diagnostic hemoglobinopathieslaboratory methods and case studies

7/22/2019 Diagnostic HemoglobinopathiesLaboratory Methods and Case Studies

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Diagnostic HemoglobinopathiesLaboratory Methods and Case Studies

Zia Uddin, PhDSt. John Macomb-Oakland Hospital

Warren, Michigan

November 2013


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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

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Contributors and Reviewers

Antonio Amato, MDDirectorCentro Studi Microcitemie Di Roma

A.N.M.I. ONLUSVia Galla Placidia 28/3000159 Rome, RomeItaly

Erol Omer Atalay, MDProfessor, Medical FacultyPamukkale UniversityKinikli, DenizliTurkey

Celeste Bento, PhDLaboratorio de Anemias Congenitas e Hematologia MolecularServico de Hematologia, Hospital PediatricoCentro Hospitalar e Universitario de CoimbraPortugal

Aigars Brants, PhDScientific Affairs ManagerSebia, Inc400-1705 Corporate DriveNorCross, GA 30093

USA

Thomas E. Burgess, PhDTechnical Director, Quest DiagnosticsTucker, GeorgiaUSA

Shahina Daar, MD, PhDAssociate ProfessorDepartment of HematologySultan Qaboos University, Muscat

Sultanate of Oman

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Rita Ellerbrook, PhDTechnical Director EmeritusHelena Laboratories, USA1530 Lindberg DriveBeaumont, TX 77707

USA

Eitan Fibach, MDProfessor, Department of HematologyHadassah-Hebrew University Medical CenterEin-Kerem, JerusalemIsrael

Bernard G. Forget, MDProfessor Emeritus of Internal MedicineYale School of Medicine

New Haven, CT 06520USA

Piero C. Giordano, PhDHemoglobinopathies LaboratoryHuman and Clinical Genetics DepartmentLeiden University Medical CenterThe Netherlands

Dina N. Greene, PhDScientific Director, ChemistryRegional Laboratories, Northern CaliforniaThe Permanente Medical GroupBerkeley, CA 94710USA

Rosline Hassan, PhDProfessor of HematologySchool of Medical SciencesUniversity Sains Malaysia, KelanranMalaysia

David Hockings, PhDFormerly with Isolab, USA andPerkinElmer Corporation, USARaleigh-Durham, North CarolinaUSA

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Prasad Rao Koduri, MDDivision of Hematology-OncologyHektoen Institute of Medical ResearchChicago, Illinois 60612USA

Elaine Lyon, PhDAssociate Professor of PathologyUniversity of Utah School of MedicineMedical Director, Molecular Genetics

ARUP Laboratories, Salt Lake City, UTUSA

Bushra Moiz, PhDAssociate ProfessorDepartment of Pathology and Microbiology

The Agha Khan University Hospital, KarachiPakistan

Herbert L. Muncie, MDProfessor, Department of Family MedicineSchool of Medicine, Louisiana State University1542 Tulane AveNew Orleans, LA 70112USA

Gul M. Mustafa, PhDPost-Doctorate FellowDepartment of PathologyThe University of Texas Medical BranchGalveston, TX 77555USA

Diane M. Maennle, MDAssociate PathologistDepartment of PathologySt. John Macomb-Oakland Hospital

. Warren, MI 48093USA

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Jayson Miedema, MDPost-Doctorate FellowDepartment of Pathology and Laboratory MedicineUniversity of North CarolinaChapel Hill, North Carolina

USA

Christopher R. McCudden, PhDAssistant Professor, Department of Pathologyand Laboratory Medicine, University of OttawaOttawa, OntarioCanada

Michael A. Nardi, MSAssociate ProfessorDepartment of Pediatrics and Pathology

New York University School of MedicineNew York, NY 100016USA

John Petersen, PhDProfessor, Department of PathologyThe University of Texas Medical BranchGalveston, TX 77555USA

Joseph M. Quashnock, PhDLaboratory DirectorPerkinElmer Genetics, Inc90 Emerson Lane, Suite 1403P.O. Box 219Bridgeville, PA 15017USA

