Diagnostic Hemoglobinopathies
Laboratory Methods and Case Studies

Zia Uddin, PhD
St. John Macomb-Oakland Hospital
Warren,...
Editorial Board
Diane M. Maennle, MD

Chairperson

Kenneth F. Tucker, MD

Member

Rita Ellerbrook, PhD

Member

Piero C. G...
Contributors and Reviewers
Antonio Amato, MD
Director
Centro Studi Microcitemie Di Roma
A.N.M.I. ONLUS
Via Galla Placidia ...
Rita Ellerbrook, PhD
Technical Director Emeritus
Helena Laboratories, USA
1530 Lindberg Drive
Beaumont, TX 77707
USA
Eitan...
Prasad Rao Koduri, MD
Division of Hematology-Oncology
Hektoen Institute of Medical Research
Chicago, Illinois 60612
USA
El...
Jayson Miedema, MD
Post-Doctorate Fellow
Department of Pathology and Laboratory Medicine
University of North Carolina
Chap...
Maria Cristina Rosatelli, PhD
Professor, Dipartimnto di Scienze Biomediche
e Biotecnologie Universit degli Studi di Caglia...
Elizabeth Sykes, MD
Clinical Pathologist
William Beaumont Hospital
Royal Oak, Michigan
USA
Ali Taher, MD, PhD
Professor Me...
Winfred Wang, MD
Professor of Pediatrics
University of Tennessee College of Medicine
Pediatric Hematologist & Oncologist
S...
Financial Disclosure
I neither had nor will have financial relationship
with any of the manufacturers or any other
organiz...
Dedication
This book is dedicated with heartfelt thanks to my
professors responsible for my PhD level education in
Chemist...
Preface
Higher level education is one of the blessings of God.
Unfortunately, primarily due to economic and logistic reaso...
Acknowledgement
During the past three years I contacted worldwide >200 family physicians,
clinical chemists, pathologists,...
Table of Contents

Chapter 1

Hemoglobin

1

Thomas E. Burgess, PhD
1.1
1.2

Hemoglobin Function

1.3

Hemoglobin Synthesi...
2.4

Flow Cytometry Measurements of Cellular Fetal
Hemoglobin, Oxidative Stress and Free Iron in
Hemoglobinopathies 41
Eit...
2.7

Isoelectric Focusing

83

David Hockings, PhD
2.7.1 IEF of Normal Adult Hemoglobin: HbA (Adult),
HbF ( Fetal), HbA2
2...
3.4

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

3.5

PCR and Sanger Sequenci...
Chapter 5

Neonatal Screening for
Hemoglobinopathies 178
Zia Uddin, PhD
5.1 Introduction
5.2 Methodologies
5.3 Laboratory ...
Case Studies

244

Introduction
Case # 1

Normal Adult

247

Case # 2

Hemoglobin S trait

Case # 3

Hemoglobin S homozygo...
Case # 14

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

339

Case # 14 b Hemoglobin E homozygous
...
Chapter 1
Hemoglobin
Thomas E. Burgess, PhD
To attempt a full treatise on hemoglobin in this textbook would be an effort i...
Figure 1. Globin chains concentration changes in embryonic, fetal and post-natal
stages of life (Huehns ER, Dance N, Hecht...
provides a hydrophobic environment in which the heme molecules reside. This
pocket protects the heme from oxidation and fa...
Figure 2. Tertiary structure of a β globin chain and the quaternary structure of
hemoglobin molecule (Adopted with permiss...
3

via proton scavenging, keep CO2 in the soluble bicarbonate form . Nitrous oxide is
handled in two different ways by hem...
1.3 Hemoglobin Synthesis
The synthesis of hemoglobin, as mentioned before, is under the control of
gene loci on two chromo...
As mentioned before, the true reason for identifying the abnormal hemoglobin
or hemoglobins in patients is to identify any...
during the interpretation of the hemoglobinopathy. This data includes, but is not
limited to, pregnancy, transfusion histo...
may appear as a problem, the SG phenotype is no more of a clinical issue than a
simple AS trait. Without the exact identif...
Chapter 2
Diagnostic Laboratory Methods
2.1

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

2.1.1 Un...
hemoglobins will give abnormal results on HPLC or electrophoresis and/or these results
can be somewhat non-specific, more ...
etc., are known in the literature for altered affinity for oxygen. Many of these are
probably clinically insignificant but...
thrived in the gene pool because of its protective effects against malaria, it affects a
large number of people of African...
monoclonal proteins (dysproteinemia). Both positive and negative controls should be
used as results can be somewhat subjec...
premenopausal women. Accordingly, body stores depend on controlling iron uptake in
the GI tract and recycling.
Disorders o...
the stoichiometric relationship between transferrin and iron (2 molecules of iron are
bound to each molecule of transferri...
useful diagnostic marker of iron homeostasis. While considered the most useful of the
currently available tests for non-in...
Table 1. Iron Tests in Different Disorders
Disorder

Serum

TIBC

%

Iron
Chronic

Anemia

Transferrin

Ferritin

Saturati...
2.1.6 Hepcidin
Discovered in 2000, hepcidin is a hormone involved in iron homeostasis.
Hepcidin is produced by the liver a...
Rees DC, Williams TN, Gladwin MT. Sickle-cell disease. The Lancet. 2010;376:20182031.
Steinberg MH. Genetic disorders of h...
Chapter 2
Diagnostic Laboratory Methods
2.2 Microcytosis
Diane Maennle, MD, and Kimberly Russell, MT (ASCP), MBA

Smaller-...
Iron deficiency anemia, α-thalassemia trait, and β-thalassemia trait are the most common
causes of microcytosis. However, ...
Table III Laboratory Tests in the Differential Diagnosis of Microcytosis
Suggested diagnosis
Test

Iron deficiency anemia ...
24
Clinical observations of Kenneth F. Tucker, MD, FACP, a practicing hematologist for the
last forty years:
Ordinary hemoglo...
seen in refractory anemias, all types of chronic inflammatory conditions, etc. Since this test
is an acute phase reactant ...
8. Hematologic diseases. In: Wallach J. Interpretation of Diagnostic Tests. 8 th ed. Boston,
Mass.: Little Brown and Compa...
Chapter 2
Diagnostic Laboratory Methods
2.3

Hereditary Persistence of Fetal Hemoglobin
Bernard G. Forget, MD

2.3.1 Intro...
Figure 1. Deletions of the β-globin gene cluster associated with fusion proteins and
HPFH. The circle 3’ to the β-globin g...
due in great part to the quantitative differences in the amount of Hb F per cell and the
sensitivity of the methods used t...
β-gene cluster. However, whereas the Lepore crossover occurred between the δ- and
β-globin genes, the Kenya gene resulted ...
HPFH 3' deletion breakpoints, that become juxtaposed to the γ genes as a result of the
deletion events, may influence γ-ge...
erythroid cells (10). This region of DNA also appears to serve as a boundary region
between fetal and adult domains of the...
in their ChIP experiments. These findings provide very strong evidence for a γ-gene
silencer element in the β-gene cluster...
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...
the "cap site" or site of transcription initiation of the γ genes (at least five different point
mutations involving singl...
change disrupts a pentanucleotide sequence, YYTTGA (Y = pyrimidine), that is highly
conserved immediately upstream of the ...
above on Nondeletion HPFH). These loci are located on chromosome 2 and 6 (16, 17).
The locus on chromosome 2 corresponds t...
2.3.6 Hemoglobin F Quantification
Hb F can be quantified by several methods, and the most commonly used
procedures in a cl...
10. Chakalova L, Osborne CS, Dai YF, et al. The Corfu δβ thalassemia deletion disrupts
γ-globin gene silencing and reveals...
Chapter 2
Diagnostic Laboratory Methods
2.4 Flow Cytometry Measurements of Cellular Fetal Hemoglobin, Oxidative
Stress and...
hydro-dynamically focused in a narrow sheath of physiological solution before
being intercepted by one or more laser beams...
FC is especially fitting for analysis of blood cells: (I) These cells which can be easily
obtained by blood drawing are pr...
PMN

RBC

Monocytes
Lymphocytes
CD45

Fig. 1. Flow cytometry of blood cells. A dot plot of blood cells with respect CD45 (...
spectrofluorometric measurements following treatment with alkaline (to destroy non-fetal
hemoglobins) and staining with be...
blood to a quantity that allows differences before and after treatment to become
apparent. Measuring differences in F-RBC ...
the antibodies are fluorochrome-conjugated, the cells are resuspended in PBS and
analyzed directly. In the case of unconju...
Fig. 2. Flow cytometry analysis of F-RBC and F-Retics. Blood cells stained with thiazol-orange
(T.O) and anti-HbF. A. Forw...
Recently, in order to increase the sensitivity, reproducibility and accuracy of the assay,
another marker was introduced –...
generated in most cells mainly during energy production. Although important for various
aspects of normal physiology (e.g....
right away, but form the soluble tetramers γ4 (Hb Bart’s) and later the β4 (HbH), which
are less stable than HbA and have ...
and t-butylhydroxyperoxide and with the catalase inhibitor sodium azide, while treatment
with ROS scavengers such as N-ace...
2.4.6 Staining Protocols for ROS and GSH
ROS Assay – Blood cells are incubated with 2'-7'-dichlorofluorescin diacetate, di...
Fig. 3. Flow cytometry of ROS and GSH in normal and thalassemic RBC. Blood cells derived
from a normal donor (A,C) and a t...
hemin) that are released during hemolysis can add to the iron load and further
aggravate the hemolysis.
Normally, iron is ...
provided a significant advance in monitoring iron overload, although, similarly to serum
ferritin, substantial changes in ...
Fig. 4. Flow cytometry of labile iron pool (LIP) in RBC. Blood cells were loaded with calcein, then
washed and treated wit...
5.
6.
7.
8.
9.
10.

