radial immunodiffusion, b) Elisa method, c) HPLC, and d) capillary zone electrophoresis. References 1. Bollekens JA, Forget BG. Delta beta thalassemia and hereditary persistence of fetal hemoglobin. Hematol Oncol Clin North Am 1991;5(3):399-422. 2. Forget BG. Molecular basis of hereditary persistence of fetal hemoglobin. Ann N Y Acad Sci 1998; 850:38-44. 3 Weatherall DJ, Clegg JB. The Thalassaemia Syndromes. 4th ed. Oxford ; Malden, MA: Blackwell Science; 2001. 4. Sankaran VG, Xu J, Byron R, et al. Functional element necessary for fetal hemoglobin silencing. N Engl J Med 2011; 365(9):807-14. 5. Feingold, EA, Forget BG. The breakpoint of a large deletion causing hereditary persistence of fetal hemoglobin occurs within an erythroid DNA domain remote from the β-globin gene cluster. Blood 1989; 74: 2178–2186. 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 gamma- globin gene in transgenic mice. J. Biol Chem 1995; 270: 10256-63. 8. Huisman TH, Schroeder WA, Efremov GD, et al. The present status of the heterogeneity of fetal hemoglobin in beta-thalassemia: an attempt to unify some observations in thalassemia and related conditions. Ann N Y Acad Sci 1974;232(0):107- 24. 9. Bank A, O'Neill D, Lopez R, et al. Role of intergenic human γ-δ -globin sequences in human hemoglobin switching and reactivation of fetal hemoglobin in adult erythroid cells. Ann N Y Acad Sci 2005;1054:48-54.
  • 95. 74 10. Chakalova L, Osborne CS, Dai YF, et al. The Corfu δβ thalassemia deletion disrupts γ-globin gene silencing and reveals post-transcriptional regulation of HbF expression. Blood 2005;105:2154-60. 11. Sankaran VG, Xu J, Orkin SH. Transcriptional silencing of fetal hemoglobin by BCL11A. Ann N Y Acad Sci. 2010;1202:64-8. 12. Sankaran VG, Xu J, Ragoczy T, et al. Developmental and species-divergent globin switching are driven by BCL11A. Nature 2009;460(7259):1093-7. 13. Sankaran VG, Nathan DG. Reversing the hemoglobin switch. N Engl J Med 2010; 363(23):2258-60. 14. Sankaran VG, Menne TF, Xu J, et al. Human fetal hemoglobin expression is regulated by the developmental stage-specific repressor BCL11A. Science 2008; 322(5909):1839-42. 15. Xu J, Sankaran VG, Ni M, et al. Transcriptional silencing of γ-globin by BCL11A involves long-range interactions and cooperation with SOX6. Genes Dev 2010; 24:783- 98. 16. Thein SL, Menzel S, Lathrop M, Garner C. Control of fetal hemoglobin: new insights emerging from genomics and clinical implications. Hum Mol Genet 2009;18(R2):R216- 23. 17. Galarneau G, Palmer CD, Sankaran VG, Orkin SH, Hirschhorn JN, Lettre G. Fine mapping at three loci known to affect fetal hemoglobin levels explains additional genetic variation. Nat Genet 2010;42(12):1049-51. 18. Borg J, Papadopoulos P, Georgitsi M, et al. Haploinsufficiency for the erythroid transcription factor KLF1 causes hereditary persistence of fetal hemoglobin. Nat Genet 2010;42(9):801-5. 19. Borg J, Patrinos GP, Felice AE, Philipsen S. Erythroid phenotypes associated with KLF1 mutations. Haematologica 2011; 96:635-8. 20. Zhou D, Liu K, Sun CW, Pawlik KM, Townes TM. KLF1 regulates BCL11A expression and γ- to β-globin gene switching. Nat Genet 2010; 42:742-4.
  • 96. 75 Chapter 3 Diagnostic Laboratory Methods 3.4 Flow CytometryMeasurements of Cellular Fetal Hemoglobin,Oxidative Stress and Free Iron in Hemoglobinopathies Eitan Fibach, MD 3.4.1 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
  • 97. 76 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.