Semyon A. Risin, MD, PhDProfessor of Pathology & Laboratory MedicineDirector of Laboratory Medicine Restructuring& Strategic Planning ProgramUniversity of Texas Health Science Center-Houston Medical School6431 Fannin Street, MSB, 2.290Houston, TX 77030USA

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Maria Cristina Rosatelli, PhDProfessor, Dipartimnto di Scienze Biomedichee Biotecnologie Universit degli Studi di Cagliari09121 Cagliari, SardinaItaly

Donald L Rucknagel, MD, PhDProfessor EmeritusDepartment of Human GeneticsUniversity of Michigan, School of Medicine

Ann Arbor, MichiganUSA

Kimberly Russell, MT (ASCP), MBAManager & Operations CoordinatorHematology and Blood Bank

St. John Hospital & Medical Centerand affiliated hospitals of St. JohnProvidence Health System, MichiganUSA

Luisella Saba, PhDProfessor, Dipartimnto di Scienze Biomedichee Biotecnologie Universit degli Studi di Cagliari09121 Cagliari, SardinaItaly

Dror Sayar, MD, PhDDepartment of Pediatrics,Hematology-OncologyTel Hashmer Medical CenterRamat GanIsrael

Upendra Srinivas, MDDepartment of HematologyKokilaben Dhirubhai Ambani Hospital& Medical Research InstituteMumbai, MaharashtraIndia

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Elizabeth Sykes, MDClinical PathologistWilliam Beaumont HospitalRoyal Oak, MichiganUSA

Ali Taher, MD, PhDProfessor Medicine, Hematology & Oncology

American University of Beirut Medical CenterBeirutLebanon

Kenneth F. Tucker, MDDirector, Hematology & Oncology ServicesWebber Cancer CenterSt. John Macomb-Oakland Hospital

Warren, MichiganUSA

Zia Uddin, PhDConsultant, Clinical ChemistryDepartment of PathologySt John Macomb-Oakland HospitalWarren, MichiganUSA

Vip Viprakasit, MD, D. PhilProfessorDepartment of Paediatrics & Thalassemia CenterFaculty of Medicine Siriraj Hospital, Mahidol University2 Prannok Road, BangkoiBangkok 10700Thailand

Dr. Henri WajcmanDirector of Research EmeritusEditor-in-Chief HemoglobinINSERM U955 (Team 11)Hospital Henri Mondor94010 CreteilFrance

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Winfred Wang, MDProfessor of PediatricsUniversity of Tennessee College of MedicinePediatric Hematologist & OncologistSt Jude Childrens Research Hospital

Memphis, TennesseeUSA

Andrew N Young, MD, PhDDepartment of Pathology & Laboratory MedicineEmory University School of Medicine

Atlanta, GA 30303USA

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Financial Disclosure

I neither had nor will have financial relationshipwith any of the manufacturers or any otherorganization mentioned in the book.

Similarly all the contributors and reviewersof the book have worked with gratis to furtherthe cause of education.

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

November 2013 Zia Uddin, PhD

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Dedication

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

Illinois Institute of Technology, Chicago, Illinois

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

University of Illinois Medical Center, Chicago, Illinois

Professor Newton Ressler, PhD


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Preface

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

Internet has the potential of mass education at aninfinitesimal cost. This is the 3rd book launched via Internet

by me at no charge.

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

Love God

Love People

Serve The World


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Acknowledgement

During the past three years I contacted worldwide >200 family physicians,clinical chemists, pathologists, hematologists, public health officials and experts in

diagnostic hemoglobinopathy for formatting this book. The contribution of all of theseindividuals is heartfelt and very much appreciated.