11.

12.
13.
14.
15.
16.
17.
18.

19.
20.

21.
22.
23.
24.

Peterson KR. Hemoglobin switching: new ins...
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Diagnostic Hemoglobinopathies‏

  1. 1. Diagnostic Hemoglobinopathies Laboratory Methods and Case Studies Zia Uddin, PhD St. John Macomb-Oakland Hospital Warren, Michigan November 2013
  2. 2. 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
  3. 3. 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
  4. 4. Rita Ellerbrook, PhD Technical Director Emeritus Helena Laboratories, USA 1530 Lindberg Drive Beaumont, TX 77707 USA Eitan Fibach, MD Professor, Department of Hematology Hadassah-Hebrew University Medical Center Ein-Kerem, Jerusalem Israel Bernard G. Forget, MD Professor Emeritus of Internal Medicine Yale School of Medicine New Haven, CT 06520 USA Piero C. Giordano, PhD Hemoglobinopathies Laboratory Human and Clinical Genetics Department Leiden University Medical Center The Netherlands Dina N. Greene, PhD Scientific Director, Chemistry Regional Laboratories, Northern California The Permanente Medical Group Berkeley, CA 94710 USA Rosline Hassan, PhD Professor of Hematology School of Medical Sciences University Sains Malaysia, Kelanran Malaysia David Hockings, PhD Formerly with Isolab, USA and PerkinElmer Corporation, USA Raleigh-Durham, North Carolina USA III
  5. 5. Prasad Rao Koduri, MD Division of Hematology-Oncology Hektoen Institute of Medical Research Chicago, Illinois 60612 USA Elaine Lyon, PhD Associate Professor of Pathology University of Utah School of Medicine Medical Director, Molecular Genetics ARUP Laboratories, Salt Lake City, UT USA Bushra Moiz, PhD Associate Professor Department of Pathology and Microbiology The Agha Khan University Hospital, Karachi Pakistan Herbert L. Muncie, MD Professor, Department of Family Medicine School of Medicine, Louisiana State University 1542 Tulane Ave New Orleans, LA 70112 USA Gul M. Mustafa, PhD Post-Doctorate Fellow Department of Pathology The University of Texas Medical Branch Galveston, TX 77555 USA . Diane M. Maennle, MD Associate Pathologist Department of Pathology St. John Macomb-Oakland Hospital Warren, MI 48093 USA IV
  6. 6. Jayson Miedema, MD Post-Doctorate Fellow Department of Pathology and Laboratory Medicine University of North Carolina Chapel Hill, North Carolina USA Christopher R. McCudden, PhD Assistant Professor, Department of Pathology and Laboratory Medicine, University of Ottawa Ottawa, Ontario Canada Michael A. Nardi, MS Associate Professor Department of Pediatrics and Pathology New York University School of Medicine New York, NY 100016 USA John Petersen, PhD Professor, Department of Pathology The University of Texas Medical Branch Galveston, TX 77555 USA Joseph M. Quashnock, PhD Laboratory Director PerkinElmer Genetics, Inc 90 Emerson Lane, Suite 1403 P.O. Box 219 Bridgeville, PA 15017 USA Semyon A. Risin, MD, PhD Professor of Pathology & Laboratory Medicine Director of Laboratory Medicine Restructuring & Strategic Planning Program University of Texas Health Science CenterHouston Medical School 6431 Fannin Street, MSB, 2.290 Houston, TX 77030 USA V
  7. 7. Maria Cristina Rosatelli, PhD Professor, Dipartimnto di Scienze Biomediche e Biotecnologie Universit degli Studi di Cagliari 09121 Cagliari, Sardina Italy Donald L Rucknagel, MD, PhD Professor Emeritus Department of Human Genetics University of Michigan, School of Medicine Ann Arbor, Michigan USA Kimberly Russell, MT (ASCP), MBA Manager & Operations Coordinator Hematology and Blood Bank St. John Hospital & Medical Center and affiliated hospitals of St. John Providence Health System, Michigan USA Luisella Saba, PhD Professor, Dipartimnto di Scienze Biomediche e Biotecnologie Universit degli Studi di Cagliari 09121 Cagliari, Sardina Italy Dror Sayar, MD, PhD Department of Pediatrics, Hematology-Oncology Tel Hashmer Medical Center Ramat Gan Israel Upendra Srinivas, MD Department of Hematology Kokilaben Dhirubhai Ambani Hospital & Medical Research Institute Mumbai, Maharashtra India VI
  8. 8. Elizabeth Sykes, MD Clinical Pathologist William Beaumont Hospital Royal Oak, Michigan USA Ali Taher, MD, PhD Professor Medicine, Hematology & Oncology American University of Beirut Medical Center Beirut Lebanon Kenneth F. Tucker, MD Director, Hematology & Oncology Services Webber Cancer Center St. John Macomb-Oakland Hospital Warren, Michigan USA Zia Uddin, PhD Consultant, Clinical Chemistry Department of Pathology St John Macomb-Oakland Hospital Warren, Michigan USA Vip Viprakasit, MD, D. Phil Professor Department of Paediatrics & Thalassemia Center Faculty of Medicine Siriraj Hospital, Mahidol University 2 Prannok Road, Bangkoi Bangkok 10700 Thailand Dr. Henri Wajcman Director of Research Emeritus Editor-in-Chief Hemoglobin INSERM U955 (Team 11) Hospital Henri Mondor 94010 Creteil France VII
  9. 9. 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
  10. 10. Financial Disclosure I neither had nor will have financial relationship with any of the manufacturers or any other organization mentioned in the book. Similarly all the contributors and reviewers of the book have worked with gratis to further the cause of education. This book and its translations into several languages are provided at no charge. November 2013 Zia Uddin, PhD IX
  11. 11. Dedication This book is dedicated with heartfelt thanks to my professors responsible for my PhD level education in Chemistry at the Illinois Institute of Technology, Chicago, Illinois, and post-doctoral education and training in Clinical Chemistry at the University of Illinois Medical Center, Chicago, Illinois. Illinois Institute of Technology, Chicago, Illinois Professor Kenneth D. Kopple, PhD Professor Paul E. Fanta, PhD Professor Robert Filler, PhD Professor Sidney I. Miller, PhD University of Illinois Medical Center, Chicago, Illinois Professor Newton Ressler, PhD November 2013 Zia Uddin, PhD X
  12. 12. Preface Higher level education is one of the blessings of God. Unfortunately, primarily due to economic and logistic reasons a vast majority of the qualified candidates are denied this opportunity. Internet has the potential of mass education at an infinitesimal cost. This is the 3rd book launched via Internet by me at no charge. All the MD/PhD degree holders are most respectfully requested to utilize the Internet as a means of communication to launch books at no charge in their areas of expertise. Love God Love People Serve The World November 2013 Zia Uddin, PhD XI
  13. 13. Acknowledgement During the past three years I contacted worldwide >200 family physicians, clinical chemists, pathologists, hematologists, public health officials and experts in diagnostic hemoglobinopathy for formatting this book. The contribution of all of these individuals is heartfelt and very much appreciated. I am highly indebted to the following persons for their technical support: Diane M. Maennle, MD Rita Ellerbrook, PhD Kimberly R. Russell, MT (ASCP), MBA Jennifer Randazzo, MS (Information Technology) The following manufacturers and organizations provided technical support, and facilities for the collection of data for the book: Helena Laboratories, USA Sebia, France PerkinElmer Corporation, USA Bio-Rad, USA ARUP Laboratories, USA Quest Diagnostics, USA College of American Pathologists, USA Seven Universities and four Newborn Screening Laboratories, USA (names are with held as per their request) Mr. Mathew Garrin, Biomedical Communications and Graphic Arts Department, Wayne State University, School of Medicine, Detroit has worked on the figures, scans, and layout of the book. I am very grateful to him for his contribution. Finally, I would like to thank the following persons for facilitating my work: Adrian J. Christie, MD, Medical Director of Laboratories St. John Macomb-Oakland Hospital, Warren, Michigan, USA Anoop Patel, MD, Assistant Systems Medical Director St John Providence Health System Laboratories, Warren, Michigan, USA Mr. Tipton Golias, President & CEO Helena Laboratories, Beaumont, Texas, USA November 2013 Zia Uddin, PhD XII
  14. 14. Table of Contents Chapter 1 Hemoglobin 1 Thomas E. Burgess, PhD 1.1 1.2 Hemoglobin Function 1.3 Hemoglobin Synthesis 1.4 Chapter 2 Hemoglobin Structure Hemoglobin Variants Diagnostic Laboratory Methods 2.1 Basic Concepts 10 Jayson Miedema, MD and Christopher R. McCudden, PhD 2.1.1 2.1.2 2.1.3 2.1.4 2.1.5 2.1.6 2.2 Unstable Hemoglobins Altered Affinity Hemoglobins Sickle Solubility Test Serum Iron, TIBC, Transferrin, Ferritin Soluble Transferrin Receptor Hepcidin Microcytosis 21 Diane Maennle, MD and Kimberly Russell, MT (ASCP), MBA 2.3 Hereditary Persistence of Fetal Hemoglobin Bernard G. Forget, MD 2.3.1 2.3.2 2.3.3 2.3.4 2.3.5 2.3.6 Introduction Deletions Associated with the HPFH Phenotype Non-Deletion Forms of HPFH HPFH Unlinked to the β-Globin Gene Cluster Conclusion Hemoglobin F Quantification XIII 28