  • 98. 77 FC is especially fitting for analysis of blood cells: (I) These cells which can be easily obtained by blood drawing are present as single cells, thus in contrast to cells of solid tissues, their use does not require harsh procedures for tissue disaggregation (e.g., trypsinization). (II) They are present as a mixture of various cell types, including numerous subtypes (e.g., lymphocytes), with very large (e.g., RBC) to very small (hematopoietic stem cells) representation. Cells of these sub-types can be identified and "gated" based on differences in their size (forward light scattering), granularity (side light scattering) and expression of surface antigens, and can be measured simultaneously. For measurements of various characteristics (HbF content, oxidative stress parameters and LIP content), the blood sample is stained with specific probes (as detailed below), and then with fluorescent reagents (usually antibodies) against surface markers which identify a specific subpopulation. Such markers are glycophorin A for RBC, CD61 for platelets, CD15 for neutrophils, CD19 for B-lymphocytes and CD3 for T- lymphocytes. CD45 is particularly useful since it is differentially expressed on various nucleated blood cells (Fig. 1).
  • 99. 78 Fig. 1. Flow cytometry of blood cells. A dot plot of blood cells with respect CD45 (FL3-H) and side light scatter (SSC-H). 3.4.2 Measurement of Fetal Hemoglobin-Containing Erythroid Cells Fetal hemoglobin (HbF, α2γ2) is the major hemoglobin (Hb) in the prenatal period that is largely replaced after birth by the adult Hb (HbA, α2β2) (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 RBC PMN Monocytes CD45 Lymphocytes
  • 100. 79 spectrofluorometric measurements following treatment with alkaline (to destroy non-fetal hemoglobins) and staining with benzidine (8), chromatography (ion-exchange HPLC for hemoglobins and reverse-phase HPLC for globin chains) (9), as well as immunological techniques, such as Elisa, based on antibodies against HbF (10). However, quantitative FC measurement of RBC, fluorescently stained with antibodies to HbF (as well as for the other hemoglobins), has several advantages. For example, in the differential diagnosis of Hereditary Persistence of Fetal Hemoglobin (11). This condition encompasses a heterogeneous group of disorders with marked increased levels of HbF. Based on the cellular distribution of HbF, they are characterized as pan-cellular, where all RBCs have increased levels of HbF, albeit not always uniformly so; and hetero- cellular, where nearly all the HbF is confined to a minor, distinct subpopulation of RBCs. This important distinction is most reliably ascertained by FC. Epidemiological studies have indicated that high levels of HbF improve the clinical symptoms of the underlying disease. In sickle cell anemia not only do HbF- containing cells have a lower concentration of sickle hemoglobin, but HbF inhibits polymerization of HbS directly, accounting for the lower propensity of such cells to undergo sickling (12). In β-thalassemia, elevated HbF should compensate partially for the deficiency of β-globin chains and reduce the excess of α-globin chains. Several pharmacological agents have been used to stimulate HbF production (13). Hydroxyurea (HU) is currently the drug of choice (14). When patients are monitored during HU treatment by measuring HbF in the hemolysate, an increase is usually observed after 2- 3 months (10). HU acts by a still unknown mechanism on the early erythroid precursors in the bone marrow. It takes several weeks for HbF to accumulate in the peripheral
  • 101. 80 blood to a quantity that allows differences before and after treatment to become apparent. Measuring differences in F-RBC by FC may be more sensitive, and measuring F-reticulocytes (retics) may provide early indication of treatment efficacy (15): Retics have a very short life-span (1-2 days) compared to mature RBC (120 days in normal subjects) and therefore measuring peripheral blood F-retics more closely characterizes the current status of HbF production in the bone marrow. Measuring F- retics can indicate the efficacy of the drug and/or the patient’s compliance several days after treatment initiation. Such follow up is very important since about 30% of the patients are non-responders. It is imperative that such patients be identified as early as possible and the treatment (that is not without potential risks) be discontinued and replaced by treatment with another drug (e.g., butyroids). 3.4.3 Staining Protocols for F-RBC and F-Retics (15) Heparinized blood is washed three times in phosphate buffered saline (PBS). For fixation, 50μl of the packed cells are resuspended in 10 ml of PBS containing 4% formaldehyde for 15-min at room temperature under constant agitation in polypropylene tubes. For permeabilization, the cells are centrifuged for 3 min at 1,500 g, and 2 ml methanol-acetone are added to the pellet, mixed and incubated for 1-min at room temperature. The cells are then washed three times and resuspended in PBS to a final volume of 0.5 ml (10% suspension). Anti-HbF monoclonal antibodies (the amount depends on the Manufacturer’s instructions or on a pre-performed titration) are added to 5x106 cells (5 μl of the 10% suspension) and incubated for 1-hr at 370C, after which the cells are washed in PBS. If