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

Diane M. Maennle, MDRita Ellerbrook, PhDKimberly R. Russell, MT (ASCP), MBAJennifer Randazzo, MS (Information Technology)

The following manufacturers and organizations provided technical support,

and facilities for the collection of data for the book:

Helena Laboratories, USASebia, FrancePerkinElmer Corporation, USABio-Rad, USA

ARUP Laboratories, USAQuest Diagnostics, USACollege of American Pathologists, USASeven 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 LaboratoriesSt. John Macomb-Oakland Hospital, Warren, Michigan, USA

Anoop Patel, MD, Assistant Systems Medical DirectorSt John Providence Health System Laboratories, Warren, Michigan, USA

Mr. Tipton Golias, President & CEOHelena Laboratories, Beaumont, Texas, USA


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Table of Contents

Chapter 1 Hemoglobin 1Thomas E. Burgess, PhD

1.1 Hemoglobin Structure

1.2 Hemoglobin Function

1.3 Hemoglobin Synthesis

1.4 Hemoglobin Variants

Chapter 2 Diagnostic Laboratory Methods

2.1 Basic Concepts 10Jayson Miedema, MDand Christopher R. McCudden, PhD

2.1.1 Unstable Hemoglobins2.1.2 Altered Affinity Hemoglobins2.1.3 Sickle Solubility Test2.1.4 Serum Iron, TIBC, Transferrin, Ferritin

2.1.5 Soluble Transferrin Receptor2.1.6 Hepcidin

2.2 Microcytosis 21Diane Maennle, MDand Kimberly Russell, MT (ASCP), MBA

2.3 Hereditary Persistence of Fetal Hemoglobin 28Bernard G. Forget, MD

2.3.1 Introduction

2.3.2 Deletions Associated with the HPFH Phenotype2.3.3 Non-Deletion Forms of HPFH2.3.4 HPFH Unlinked to the -Globin Gene Cluster2.3.5 Conclusion2.3.6 Hemoglobin F Quantification

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2.4 Flow Cytometry Measurements of Cellular FetalHemoglobin, Oxidative Stress and Free Iron inHemoglobinopathies 41Eitan Fibach, MD

2.4.1 Flow Cytometry of Blood Cells2.4.2 Measurement of Fetal Hemoglobin-Containing

Erythroid Cells2.4.3 Staining Protocols for F-RBCs and F-Retics (15)2.4.4 F-Cell Determination for Fetal-Maternal Hemorrhage

(FMH) in Pregnant Patients wit -Thalassemia- Asingle Case and General Conclusion (16)

2.4.5 Oxidative Stress2.4.6 Staining Protocols for ROS and GSH2.4.7 Intracellular Free Iron

2.4.8 Staining Protocol for LIP

2.5 Solid Phase Electrophoretic Separation 61Rita Ellerbrook, PhD, and Zia Uddin, PhD

2.5.1 Introduction

2.5.2 Cellulose Acetate Electrophoresis (alkaline pH)

2.5.3 Agarose Gel Electrophoresis (alkaline pH)

2.5.4 Agar Electrophoresis (acid pH)

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

2.5.6 Requirements for the Identification of ComplexHemoglobinopathies

2.6 Capillary Zone Electrophoresis 73Zia Uddin, PhD

2.6.1 Introduction2.6.2 Basic Principle2.6.3 Application of CZE in Diagnostic Hemoglobinopathies2.6.4 Interpretation of CZE Results

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2.7 Isoelectric Focusing 83David Hockings, PhD

2.7.1 IEF of Normal Adult Hemoglobin: HbA (Adult),

HbF ( Fetal), HbA2

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

2.7.3 IEF of Beta-Chain Variant Hemoglobins2.7.4 IEF of Alpha Chain Variant Hemoglobins2.7.5 IEF of Thalassemias

2.8 High Performance Liquid Chromatography 95Zia Uddin, PhD

2.8.1 Introduction2.8.2 Basic Principle2.8.3 Illustrations

Chapter 3 Globin Chain Analysis

3.1 Solid Phase Electrophoretic Separation 102Zia Uddin, PhD

3.1.1 Cellulose Acetate Electrophoresis(Alkaline and Acid pH)

3.2 Reverse Phase High Performance Liquid 106ChromatographyZia Uddin, PhD, and Rita Ellerbrook, PhD

3.3 Globin Chain Gene Mutations: DNA Studies 115Joseph M. Quashnock, PhD

3.3.1 Introduction

3.3.2 Genotyping-PCR Methodology3.3.3 Mutations

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3.4 Electrospray Ionization-Mass Spectrometry 132Gul M. Mustafa, PhD and John R. Petersen, PhD

3.5 PCR and Sanger Sequencing 147Elaine Lyon, PhD

3.5.1. Alpha Globin3.5.2 Beta Globin3.5.3 Sequencing3.5.4 Reporting Sequence variants3.5.5 DNA Sequence Traces3.5.6 Conclusion

Chapter 4 Alpha and Beta Thalassemia 157Herbert L. Muncie, MD.