  15. 15. 2.4 Flow Cytometry Measurements of Cellular Fetal Hemoglobin, Oxidative Stress and Free Iron in Hemoglobinopathies 41 Eitan Fibach, MD 2.4.1 Flow Cytometry of Blood Cells 2.4.2 Measurement of Fetal Hemoglobin-Containing Erythroid Cells 2.4.3 Staining Protocols for F-RBCs and F-Retics (15) 2.4.4 F-Cell Determination for Fetal-Maternal Hemorrhage (FMH) in Pregnant Patients wit β-Thalassemia- A single Case and General Conclusion (16) 2.4.5 Oxidative Stress 2.4.6 Staining Protocols for ROS and GSH 2.4.7 Intracellular Free Iron 2.4.8 Staining Protocol for LIP 2.5 Solid Phase Electrophoretic Separation 61 Rita Ellerbrook, PhD, and Zia Uddin, PhD 2.5.1 Introduction 2.5.2 Cellulose Acetate Electrophoresis (alkaline pH) 2.5.3 Agarose Gel Electrophoresis (alkaline pH) 2.5.4 Agar Electrophoresis (acid pH) 2.5.5 Interpretation of Hemoglobin Agarose Gel (pH 8.6) and Agar Gel (pH 6.2) Electrophoresis 2.5.6 Requirements for the Identification of Complex Hemoglobinopathies 2.6 Capillary Zone Electrophoresis 73 Zia Uddin, PhD 2.6.1 2.6.2 2.6.3 2.6.4 Introduction Basic Principle Application of CZE in Diagnostic Hemoglobinopathies Interpretation of CZE Results XIV
  16. 16. 2.7 Isoelectric Focusing 83 David 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 Hemoglobins 2.7.4 IEF of Alpha Chain Variant Hemoglobins 2.7.5 IEF of Thalassemias 2.8 High Performance Liquid Chromatography 95 Zia Uddin, PhD 2.8.1 Introduction 2.8.2 Basic Principle 2.8.3 Illustrations Chapter 3 Globin Chain Analysis 3.1 Solid Phase Electrophoretic Separation 102 Zia Uddin, PhD 3.1.1 Cellulose Acetate Electrophoresis (Alkaline and Acid pH) 3.2 Reverse Phase High Performance Liquid Chromatography 106 Zia Uddin, PhD, and Rita Ellerbrook, PhD 3.3 Globin Chain Gene Mutations: DNA Studies Joseph M. Quashnock, PhD 3.3.1 Introduction 3.3.2 Genotyping-PCR Methodology 3.3.3 Mutations XV 115
  17. 17. 3.4 Electrospray Ionization-Mass Spectrometry Gul M. Mustafa, PhD and John R. Petersen, PhD 3.5 PCR and Sanger Sequencing 147 Elaine Lyon, PhD 3.5.1. Alpha Globin 3.5.2 Beta Globin 3.5.3 Sequencing 3.5.4 Reporting Sequence variants 3.5.5 DNA Sequence Traces 3.5.6 Conclusion Chapter 4 Alpha and Beta Thalassemia Herbert L. Muncie, MD. 4.1 Epidemiology 4.2 Pathophysiology 4.3 Alpha Thalassemia 4.4 Beta Thalassemia 4.5 Diagnosis 4.6 Treatment 4.7 Complications 4.8 Other Treatment Issues 4.8.1 Hypersplenism 4.8.2 Endocrinopathies 4.8.3 Pregnancy 4.8.4 Cardiac 4.8.5 Hypercoagulopathy 4.8.6 Psychosocial 4.8.7 Vitamin Deficiencies 4.8.8 Prognosis XVI 157 132
  18. 18. Chapter 5 Neonatal Screening for Hemoglobinopathies 178 Zia Uddin, PhD 5.1 Introduction 5.2 Methodologies 5.3 Laboratory Reports Format & Interpretation 5.4 Examples of Neonatal Screening 5.4.1. 5.4.2 5.4.3 5.4.4 Capillary Zone Electrophoresis Isoelectric focusing Isoelectric focusing and High Performance Liquid Chromatography Isoelectric focusing, High Performance Liquid Chromatography and DNA studies 5.5 Genetic Counseling & Screening Chapter 6 Prenatal Diagnosis of Beta-Thalassemia and Hemoglobinopathies 202 Maria Cristina Rosatelli, PhD, and Luisella Saba, PhD Chapter 7 Hemoglobin A1c 232 Zia Uddin, PhD 7.1 Introduction 7.2 HbA1c Diagnostic Role in Diabetes Mellitus, and Glycemic Control in Adults 7.3 Measurement of HbA1c 7.4 Factors Affecting the Accuracy of Hb A1c Assay XVII
  19. 19. Case Studies 244 Introduction Case # 1 Normal Adult 247 Case # 2 Hemoglobin S trait Case # 3 Hemoglobin S homozygous Case # 4 Hemoglobin S with hereditary persistence of fetal hemoglobin (HPFH) 264 Case # 5 Hemoglobin G-Philadelphia trait Case # 6 Hemoglobin S-G Philadelphia 279 Case # 7 Hemoglobin G-Coushatta trait 287 Case # 8 Hemoglobin C trait Case # 9 Hemoglobin C homozygous Case # 10 Hemoglobin C with hereditary persistence of fetal hemoglobin (HPFH) 306 Case # 11 Hemoglobin S-C disease Case # 12 Hemoglobin D-Los Angeles (D-Punjab) trait Case # 13 Hemoglobin S-D disease 252 258 272 293 XVIII 299 312 326 319
  20. 20. Case # 14 Hemoglobin E and Associated Disorders Case # 14 a Hemoglobin E trait 339 Case # 14 b Hemoglobin E homozygous Case # 14 c Hemoglobin S-E disease Case # 15 Hemoglobin S-Korle Bu (G-Accra) Case # 16 Hemoglobin O-Arab trait Case # 17 β-Thalassemia trait Case # 18 Hemoglobin S-β - thalassemia Case # 19 Hemoglobin C-β – thalassemia Case # 20 Hemoglobin Hasharon trait Case # 21 Hemoglobin Zurich trait Case # 22 Hemoglobin Lepore trait Case # 23 Hemoglobin J-Oxford trait Case # 24 Hemoglobin J-Baltimore trait Case # 25 Hemoglobin Malmo trait Case # 26 Hemoglobin Koln trait 368 374 o 381 387 394 400 408 421 432 Case # 27 Hemoglobin Q-India trait 441 Case # 28 Hemoglobin Dhofar trait 454 XIX 356 362 + 333 415 344 350
  21. 21. 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 1 such as Disorders of Hemoglobin edited by Steinberg, Forget, Higgs and Nagel should be consulted. 1.1 Hemoglobin Structure Composed of 2 distinct globin chains, the complex protein molecule known as hemoglobin (“heme” + “globin”) is arguably THE primary component of the red blood cell in human beings. In “normal” adults, the globin chains are either alpha (α), beta (β), gamma (ϒ) or delta (δ). In addition, during embryonic life in utero, zeta (ζ) and epsilon (ε) chains are present in the first several weeks of life, being rapidly converted to alpha, beta and gamma chains as development occurs. 1
  22. 22. Figure 1. Globin chains concentration changes in embryonic, fetal and post-natal stages of life (Huehns ER, Dance N, Hecht S, Motulsky AG. Human embryonic hemoglobins. Cold Spring Harbor Symp Quant Biol 1969; 29: 327-331). Adopted with permission from Blackwell Publishing (Barbara J. Bain, Haemoglobinopathy Diagnosis, 2nd Edition, 2006). Each of these globin chains has associated with it a porphyrin molecule known as heme whose primary function in the red blood cell is the facilitation of transport of oxygen to the tissues of the human body. The globin portion of the molecule serves several functions, not the least of which is protection. The internal pocket of the molecule formed from the convergence of the four globin chains, 2
  23. 23. 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 2 helices separated by non-helical turns . The tertiary structure is the 3-dimensional arrangement of these globin chains forming the “pocket” of hemoglobin that cradles the iron molecule in its grasp. The quaternary structure is the moving structure of the molecule that facilitates the oxygenation of the heme molecules in response to the physiological needs of the human body. 3
  24. 24. 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, 4
  25. 25. 3 via proton scavenging, keep CO2 in the soluble bicarbonate form . Nitrous oxide is handled in two different ways by hemoglobin: one as a transporter and the other as a scavenger. Blood levels of NO are therefore, by definition, a balance between NO production and NO removal by binding to oxyhemoglobin. Since NO is an extremely potent vasodilator, hypoxic patients will have lower oxyhemoglobin and therefore higher amounts of free NO. This free NO can cause significant vasodilation, a physiological effect that is very desirable in hypoxia. All hemoglobin molecules, either normal or variant, share the same functionality in the human body. Therefore, the primary structural differences mentioned above and in more complete treatises (i.e., amino acid substitutions/deletions) will be the prime reason for functional differences. It is these amino acid variances that, along with the secondary, tertiary and quaternary structural differences, will determine if the variant hemoglobin is either benign or clinically important. The bottom line is this – whether the hemoglobin is normal or variant in nature, the prime reason for determining the hemoglobin phenotype of the patient is to assess the functionality of the hemoglobin. If the variant is normally functioning in both the heterozygous and homozygous states, the clinical picture is benign. If, however, the variant has normal properties in the heterozygous state (i.e., “trait”) but clinical issues in the homozygous state (i.e., “disease”), the phenotypic analysis and subsequent interpretation becomes ultimately important to the patient. 5
  26. 26. 1.3 Hemoglobin Synthesis The synthesis of hemoglobin, as mentioned before, is under the control of gene loci on two chromosomes: chromosome 11 (the beta globin or “non-alpha” gene) and chromosome 16 (the alpha globin gene). Hemoglobin variants (alpha, beta, gamma, delta and fusion) are the result of alterations in the nucleotide sequences of the globin genes and can occur for more than one reason. Mutations such as point mutations, insertions and deletions can have major, minor or no influences on hemoglobin function or structure. That being said, the site of the synthetic variance can in some cases alter the ability of the hemoglobin molecule to function in a normal manner, i.e., stability, oxygen affinity, solubility or other critical functions. These alterations truly determine whether the variant hemoglobin is classified as benign (i.e., no abnormal or pathological effect) or pathological (a significant physiological effect). The actual nature of the alteration is not of initial importance to the hemoglobinopathy interpreter. However, once assigned, the identity of the variant hemoglobin may become of importance when looking at second generation offspring from the variant carrier, i.e., the pregnant female. For most hemoglobin variants, the synthetic pathway is of no clinical interest in that the resulting hemoglobin is benign. It may, however, be of academic interest in that the identification of the synthetic anomaly can, indeed point to the genetic locus or loci involved in the alteration, thus giving information to the genetic counselor as to possible genetic details of the hemoglobinopathy. 6
  27. 27. 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 7
  28. 28. during the interpretation of the hemoglobinopathy. This data includes, but is not limited to, pregnancy, transfusion history and ethnicity. All of these pieces of information can be critical to the proper identification/interpretation of the hemoglobin variant in the patient’s specimen. For example, an elevation of hemoglobin F in a female patient with a normal hemogram may be evidence of hereditary persistence of fetal hemoglobin; whereas, if this female is pregnant, the elevation may be a normal physiological response to the fetal presence in her body. These data may not be readily available and may require contact with the ordering healthcare professional to obtain these facts. However obtained, they are necessary for the proper identification of the hemoglobin variant or variants in the patient’s bloodstream and therefore are important in the assignment of a benign or pathological assessment of the variant hemoglobin. The variants described in the following chapters all obey the aforementioned differences, i.e., amino acid substitutions, genetic deletions, sequence modifications, etc. While not critical, the exact identification of the variant in and of itself is not normally life-threatening, especially in the heterozygous state, i.e., “trait”. It is essential that the variant be properly identified as a mis-identification can lead to other issues. For example, a mis-interpretation of a hemoglobin G trait (AG) as a sickle trait (AS), while not in and of itself is clinically an issue, presents real difficulties for a couple expecting a child. If both partners are AS, there is a 1 in 4 chance that a child born to this couple could be homozygous SS or sickle cell disease. In the case of an AS mother and an AG father (or vice versa), there is a 1 in 4 chance of a child being born with a phenotype of SG. While on the surface this 8
  29. 29. 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. 9
  30. 30. Chapter 2 Diagnostic Laboratory Methods 2.1 Basic Concepts Jayson Miedema, MD, and Christopher R. McCudden, PhD 2.1.1 Unstable Hemoglobins Unstable hemoglobins are characterized by disorders in globin production which affect the lifespan of the hemoglobin molecule and subsequently the cell leading to decreased cell stability and increased cell turnover. There are a large number of specific variants which can result in abnormal hemoglobin production, the most commonly reported of which is Hb Koln. Many of these abnormal globin chains are a result of single mutations in the form of deletions (e.g. Hb Gun Hill), insertions (e.g. Hb Montreal), or substitutions (e.g. Hb Koln) and can result in weakened heme-globin interactions, subunit interactions, or abnormal folding. These disorders are most commonly expressed in the heterozygous form, most homozygous situations result in preterm lethality. Clinically, these patients often present with symptoms of hemolytic anemia which can be of varying severity. Symptoms of hemolytic anemia include hyperbilirubinemia, jaundice, splenomegaly, hyperbilirubinuria or pigmenturia as well as the formation of Heinz bodies. This pheonotype can present or be exacerbated by infections as well as certain types of drugs. Specifically sulfonamides, pyridium, and antimalarials are known to cause exacerbation. Parvovirus can also induce aplastic crisis andHbA2 and HbF may be increased. The peripheral smear often shows anisocytosis, poikilocytosis, basophilic stippling, polychromasia and, hypochromasia. Since not all unstable 10
  31. 31. 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 2.1.2 Altered Affinity Hemoglobins Similar to how certain types of mutations can cause instability of the hemoglobin molecule, other mutations can cause hemoglobins to have altered affinity for oxygen. These mutations can be single point mutations, insertions, deletions, elongation, deletion/insertion mutations and are often named after the city in which they were discovered (Chesapeake, Capetown, Syracuse, etc.). Both alpha-chain variants, e.g. Hb Chesapeake, and beta-chain variants, e.g. Hb Olser, Hiroshima, Andrew-Minneapolis, 11
  32. 32. etc., are known in the literature for altered affinity for oxygen. Many of these are probably clinically insignificant but when significant most commonly present phenotypically as an increase in oxygen affinity often times resulting clinically in polycythemia (secondary to the bodies perceived lack of oxygen and subsequent increase in erythropoietin). Measurement of hemoglobin affinity (p50) is critical to the diagnosis. Conversely and less frequently described, a decreased affinity for oxygen can lead to clinical cyanosis. Testing for altered affinity hemoglobins relies on subsequent changes to the oxygen dissociation curve and the partial pressure of oxygen at which hemoglobin is 50% saturated, the p50. Because most types of altered affinity hemoglobins cause an increase in oxygen binding, a left shift in the oxygen dissociation curve results. Automated systems are available for recording the oxygen dissociation curve and rely on a Clarke electrode to measure oxygen tension while oxyhemoglobin fraction is measured by dual wavelength spectrophotometer. Abnormal oxygen dissociation curves are primarily caused by altered affinity hemoglobins but can also be caused by such factors as pH, temperature, pCO2, and 2,3-diphosphoglycerate (2,3-DPG). Measurement of pO2, pCO2, pH and SO2 allows for an estimation of p50 to be calculated. 2.1.3 Sickle Solubility Testing Sickle cell anemia is a disease resulting in anemia and painful crises, seen almost exclusively in African Americans. These crises are caused by inappropriate aggregation of deformed blood cells in small blood vessels. Widely believed to have 12
  33. 33. thrived in the gene pool because of its protective effects against malaria, it affects a large number of people of African descent in its homozygous and clinically significant form. An even greater number of people have sickle cell trait (approximately 8-10% of African Americans), the heterozygous form, which is largely insignificant from a clinical standpoint. Sickle cell testing can be performed in a variety of ways and is currently most commonly tested via hemoglobin electrophoresis when necessary. However, another form of testing is known as sickle solubility testing which relies on the property of increased cell fragility as a result of the glutamic acid to valine substitution at the 6 th position of the beta globin gene, the most common genetic abnormality of sickle cell anemia. Sickled red blood cells are soluble when oxygenated but upon deoxygenation tend toward sickling, polymerization, and precipitation. The addition of sodium metabisulfite reagent to a sample with hemoglobin S promotes deoxygenating and cell lyses, creating turbidity in the solution. This turbidity makes it difficult to read a card through the test tube. A negative test is one in which a card can be read through the tube, a positive test is one in which the card cannot be read. Several types of hemoglobins can cause false positives (for example some types of hemoglobin C) so results should be confirmed by electrophoresis; in other words, when used, solubility testing should be used as a screening test. The test also fails to differentiate sickle cell trait (a single copy of the sickle cell gene, heterozygous) from true sickle cell anemia (both copies are sickle cell, homozygous). Samples with low hemoglobin concentration (<8%) should be doubled as this low concentration can lead to false negatives. False positives can occur in the settings of lipemia or samples with 13
  34. 34. monoclonal proteins (dysproteinemia). Both positive and negative controls should be used as results can be somewhat subjective 2.1.4 Serum Iron, TIBC, Transferrin, and Ferritin Iron is essential for numerous metabolic functions in the body through its incorporation into proteins involved in oxygen delivery (hemoglobin, myoglobin) and electron transport and exchange (cytochromes, catalases). While a detailed description of iron metabolism is beyond the scope of this compendium (interested readers should seek the references below), it is worth considering the major mechanisms of iron homeostasis in the context of erythropoiesis. Iron intake in the diet occurs either as free iron or as heme. Free iron, in the form of Fe3+, requires reduction to Fe2+ by enzymes and transporters to cross the intestinal mucosa; heme iron is absorbed directly by mucosal cells where it is split from heme intracellularly. Once absorbed by the GI tract, iron is either stored in association with ferritin or transported into the circulation in the ferric (Fe3+) form. Because of the toxicity of ferric iron, it is transported in the circulation bound to transferrin. The main target of transferrin-bound iron is erythroid tissue, which takes up iron through receptor-mediated endocytosis. As dietary absorption accounts for <20% of the daily requirement, iron recycling plays an essential role in maintaining iron stores. During recycling, senescent red blood cells are phagocytosed by macrophages in the spleen, liver, and bone marrow. Macrophages store some iron (bound to ferritin), but most is returned to red cell precursors via transferrin. Unlike dietary absorption, iron excretion is largely unregulated, where losses occur via epithelial cell sloughing in the skin and GI tract or through menstrual bleeding in 14