4.1 Epidemiology

4.2 Pathophysiology

4.3 Alpha Thalassemia

4.4 Beta Thalassemia

4.5 Diagnosis

4.6 Treatment

4.7 Complications

4.8 Other Treatment Issues4.8.1 Hypersplenism4.8.2 Endocrinopathies4.8.3 Pregnancy4.8.4 Cardiac4.8.5 Hypercoagulopathy4.8.6 Psychosocial4.8.7 Vitamin Deficiencies4.8.8 Prognosis

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Chapter 5 Neonatal Screening forHemoglobinopathies 178Zia Uddin, PhD

5.1 Introduction

5.2 Methodologies

5.3 Laboratory Reports Format & Interpretation

5.4 Examples of Neonatal Screening

5.4.1. Capillary Zone Electrophoresis5.4.2 Isoelectric focusing5.4.3 Isoelectric focusing and High Performance

Liquid Chromatography5.4.4 Isoelectric focusing, High Performance Liquid

Chromatography and DNA studies

5.5 Genetic Counseling & Screening

Chapter 6 Prenatal Diagnosis of Beta-Thalassemiaand Hemoglobinopathies 202Maria Cristina Rosatelli, PhD, and Luisella Saba, PhD

Chapter 7 Hemoglobin A1c 232Zia Uddin, PhD

7.1 Introduction

7.2 HbA1cDiagnostic Role in Diabetes Mellitus, and

Glycemic Control in Adults

7.3 Measurement of HbA1c

7.4 Factors Affecting the Accuracy of Hb A1cAssay

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Case Studies 244

Introduction

Case # 1 Normal Adult 247

Case # 2 Hemoglobin S trait 252

Case # 3 Hemoglobin S homozygous 258

Case # 4 Hemoglobin S with hereditary persistenceof fetal hemoglobin (HPFH) 264

Case # 5 Hemoglobin G-Philadelphia trait 272

Case # 6 Hemoglobin S-G Philadelphia 279

Case # 7 Hemoglobin G-Coushatta trait 287

Case # 8 Hemoglobin C trait 293

Case # 9 Hemoglobin C homozygous 299

Case # 10 Hemoglobin C with hereditary persistenceof fetal hemoglobin (HPFH) 306

Case # 11 Hemoglobin S-C disease 312

Case # 12 Hemoglobin D-Los Angeles (D-Punjab) trait 319

Case # 13 Hemoglobin S-D disease 326

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Case # 14 Hemoglobin E and Associated Disorders 333

Case # 14 a Hemoglobin E trait 339

Case # 14 b Hemoglobin E homozygous 344

Case # 14 c Hemoglobin S-E disease 350

Case # 15 Hemoglobin S-Korle Bu (G-Accra) 356

Case # 16 Hemoglobin O-Arab trait 362

Case # 17 -Thalassemia trait 368

Case # 18 Hemoglobin S-+

- thalassemia 374

Case # 19 Hemoglobin C-othalassemia 381

Case # 20 Hemoglobin Hasharon trait 387

Case # 21 Hemoglobin Zurich trait 394

Case # 22 Hemoglobin Lepore trait 400

Case # 23 Hemoglobin J-Oxford trait 408

Case # 24 Hemoglobin J-Baltimore trait 415

Case # 25 Hemoglobin Malmo trait 421

Case # 26 Hemoglobin Koln trait 432

Case # 27 Hemoglobin Q-India trait 441

Case # 28 Hemoglobin Dhofar trait 454

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1

Chapter 1

HemoglobinThomas 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 Hemoglobin1edited 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 THEprimary component of the red blood cell

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

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

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

beta and gamma chains as development occurs.