  35. 35. premenopausal women. Accordingly, body stores depend on controlling iron uptake in the GI tract and recycling. Disorders of iron homeostasis fall into diseases of excess or deficiency. Iron deficiency is common, particularly in women, and may result from inadequate intake, blood loss, and pregnancy; in chronic disease iron deficiency is also common. Iron excess may occur in hemochromatosis or as a result of repeated transfusions. Clinically, iron status is assessed by measurement of serum iron, ferritin, transferrin, and total iron binding capacity (TIBC). Serum or plasma iron levels can be directly measured using several different methods. Most commonly, a colorimeteric reaction scheme is used in which iron is separated from transferrin at low pH (~4) and then reduced to Fe 2+ for dye binding; the color-complex is detected between 530-600 nm spectrophotometrically. Although iron is typically increased in cases of iron excess and decreased in cases of deficiency, serum iron measurement by itself is not particularly useful for diagnosis of iron homeostasis disorders because of the high intra-individual variation in circulating iron levels. Total iron binding capacity (TIBC) is another test used to assess iron homeostasis. TIBC can be measured or calculated. TIBC is measured by adding excess iron to saturate transferrin (usually transferrin is 30% saturated). Unbound iron is chelated and removed and then the remaining transferrin-bound iron is measured as described above yielding the total capacity. This method can be affected by the presence proteins, of non-transferrin iron binding particularly in cases of hemochromatosis and thalassemias. Alternatively, TIBC may be calculated based on 15
  36. 36. 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 to clinical and morphological manifestations of anemia (e.g. microcytosis) making it a 16
  37. 37. useful diagnostic marker of iron homeostasis. While considered the most useful of the currently available tests for non-invasively assessing iron stores, ferritin is also an acute phase reactant and may be normal or even increased when chronic infection or inflammation occurs in combination with underlying iron deficiency anemia. In thalassemias, ferritin is typically elevated reflecting a state of iron overload; in contrast, ferritin is decreased in iron deficiency making it a useful marker to differentiate causes of microcytosis. Transferrin is an iron transporting protein and negative acute phase reactant produced primarily by the liver. As with ferritin, transferrin is routinely measured by immunoassay. Most circulating iron is bound to transferrin, binding to Fe 3+ with very high affinity. Transferrin transports iron absorbed in the GI tract to cells containing specific receptors, in particular erythroid tissue. Transferrin delivers iron to cells via the ubiquitously distributed transferrin receptor. Clinically, measurement of transferrin is useful for hypochromic microcytic anemia workups. Transferrin is increased in iron deficiency anemias, but normal or decreased in chronic anemia of disease, iron overload, and thalassemias. Transferrin is decreased in cases of liver disease, nephropathy (or other protein loss or malabsorption), and inflammation. 17
  38. 38. Table 1. Iron Tests in Different Disorders Disorder Serum TIBC % Iron Chronic Anemia Transferrin Ferritin Saturation ↓ ↓ ↓ ↔ or ↓ ↔ or ↑ ↓ ↑ ↓ ↑ ↓ ↔ or ↑ ↔ ↔ or ↑ ↔ or ↓ ↔ or ↑ ↑ ↓ ↑↑ ↔ or ↓ ↑↑ of Disease Iron Deficiency Thalassemia Hemochromatosis ↓decreased; ↔ within reference interval; ↑ increased 2.1.5 Soluble Transferrin Receptor An additional test that is useful for diagnosis of anemia is the soluble transferrin receptor (sTfR). The sTfR consists of the N-terminus of the membrane receptor that can be measured in circulation. Circulating levels reflect the activity of the erythroid bone marrow, where sTfR levels are decreased in cases of low red cell synthesis (renal failure and aplastic anemia) and increased in patients with hemoglobinopathies. The utility of sTfR measurement is that it can differentiate iron deficiency in cases of acute inflammation because sTfR levels are not affected by inflammatory cytokines. In thalassemias, sTfR levels are generally increased in proportion to the severity of the genotype. Despite the apparent advantages, sTfR testing is not widely used and is not currently standardized. 18
  39. 39. 2.1.6 Hepcidin Discovered in 2000, hepcidin is a hormone involved in iron homeostasis. Hepcidin is produced by the liver and negatively regulates iron balance by inhibiting macrophage recycling and decreasing intestinal absorption. Thus, when iron stores are replete, hepcidin levels are increased and when iron stores are low, hepcidin is elevated. Similar to ferritin, hepcidin is an acute phase reactant, making interpretation of circulating levels in patients with inflammation more challenging. At the time of writing, hepcidin testing was not available commercially. The hepcidin in human iron stores and its diagnostic implications has been recently reviewed (Kroot JJC, Tjalsma H, Fleming RE, Swinkels DW. Hepcidin in Human Iron Disorders: Diagnostic Implications: Clin Chem 2011; 57(12): 1650-1669). Additional Readings Fairbanks VF, Klee GG. Biochemical aspects of hematology. In Fundamentals of Clinical Chemistry. Edited by Tietz N. Saunders,1987,789-818. Guarnone R, Centenara E, Barosi G. Performance characteristics of hemox-analyzer for assessment of the hemoglobin dissociation curve. Haematologica 1995;80:426-430. Pincus MR and Abraham NZ. Interpreting laboratory results. In: Henry's Clinical Diagnosis and Management by Laboratory Methods (Clinical Diagnosis & Management by Laboratory Methods) Edited by McPherson RA and Pincus MR. 21st Edition. Higgins T, Beutler E, Doumas BT. Hemoglobin, Iron and Bilirubin. In Tietz textbook of clinical chemistry and molecular diagnostics. Edited by Burtis CA, Ashwood ER, Bruns DE. Elsevier Saunders, 2006,1165-1208. Marengo-Rowe AJ. Structure-function relations of human hemoglobins. Proc (Bayl Univ Med Cent) 2006;19:239-245. Mayomedicallaboratories.com/test-catalog. Accessed April 20, 2011. 19
  40. 40. Rees DC, Williams TN, Gladwin MT. Sickle-cell disease. The Lancet. 2010;376:20182031. Steinberg MH. Genetic disorders of hemoglobin oxygen affinity. www.uptodate.com. Accessed April 28, 2011. Steinberg MH. Unstable hemoglobin variants. www.uptodate.com. Accessed April 28, 2011. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. Edited by Burtis CA, Ashwood ER, and Bruns DE. 5th Edition. Vichinsky EP. Sickle cell trait. www.uptodate.com. Accessed April 28, 2011. 20
  41. 41. Chapter 2 Diagnostic Laboratory Methods 2.2 Microcytosis Diane Maennle, MD, and Kimberly Russell, MT (ASCP), MBA Smaller-than-normal size of Red Blood Cells (RBCs) is defined as microcytosis. This is quantified by calculating the mean corpuscular volume (MCV) using the following formula employing the values of hematocrit and RBC count: MCV = Hematocrit (HCT) X 10 / RBC Count (Million) In adults, a MCV value of less than 80fL is defined as microcytosis. In pediatric subjects, the MCV and hemoglobin range distinctly vary with age (Table I). Table I Age Dependent Mean Hemoglobin and MCV Values Age Mean Hemoglobin (g/dL) Mean MCV (fL) 3 to 6 months 11.5 91 6 months to 2 years 12.0 78 2 to 6 years 12.5 81 6 to 12 years 13.5 86 12 to 18 years (female) 14.0 90 12 to 18 years (male) 14.5 88 > 18 years (female) 14.0 90 > 18 years (male) 15.75 90 21 1,2,3,4
  42. 42. 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 ironbinding capacity (TIBC), transferrin saturation, hemoglobin electrophoresis, and the examination of the peripheral blood smears (by a pathologist or hematologist), are employed to provide insight and etiologies of microcytosis (Table III). 22 3,8
  43. 43. 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 distribution width (RDW) Normal to increased Normal Increased Serum iron Decreased Normal to increased Normal to decreased Normal to increased Total ironbinding capacity Increased Normal Slightly increased Normal Transferrin saturation Decreased Normal to increased Normal to slightly decreased Normal to increased 3 Van Vranken has recently suggested a protocol for diagnosing the cause of microcytosis (Figure 1). If the cause remains unclear, the diagnosis of hemoglobinopathy by methods besides electrophoresis alone is imperative. Note: There is a type-setting error in the presentation of 3 the protocol suggested by Van Vranken. We have corrected this error in the figure 1, and the journal (American Family Physician) editor was also informed. 23
  44. 44. 24
  45. 45. 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 bellshaped curve. When this value is 2-3% higher, it represents red cells which have varying widths. This certainly can be seen in patients who are iron deficient with microcytosis, but have normal or large cells in addition to megaloblastic or dysplastic marrows, elevated reticulocytes, vitamin B12 or folic acid deficiency, and other conditions. Despite the availability of automated cell counters, review of the peripheral film is one of the most informative and rewarding tests that should be done (by pathologist or hematologist) in any case in which the cause of anemia is not obvious, e.g., bleeding, pure iron deficiency, pure vitamin B12 deficiency, etc. It is also emphasized that the RDW test is not sensitive or specific enough to differentiate iron deficiency and β-thalassemia trait. 9 A fairly low to extremely low ferritin is an excellent measure of iron deficiency anemia. In my practice, regardless of what else is going on, any ferritin level of <10 ng/mL, means there is iron deficiency. As mentioned above (Table III), elevated ferritin levels are 25
  46. 46. 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. 26
  47. 47. 8. Hematologic diseases. In: Wallach J. Interpretation of Diagnostic Tests. 8 th ed. Boston, Mass.: Little Brown and Company; 2006: 385-419. 9. Ntalos G, Chatzinikolaou A, Saouli Z, et al. Discrimination indices as screening tests for beta-thalassemia trait. Ann Hematol. 2007; 86(7): 487-491. 27
  48. 48. Chapter 2 Diagnostic Laboratory Methods 2.3 Hereditary Persistence of Fetal Hemoglobin Bernard G. Forget, MD 2.3.1 Introduction Hereditary persistence of fetal hemoglobin or HPFH consists of a group of genetic disorders characterized by the presence of a substantial elevation of fetal hemoglobin (Hb F) in RBCs of heterozygotes, as well as of homozygotes and compound heterozygotes for HPFH and other hemoglobinopathies. Increased levels of Hb F can ameliorate the clinical course of hemoglobinopathies such as β thalassemia and sickle cell anemia. HPFH is usually due to deletions of different sizes involving the β-globin gene cluster, but nondeletion types of disorders have also been identified, usually due to point mutations in the γ-globin gene promoters (reviewed in refs. 1-3). Figure 1 diagrammatically illustrates the spatial organization of the β-like globin genes in the β-gene cluster on chromosome 11. However, as discussed later in this chapter, certain forms of nondeletion HPFH are clearly not linked to the β-globin gene cluster. 28
  49. 49. 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 socalled 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 29
  50. 50. 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. 2.3.2 Deletions Associated with the HPFH Phenotype. Classic pancellular HPFH, with absence of δ-and β-globin gene expression from the affected chromosome, is associated with large deletions in the β-globin gene cluster that remove the δ-and β-globin genes together with variable amounts of their 5’ and 3’ flanking DNA. At least nine different HPFH deletions of this type have been characterized that vary in size or length from ~13 kb to ~ 85 kb (1-4), some of which are illustrated in Fig. 1. The mechanisms by which such deletions cause marked elevation of Hb F are not well understood, but a number of theories have been proposed. One theory is based on the model of the proposed mechanism for the marked elevation of Hb F associated with Hb Kenya. Hb Kenya is a structurally abnormal hemoglobin that, like Hb Lepore, contains a "hybrid" or fused β-like globin chain resulting from a non-homologous crossing-over event between two globin genes in the 30
  51. 51. 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 βthalassemic 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 31
  52. 52. 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 deletiontype HPFH is the nature and function of the DNA sequences conserved at the 5’ breakpoint of the deletions. The 5’ breakpoints of the HPFH deletions, as well as many of the 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 32
  53. 53. 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 33
  54. 54. in their ChIP experiments. These findings provide very strong evidence for a γ-gene silencer element in the β-gene cluster that associates with a BCL11A-containing repressor complex and that this interaction is an important factor in the suppression of γ-gene expression during the perinatal switch from expression of Hb F to Hb A. 2.3.3 Nondeletion Forms of HPFH In contrast to the deletional types of HPFH syndromes, where both linked Gγ and A γ genes are over expressed, only one or the other γ gene is usually over expressed in the best characterized nondeletional types of HPFH associated with high levels of pancellular Hb F expression. However, less well characterized nondeletion forms of G A γ γ HPFH have been described that are associated with relatively low levels of heterocellular expression of both γ genes. Because of the restricted pattern of γ-globin gene expression in the 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. 34
  55. 55. Table 1 adopted from reference 2. The one patient studied was doubly heterozygous for Hb A and Hb C. About 20% of Hb F (or 8% of the total Hb) was of the G G A γ type, and the γ gene in cis to the -175 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 35
  56. 56. 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 (1525%). This region of DNA is noteworthy because it contains an octanucleotide sequence that is present in the promoter region of a number of genes and is the binding site of another ubiquitous trans-acting factor called OCT-1. In addition, the octamer consensus sequence of the 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 CCAAT box of the Aγ-globin gene promoter. The base 36
  57. 57. 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 transacting factors, including the ubiquitous factors CCAAT binding protein (CP1) and CCAAT displacement factor (CDP) as well as the erythroid-specific factor NF-E3. The unifying model by which these various mutations are thought to affect hemoglobin switching proposes that these base changes alter the binding of a number of different trans- acting factors to critical regions of the γ-gene promoters and thereby prevent the normal postnatal suppression of γ-gene expression (reviewed in refs. 1,2). The mutations could prevent the binding of negative regulatory factors or enhance the binding of positive regulatory factors. Either mechanism could be operative with one mutation or the other. 2.3.4 HPFH Unlinked to the β-Globin Gene Cluster A number of studies have identified families in which increased levels of Hb F are inherited due to a genetic determinant that is unlinked to the β-globin gene cluster. Genome-wide association studies (GWAS), using co-inheritance of single nucleotide polymorphisms (SNPs) with elevated levels of Hb F, have subsequently demonstrated the presence of two different quantitative trait loci (QTLs), unlinked to the β-globin gene cluster on chromosome 11, that are associated with inheritance of mildly elevated levels of Hb F, similar to the phenotype seen in Swiss-type heterocellular HPFH (see section 37
  58. 58. above on Nondeletion HPFH). These loci are located on chromosome 2 and 6 (16, 17). The locus on chromosome 2 corresponds to the site of the gene encoding BCL11A and its identification led to the elegant studies of Sankaran and co-workers demonstrating the role of BCL11A in the regulation of γ-gene expression. The locus on chromosome 6 is located between the genes encoding HBS1L and MYB. The mechanism by which this locus causes elevation of Hb F is thus far poorly understood. Finally, mutations in the gene on chromosome 19 encoding the erythroid-specific transcription factor EKLF1 have been shown to be associated with a form of HPFH (18, 19). The involved mechanism is probably through the regulation of BCL11A levels, because it has been demonstrated that EKLF1 binds to the promoter of the BCL11A gene and regulates the expression of the gene (20). 2.3.5 Conclusion Significant insights into the normal regulation of expression of the human βglobin gene cluster have been obtained by a detailed analysis of a group of disorders called HPFH. On the basis of this information, several important regulatory elements have been identified for the normal functioning of the individual genes in the cluster during the developmental switch from fetal to adult hemoglobin gene expression, as well as for the abnormal persistent expression of the γ-globin genes in adults with HPFH. These results provide a more sophisticated understanding of the molecular basis of these syndromes and point to certain strategies for potential future molecular and cellular therapies for globin gene disorders. 38
  59. 59. 2.3.6 Hemoglobin F Quantification Hb F can be quantified by several methods, and the most commonly used procedures in a clinical laboratory are a) radial immunodiffusion, b) Elisa method, c) HPLC, and d) capillary zone electrophoresis. References 1. Bollekens JA, Forget BG. Delta beta thalassemia and hereditary persistence of fetal hemoglobin. Hematol Oncol Clin North Am 1991;5(3):399-422. 2. Forget BG. Molecular basis of hereditary persistence of fetal hemoglobin. Ann N Y Acad Sci 1998; 850:38-44. 3 Weatherall DJ, Clegg JB. The Thalassaemia Syndromes. 4th ed. Oxford ; Malden, MA: Blackwell Science; 2001. 4. Sankaran VG, Xu J, Byron R, et al. Functional element necessary for fetal hemoglobin silencing. N Engl J Med 2011; 365(9):807-14. 5. Feingold, EA, Forget BG. The breakpoint of a large deletion causing hereditary persistence of fetal hemoglobin occurs within an erythroid DNA domain remote from the β-globin gene cluster. Blood 1989; 74: 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 gammaglobin gene in transgenic mice. J. Biol Chem 1995; 270: 10256-63. 8. Huisman TH, Schroeder WA, Efremov GD, et al. The present status of the heterogeneity of fetal hemoglobin in beta-thalassemia: an attempt to unify some observations in thalassemia and related conditions. Ann N Y Acad Sci 1974;232(0):10724. 9. Bank A, O'Neill D, Lopez R, et al. Role of intergenic human 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. 39