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Figure 1. Globin chains concentration changes in embryonic, fetal and post-natalstages of life (Huehns ER, Dance N, Hecht S, Motulsky AG. Human embryonichemoglobins. Cold Spring Harbor Symp Quant Biol 1969; 29: 327-331). Adoptedwith permission from Blackwell Publishing (Barbara J. Bain, HaemoglobinopathyDiagnosis, 2

ndEdition, 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,


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

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

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

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

embryonic life of the fetus.

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

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

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

various hemoglobin molecules from normal hemoglobins to the very rare and

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

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

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

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

helices separated by non-helical 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.


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Figure 2. Tertiary structure of a globin chain and the quaternary structure ofhemoglobin molecule (Adopted with permission from Blackwell Publishing, BarbaraJ. Bain, Haemoglobinopathy Diagnosis, 2

ndEdition, 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 CO2is facilitated by reversible carbamoylation (formation of carbamoyl

moiety, i.e., H2NCO-) of the N-terminal amino acids of the globin chains and can,


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via proton scavenging, keep CO2in 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 thiswhether the hemoglobin is normal or variant in

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

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

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

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

clinical issues in the homozygous state (i.e., disease), the pheno typic analysis and

subsequent interpretation becomes ultimately important to the patient.


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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.


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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 commonthey all involve the

hemoglobin molecule and its functionality. Whether alpha, beta, gamma, delta,

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

concentration but rather on its functionality. The amino acid variation (e.g., glutamic

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

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

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

hemoglobins (A, F, A2). Most variants therefore will have altered electrophoretic or

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

hemoglobin Chicago, are not separable by normal electrophoretic techniques and

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

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

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

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

clinical picture dramatically changes with significant physiological changes being

directly associated with the homozygous state. This therefore requires the

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


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

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

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

hemoglobin variant in the patients specimen. For example, an elevation of

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

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

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

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

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

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

patients bloodstream and therefore are important in the assignment of a benign or

pathological assessment of the variant hemoglobin.

The variants described in the following chapters all obey the aforementioned

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

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

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

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

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

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

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

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

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

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


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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, BlackwellPublishing, 2006.

3. Bain, Barbara J.. in Hemoglobinopathy Diagnosis, 2ndEd., pg. 1, Blackwell

Publishing, 2006.


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Chapter 2Diagnostic Laboratory Methods

2.1 Basic Concepts

JaysonMiedema, MD, and Christopher R. McCudden, PhD

2.1.1 Unstable Hemoglobins

Unstable hemoglobins are characterized by disorders in globin production which

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

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

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

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

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

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

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

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

preterm lethality.

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

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

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

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

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

to cause exacerbation. Parvovirus can also induce aplastic crisis andHbA2and HbF

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

basophilic stippling, polychromasia and, hypochromasia. Since not all unstable


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

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

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

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

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

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

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

(Hb4C-Hb50C)/(Hb4C)x100

Where Hb4C is the hemoglobin concentration at 4 degrees centigrade and Hb50C is

the concentration of hemoglobin at 50 degrees centigrade.

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

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

information on hemoglobinopathies associated with unstable hemoglobin can be

obtained from:

http://medtextfree.wordpress.com/2011/12/30/chapter-48-hemoglobinopathies

2.1.2 Altered Affinity Hemoglobins

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

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

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

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

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

Chesapeake, and beta-chain variants, e.g. Hb Olser, Hiroshima, Andrew-Minneapolis,
http://medtextfree.wordpress.com/2011/12/30/chapter-48-hemoglobinopathieshttp://medtextfree.wordpress.com/2011/12/30/chapter-48-hemoglobinopathieshttp://medtextfree.wordpress.com/2011/12/30/chapter-48-hemoglobinopathies


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

probably clinically insignificant but when significant most commonly present

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

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

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

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

can lead to clinical cyanosis.

Testing for altered affinity hemoglobins relies on subsequent changes to the

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

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

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

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

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

measured by dual wavelength spectrophotometer. Abnormal oxygen dissociation curves

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

factors as pH, temperature, pCO2, and 2,3-diphosphoglycerate (2,3-DPG).

Measurement of pO2, pCO2, pH and SO2 allows for an estimation of p50 to be

calculated.