  60. 60. 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; 24:78398. 16. Thein SL, Menzel S, Lathrop M, Garner C. Control of fetal hemoglobin: new insights emerging from genomics and clinical implications. Hum Mol Genet 2009;18(R2):R21623. 17. Galarneau G, Palmer CD, Sankaran VG, Orkin SH, Hirschhorn JN, Lettre G. Fine mapping at three loci known to affect fetal hemoglobin levels explains additional genetic variation. Nat Genet 2010;42(12):1049-51. 18. Borg J, Papadopoulos P, Georgitsi M, et al. Haploinsufficiency for the erythroid transcription factor KLF1 causes hereditary persistence of fetal hemoglobin. Nat Genet 2010;42(9):801-5. 19. Borg J, Patrinos GP, Felice AE, Philipsen S. Erythroid phenotypes associated with KLF1 mutations. Haematologica 2011; 96:635-8. 20. Zhou D, Liu K, Sun CW, Pawlik KM, Townes TM. KLF1 regulates BCL11A expression and γ- to β-globin gene switching. Nat Genet 2010; 42:742-4. 40
  61. 61. Chapter 2 Diagnostic Laboratory Methods 2.4 Flow Cytometry Measurements of Cellular Fetal Hemoglobin, Oxidative Stress and Free Iron in Hemoglobinopathies Eitan Fibach, MD 2.4.1 Flow Cytometry of Blood Cells Flow cytometry (FC) is a common methodology in clinical diagnostic and research laboratories. In hematology, it is mainly used for diagnosis, prognosis, determining therapy efficacy and follow up of patients with leukemia or lymphoma (1). It is also used for diagnosis of red blood cell (RBC) abnormalities such as in Paroxysmal Nocturnal Hemoglobinuria (2) and hereditary spherocytosis (3). In this review, I will summarize FC methodologies for analysis of RBC (and other blood cells) from patients with hemoglobinopathies with respect to their fetal hemoglobin (HbF) and free iron (labile iron pool, LIP) contents and parameters of the oxidative state. FC analyzes individual cells in a liquid medium. Most techniques use antibodies directed against internal (following fixation and premeabilization of the membrane) or surface antigens. The antibodies are labeled with fluorescence probes (fluochromes) either directly or indirectly (by a secondary antibody). In addition to antibodies, other fluorescent compounds can be used. For example, propidium iodide, which binds stochiometrically to nucleic acids, is commonly used for determining cell viability and their distribution in the cell cycle (4). Following staining, the cells are analyzed by a flow cytometer; they are first 41
  62. 62. 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. 42
  63. 63. FC is especially fitting for analysis of blood cells: (I) These cells which can be easily obtained by blood drawing are present as single cells, thus in contrast to cells of solid tissues, their use does not require harsh procedures for tissue disaggregation (e.g., trypsinization). (II) They are present as a mixture of various cell types, including numerous subtypes (e.g., lymphocytes), with very large (e.g., RBC) to very small (hematopoietic stem cells) representation. Cells of these sub-types can be identified and "gated" based on differences in their size (forward light scattering), granularity (side light scattering) and expression of surface antigens, and can be measured simultaneously. For measurements of various characteristics (HbF content, oxidative stress parameters and LIP content), the blood sample is stained with specific probes (as detailed below), and then with fluorescent reagents (usually antibodies) against surface markers which identify a specific subpopulation. Such markers are glycophorin A for RBC, CD61 for platelets, CD15 for neutrophils, CD19 for B-lymphocytes and CD3 for Tlymphocytes. CD45 is particularly useful since it is differentially expressed on various nucleated blood cells (Fig. 1). 43
  64. 64. PMN RBC Monocytes Lymphocytes CD45 Fig. 1. Flow cytometry of blood cells. A dot plot of blood cells with respect CD45 (FL3-H) and side light scatter (SSC-H). 2.4.2 Measurement of Fetal Hemoglobin-Containing Erythroid Cells Fetal hemoglobin (HbF, 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 44
  65. 65. spectrofluorometric measurements following treatment with alkaline (to destroy non-fetal hemoglobins) and staining with benzidine (8), chromatography (ion-exchange HPLC for hemoglobins and reverse-phase HPLC for globin chains) (9), as well as immunological techniques, such as Elisa, based on antibodies against HbF (10). However, quantitative FC measurement of RBC, fluorescently stained with antibodies to HbF (as well as for the other hemoglobins), has several advantages. For example, in the differential diagnosis of Hereditary Persistence of Fetal Hemoglobin (11). This condition encompasses a heterogeneous group of disorders with marked increased levels of HbF. Based on the cellular distribution of HbF, they are characterized as pan-cellular, where all RBCs have increased levels of HbF, albeit not always uniformly so; and heterocellular, where nearly all the HbF is confined to a minor, distinct subpopulation of RBCs. This important distinction is most reliably ascertained by FC. Epidemiological studies have indicated that high levels of HbF improve the clinical symptoms of the underlying disease. In sickle cell anemia not only do HbFcontaining cells have a lower concentration of sickle hemoglobin, but HbF inhibits polymerization of HbS directly, accounting for the lower propensity of such cells to undergo sickling (12). In 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 23 months (10). HU acts by a still unknown mechanism on the early erythroid precursors in the bone marrow. It takes several weeks for HbF to accumulate in the peripheral 45
  66. 66. blood to a quantity that allows differences before and after treatment to become apparent. Measuring differences in F-RBC by FC may be more sensitive, and measuring F-reticulocytes (retics) may provide early indication of treatment efficacy (15): Retics have a very short life-span (1-2 days) compared to mature RBC (120 days in normal subjects) and therefore measuring peripheral blood F-retics more closely characterizes the current status of HbF production in the bone marrow. Measuring Fretics can indicate the efficacy of the drug and/or the patient’s compliance several days after treatment initiation. Such follow up is very important since about 30% of the patients are non-responders. It is imperative that such patients be identified as early as possible and the treatment (that is not without potential risks) be discontinued and replaced by treatment with another drug (e.g., butyroids). 2.4.3 Staining Protocols for F-RBC and F-Retics (15) Heparinized blood is washed three times in phosphate buffered saline (PBS). For fixation, 50μl of the packed cells are resuspended in 10 ml of PBS containing 4% formaldehyde for 15-min at room temperature under constant agitation in polypropylene tubes. For permeabilization, the cells are centrifuged for 3 min at 1,500 g, and 2 ml methanol-acetone are added to the pellet, mixed and incubated for 1-min at room temperature. The cells are then washed three times and resuspended in PBS to a final volume of 0.5 ml (10% suspension). Anti-HbF monoclonal antibodies (the amount depends on the Manufacturer’s instructions or on a pre-performed titration) are added to 5x106 cells (5 μl of the 10% suspension) and incubated for 1-hr at 370C, after which the cells are washed in PBS. If 46
  67. 67. the antibodies are fluorochrome-conjugated, the cells are resuspended in PBS and analyzed directly. In the case of unconjugated antibodies, a secondary antibody (fluorochrome-conjugated rabbit F(ab’)2 anti-mouse IgG) is added for 30-min at room temperature. For the F-retic count, the blood cells are double labeled with phycoerythrin-conjugated antibodies to HbF and thiazol orange, a specific nucleic acid binding green fluorescence dye. Following staining, the cells are washed and resuspended in PBS and analyzed by FC. For "acquisition", the threshold is set on forward light scatter to exclude debris and platelets. Cells are run at about 1000 cells/sec using logarithmic amplification, and data of 104-105 cells are accumulated. RBC are gated based on their forward light scatter and side light scatter. When the sample is also stained with thiazol orange, RBC are gated based on their negative staining with thiazol orange, retics - based on their weak staining (because they contain remnants of RNA) and nucleated cells (including normoblasts) – based on their intense staining; HbF is then specifically determined for each cell population (Fig. 2). 47
  68. 68. Fig. 2. Flow cytometry analysis of F-RBC and F-Retics. Blood cells stained with thiazol-orange (T.O) and anti-HbF. A. Forward light scatter (FSC) vs. T.O. RBC (negative T.O staining) and retics (intermediate T.O staining) were gated and their HbF determined (B and C), respectively. 2.4.4 F-Cell Determination for Fetal-Maternal Hemorrhage (FMH) in Pregnant Patients with β-Thalassemia – A Single Case and General Conclusion (16) F-cell analysis is commonly used to detect fetal-maternal hemorrhage (FMH) – where fetal RBC enter the maternal blood circulation due to fetal or maternal trauma or a placental defect (17). These RBC of fetal origin can be distinguished from the maternal adult RBC by their fluorescence following staining with an antibody to HbF. 48