2.1.3 Sickle Solubility Testing

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

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

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


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

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

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

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

standpoint.

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

commonly tested via hemoglobin electrophoresis when necessary. However, another

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

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

th

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

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

tend toward sickling, polymerization, and precipitation. The addition of sodium

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

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

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

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

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

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

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

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

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

hemoglobin concentration (


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

used as results can be somewhat subjective

2.1.4 Serum Iron, TIBC, Transferrin, and Ferritin

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

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

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

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

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

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

iron or as heme. Free iron, in the form of Fe3+, requires reduction to Fe2+by enzymes

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

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

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

ferric (Fe3+) form. Because of the toxicity of ferric iron, it is transported in the circulation

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

takes up iron through receptor-mediated endocytosis. As dietary absorption accounts

for


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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

hemochromatosis and thalassemias. Alternatively, TIBC may be calculated based on


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

bound to each molecule of transferrin). TIBC is calculated from measured transferrin

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

concentration of transferrin may be calculated from measured TIBC as follows:

Transferrin (mg/dL) = 0.7 TIBC (g/dL). TIBC is increased in iron deficiency and

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

decreased in thalassemia).

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

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

saturation = serum Fe (g/dL) / TIBC (g/dL) 100. The percent saturation is usually

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

overload, the % saturation increases dramatically. Saturation is moderately increased

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

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

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

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

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

10 mg total storage iron). In the blood, ferritin is generally poor in iron content and is

referred to as apoferritin. Circulating ferritin (or apoferritin) is measured using specific

antibodies, commonly by chemiluminescent immunoassay. Serum or plasma ferritin

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

with iron overload and decreased in iron deficiency. Serum ferritin levels change prior

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


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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.


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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

2.1.5 Soluble Transferrin Receptor

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

receptor (sTfR). The sTfR consists of the N-terminus of the membrane receptor that

can be measured in circulation. Circulating levels reflect the activity of the erythroid

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

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

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

inflammation because sTfR levels are not affected by inflammatory cytokines. In

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

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

currently standardized.


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2.1.6 Hepcidin

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

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

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

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

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

of circulating levels in patients with inflammation more challenging. At the time of

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

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

H, Fleming RE, Swinkels DW. Hepcidin in Human Iron Disorders: Diagnostic

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

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

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

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

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

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

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


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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. 5thEdition.

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


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2.2 Microcytosis

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

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

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

formula employing the values of hematocrit and RBC count:

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

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

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

Table I Age Dependent Mean Hemoglobin and MCV 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


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Iron deficiency anemia, -thalassemia trait, and -thalassemia trait are the most commo

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

(Table II).1,3,5,6

In addition to decreased MCV, the patients with -thalassemia trait usually have

increased hemoglobin A2. It is pointed out that lower hemoglobin A2is also observed in patients

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

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

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

exhibit a normal range for MCV.7

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

Children and adolescents Menstruating women 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 examinatio

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

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


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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 Increaseddistribution width increased(RDW)

Serum iron Decreased Normal to Normal to Normal toincreased decreased increased

Total iron- Increased Normal Slightly increased Normalbinding capacity

Transferrin Decreased Normal to Normal to slightly Normal tosaturation increased decreased increased

Van Vranken3has recently suggested a protocol for diagnosing the cause of microcytos

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

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

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

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


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

Ordinary hemoglobin electrophoresis (cellulose acetate or agarose gel

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

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

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

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

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

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

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

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


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

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

level may be normal in these clinical conditions.

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

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

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

rises.

Severe anemia in childhood is usually due to the lack of iron in food, since cows

milk does not contain iron.

A nave reader is advised to also review the Full Colorpdfof 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]. PediatrRev. 2007; 28(1): 5-14.

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

3. Van Vranken ML. Evaluation of Microcytosis. Am Fam Physician. 2010; 80(9): 1117-1124. 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 anem

in persons 65 years and older in the United States: evidence for a high rate ofunexplained anemia. Blood. 2004; 104(8): 2263-2268.

7. Bain BJ. Hemoglobinopathy Diagnosis. 2nded. Malden, Mass.: BlackwellPublishing; 2006: 94-106.
http://www.bpac.org.nz/http://www.bpac.org.nz/http://www.bpac.org.nz/http://www.bpac.org.nz/


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

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


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2.3 Hereditary Persistence of Fetal Hemoglobin

Bernard G. Forget, MD

2.3.1 Introduction

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

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

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

compound heterozygotes for HPFH and other hemoglobinopathies. Increased levels of

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

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

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

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

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

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

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


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Figure 1. Deletions of the -globin gene cluster associated with fusion proteins andHPFH. The circle 3 to the -globin gene indicates the 3 -globin gene enhancer. Thefilled 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 (adoptedfrom reference 2). The references for the individual mutations are cited in references 1,3 and 6.

HPFH is frequently contrasted with thalassemia, which is another genetic

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

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

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

defy classification as one syndrome or the other. The following is a working definition

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

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

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

heterozygotes, is heterogeneously distributed among red cells. In contrast, HPFH

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

is usually no associated phenotype of hypochromia and microcytosis. In addition, the

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

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

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

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

and Hb A2, indicating absence of - and -globin gene expression in cis to the

thalassemia and HPFH determinants. Although the apparent striking qualitative

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


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

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

appear that the increased amount of Hb F in HPFH is caused by a genetically

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

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

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

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

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

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

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

2.3.2 Deletions Associated with the HPFH Phenotype.

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

between theA to the gene. Unlike Hb Lepore, that is associated with a -

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

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

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

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

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

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

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

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

in enhanced activity of these promoters.

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

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

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

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

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

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

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

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

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


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HPFH 3' deletionbreakpoints, that become juxtaposed to the genes as a result of thedeletion events, may influence -gene expression, in a manner analogous to the

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

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

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

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

expression of the neighboring -globin genes.

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

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

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

of the -thalassemia deletions, are located in the DNA between the and genes,

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

have extensively characterized a negative regulatory transcription factor, called

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

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

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

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

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

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

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

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

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

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

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

characterized two important deletion mutants with nearly identical distal breakpoints but

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

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

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

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

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

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


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

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

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

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

2.3.3 Nondeletion Forms of HPFH

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

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

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

pancellular Hb F expression. However, less well characterized nondeletion forms ofGA HPFH have been described that are associated with relatively low levels of

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

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

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

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

these disorders.


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

type, and theG

gene in cis to the -175A

mutation carried the -158 C T change.The references for the individual mutations are cited in references 1 and 3.

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

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

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

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

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


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

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

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

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

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

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

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

at least one other ubiquitous DNA binding protein.

The second region containing a mutation associated with nondeletion HPFH is

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

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 -gene promoters is flanked on either side by a consensus

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

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

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

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

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

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

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

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


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

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

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

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

deletions. The CCAAT box region is known to be the binding site of a number of 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 -gene promoters and thereby

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

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

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

mutation or the other.

2.3.4 HPFH Unlinked to the -Globin Gene Cluster

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

expression of the gene (20).

2.3.5 Conclusion

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

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

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

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

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

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

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

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

cellular therapies for globin gene disorders.


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2.3.6 Hemoglobin F Quantification

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

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

c) HPLC, and d) capillary zone electrophoresis.

References

1. Bollekens JA, Forget BG. Delta beta thalassemia and hereditary persistence of fetalhemoglobin. 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 fetalhemoglobin silencing. N Engl J Med 2011; 365(9):807-14.5. Feingold, EA, Forget BG. The breakpoint of a large deletion causing hereditarypersistence of fetal hemoglobin occurs within an erythroid DNA domain remote from the

-globin gene cluster. Blood 1989; 74: 21782186.6. Kosteas T, Palena A, Anagnou NP.Molecular cloning of the breakpoints of thehereditary persistence of fetal hemoglobin type-6 (HPFH-6) deletion and sequenceanalysis 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 thebreakpoint of the hereditary persistence of fetal hemoglobin-3 deletion exhibit enhanceractivity 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 theheterogeneity 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 - -globin sequences inhuman hemoglobin switching and reactivation of fetal hemoglobin in adult erythroidcells.Ann N Y Acad Sci 2005;1054:48-54.
http://www.ncbi.nlm.nih.gov/pubmed/9272169http://www.ncbi.nlm.nih.gov/pubmed/9272169http://www.ncbi.nlm.nih.gov/pubmed/9272169http://www.ncbi.nlm.nih.gov/pubmed/9272169http://www.ncbi.nlm.nih.gov/pubmed/9272169http://www.ncbi.nlm.nih.gov/pubmed/16339651http://www.ncbi.nlm.nih.gov/pubmed/16339651http://www.ncbi.nlm.nih.gov/pubmed/16339651http://www.ncbi.nlm.nih.gov/pubmed/16339651http://www.ncbi.nlm.nih.gov/pubmed/16339651http://www.ncbi.nlm.nih.gov/pubmed/16339651http://www.ncbi.nlm.nih.gov/pubmed/16339651http://www.ncbi.nlm.nih.gov/pubmed/16339651http://www.ncbi.nlm.nih.gov/pubmed/16339651http://www.ncbi.nlm.nih.gov/pubmed/16339651http://www.ncbi.nlm.nih.gov/pubmed/9272169http://www.ncbi.nlm.nih.gov/pubmed/9272169http://www.ncbi.nlm.nih.gov/pubmed/9272169


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10. Chakalova L, Osborne CS, Dai YF, et al. The Corfu thalassemia deletion disrupts-globin gene silencing and reveals post-transcriptional regulation of HbF expression.Blood 2005;105:2154-60.11. Sankaran VG, Xu J, Orkin SH.Transcriptional silencing of fetal hemoglobin byBCL11A.Ann N Y Acad Sci. 2010;1202:64-8.

12. Sankaran VG, Xu J, Ragoczy T, et al. Developmental and species-divergent globinswitching 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 isregulated by the developmental stage-specific repressor BCL11A. Science 2008;322(5909):1839-42.15. Xu J, Sankaran VG, Ni M, et al. Transcriptional silencing of -globin by BCL11Ainvolves 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. Finemapping at three loci known to affect fetal hemoglobin levels explains additional geneticvariation. Nat Genet 2010;42(12):1049-51.18. Borg J, Papadopoulos P, Georgitsi M, et al. Haploinsufficiency for the erythroidtranscription factor KLF1 causes hereditary persistence of fetal hemoglobin. Nat Genet2010;42(9):801-5.19. Borg J, Patrinos GP, Felice AE, Philipsen S. Erythroid phenotypes associated withKLF1 mutations. Haematologica 2011; 96:635-8.20. Zhou D, Liu K, Sun CW, Pawlik KM, Townes TM. KLF1 regulates BCL11Aexpression and - to -globin gene switching. Nat Genet 2010; 42:742-4.
http://www.ncbi.nlm.nih.gov/pubmed/20712774http://www.ncbi.nlm.nih.gov/pubmed/20712774http://www.ncbi.nlm.nih.gov/pubmed/20712774http://www.ncbi.nlm.nih.gov/pubmed/20712774


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2.4 Flow Cytometry Measurements of Cellular Fetal Hemoglobin, OxidativeStressand Free Iron in HemoglobinopathiesEitan Fibach, MD

2.4.1 Flow Cytometry of Blood Cells

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

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

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

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

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

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

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

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

the oxidative state.

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

directed against internal (following fixation and premeabilization of the

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

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

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

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

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

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


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

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

fluorescence emission. Depending on the number of laser sources and

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

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

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

respectively, and fluorescence light emission by the fluorochromes correlates

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

below).

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

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

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

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 x105cells) 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 perm

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

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

molar concentrations, they are useful for comparative purposes.


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

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

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

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

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

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

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

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

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

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

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

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

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

blood cells (Fig. 1).


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

2.4.2 Measurement of Fetal Hemoglobin-Containing Erythroid Cells

Fetal hemoglobin (HbF, 22) is the major hemoglobin (Hb) in the prenatal period

that is largely replaced after birth by the adult Hb (HbA, 22) (5). In adults, less than 1%

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

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

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

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

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

RBC

PMN

Monocytes

CD45

Lymphocytes


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

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

hemoglobins

diagnostic hemoglobinopathieslaboratory methods and case studies

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