  69. 69. Recently, in order to increase the sensitivity, reproducibility and accuracy of the assay, another marker was introduced – carbonic anhydrase (CA) (18). The CA isoenzymes that are mainly represented by CAI and CAII (19) are fully expressed in the RBC only after birth (20,21). The "Fetal Cell Count kit" manufactured by IQ Products (Groningen, the Netherlands), which uses a combination of a murine monoclonal antibody directed to HbF and a polyclonal antibody to the CAII isoform, has significantly improved this assay (11,18). Most of the RBC of fetal origin do not express CA but highly express HbF (CA-HbF++), while RBC in adult blood express CA but do not express HbF (CA +HbF-). Some adult F-cells which express CA and HbF (CA+HbF+) can be differentiated from fetal F-cells (CA-HbF++) present in FMH based on the extent of HbF and CA expression. Until recently, β-thalassemia major was lethal. Improvements in treatment, such as the introduction of blood transfusions and iron chelation, have considerably improved the life expectancy as well as the quality of the patient’s life, including the ability of thalassemic women to give birth. Recently, we were confronted with a case of a possible FMH in a β-thalassemic woman. To establish the usefulness of the CA/HbF procedure, i.e. differentiating between fetal RBC and the maternal RBC, we screened non-pregnant β-thalassemic patients (men and women). The results demonstrated, in addition to adult non-F RBC (CA+HbF-) and adult F-RBC (CA+HbF+), two other sub-populations, CA+HbF++ and CA-HbF++. The presence in these patients of the latter RBC phenotype, which characterizes fetal cells, precludes the use of the CA/HbF method for the detection of FMH in thalassemia. 2.4.5 Oxidative Stress The oxidative status of cells is determined by the balance between pro-oxidants and antioxidants. The reactive oxygen species (ROS) are pro-oxidants which are 49
  70. 70. generated in most cells mainly during energy production. Although important for various aspects of normal physiology (e.g., signal transduction), ROS interact with and damage various cell components when they are in excess. To protect against the deleterious effects of ROS, cells maintain an effective antioxidant system consisting of water- or lipid-soluble antioxidants and enzymes that remove ROS by metabolic conversion. When the oxidant/anti-oxidant balance is tilted in favor of the oxidants, oxidative stress ensues (22). Although oxidative stress is not the primary etiology of hemoglobinopathies, it mediates several of their pathologies, including hemolysis which results in chronic anemia. Hemolysis occurs both in the bone marrow, where developing erythroid precursors undergo enhanced apoptosis (ineffective erythropoiesis) and in the peripheral blood, where mature RBC undergo lysis in the blood vessels (intra-vascular hemolysis). Destruction also occurs in reticuloendothelial tissues, such as the spleen, where mature RBC undergo phagocytosis by resident macrophages (extra-vascular hemolysis) (22). Various factors are responsible for oxidative stress in RBC of patients with hemoglobinopathies. In β-thalassemia, excess α-globin chains form unstable tetramers that dissociate into monomers and then are oxidized, first to met-Hb and then to hemichromes which precipitate intracellularly with time (23). Following the release of heme and iron, there is deposition of the protein moiety on the plasma membrane. The outcome of this chain of events is enhanced formation of ROS, catalyzed by free iron, with a variety of deleterious effects on the membrane lipids and proteins, including oxidation of the membrane protein band 4.1 and a decrease in spectrin/band3 ratio (24). In α-thalassemia, the γ- and β-globins, which are produced in excess, do not precipitate 50
  71. 71. right away, but form the soluble tetramers γ4 (Hb Bart’s) and later the β4 (HbH), which are less stable than HbA and have an increased susceptibility towards oxidation and hemichrome formation (23). In sickle cell disease, met-HbS is produced at a higher rate and is less stable than met-HbA resulting in formation of hemichromes, and release of heme and iron, with resultant denaturation and precipitation as Heinz bodies (25). Many approaches have been devised to quantify oxidative stress and its damage as well as the effects of treatment with anti-oxidants (22). Most of these methods assay the content of body fluids (mainly blood). FC can be utilized for measurements of oxidative stress parameters in various blood cells. Although the major target of oxidative stress in hemoglobinopathies is the RBC, other blood cells are affected as well. Thus, defects in the abilities of polymorphonuclear cells to adhere to, engulf and lyze bacteria may result in recurrent infections. Chronic activation of platelets may cause thromboembolic complications (26,27). In order to study the effects of oxidative stress on the spectrum of symptoms in hemoglobinopathies, all blood cell lineages should be studied. FC of oxidative stress parameters utilizes various probes: ROS can be measured by staining cells with the non-polar compound, 2’-7-dichlorofluorescein diacetate. It readily diffuses across the membrane and becomes deacetylated by esterases into a polar derivative that is trapped inside the cells. When it is oxidized by ROS (mainly peroxides), a green fluorescent product – dichlorofluorescin is produced (28). The intensity of the fluorescence is proportional to the cellular concentration of ROS. The applicability of the method was validated by the increased fluorescence measured following treatment with ROS-generating agents such as hydrogen peroxide 51
  72. 72. and t-butylhydroxyperoxide and with the catalase inhibitor sodium azide, while treatment with ROS scavengers such as N-acetyl cysteine decreased the fluorescence. ROS can also be measured by dihydrorhodamine 123, which freely enters into cells, and after oxidation by ROS to rhodamine 123 emits a bright red fluorescence (29). Reduced glutathione (GSH), the main cellular antioxidant, can be measured using mercury orange (26), which forms fluorescent adducts with GSH via the sulphydryl group, producing an S-glutathionyl derivative that emits red-orange fluorescence (30). The probe reacts more rapidly with non-protein thiols, such as GSH, compared with thiol-containing proteins, allowing specificity under controlled staining conditions (31). The validity of this method was confirmed by demonstrating that Nethylmaleimide, which totally blocks thiol groups, decreased the fluorescence in a dosedependent manner. To ascertain that non-protein thiols are being stained, cells were incubated with diethylmaleate, a specific non-protein thiol-depleting agent. This weak electrophil of the α,β-unsaturated carbonyl group, which reacts with GSH only in the presence of glutathione transferase, markedly suppressed the mercury orange fluorescence, suggesting that GSH was the principle thiol which was stained by the dye (32). Although there is no direct proof that the probe is specific for GSH, the assay measures predominantly GSH, since it is the main non-protein thiol constituent of the cellular thiol pool (33). Other parameters of oxidative stress measured by FC are membrane lipid peroxidation – by staining with fluor-DHPE (26), and externalization of phosphatidylserine (PS) moieties, a marker of damage to the membrane lipid, by fluorochrome-conjugated annexin-V (34). 52
  73. 73. 2.4.6 Staining Protocols for ROS and GSH ROS Assay – Blood cells are incubated with 2'-7'-dichlorofluorescin diacetate, dissolved in methanol, at a final concentration of 0.4 mM. After incubation at 37°C for 15 min, the cells are washed and re-suspended in PBS to the original cell concentration. GSH Assay - Blood cells are washed with PBS and then spun down. The pellet is incubated for 3 min. at room temperature with 40 M (final concentration) of mercury orange. A 100 M stock solution of mercury orange is made up in acetone and stored at 4°C. In both cases, cells are then washed and resuspended in PBS, and analyzed by FC. Fig. 3 shows FC measurements of ROS and GSH in normal and thalassemic RBC. The results indicate that thalassemic RBC have higher ROS but lower GSH contents than normal RBC. 53
  74. 74. Fig. 3. Flow cytometry of ROS and GSH in normal and thalassemic RBC. Blood cells derived from a normal donor (A,C) and a thalassemic patient (B,D) were stained for ROS (A,B) and GSH (C,D) following 1-h pre-incubation with (white) or without (pink) 2 mM H2O2. Histograms of RBC are shown. 2.4.7 Intracellular Free Iron Another contributor to oxidative stress in cells is excess of iron. Iron overload is generated in thalassemic or sickle RBC as a result of Hb-instability as discussed above. In addition, iron accumulates in these diseases as a result of increased absorption from the intestinal mucosa and by a failure to dispose of excess iron acquired by frequent therapeutic blood transfusions (35). Moreover, iron-containing compounds (Hb or 54
  75. 75. hemin) that are released during hemolysis can add to the iron load and further aggravate the hemolysis. Normally, iron is transported in the circulation bound to transferrin and is transferred into cells through the surface transferrin-receptor (36). Most of the intracellular iron is firmly bound to various components such as Hb, heme and cytochrome C; excess is stored in ferritin (37). In iron overload, serum iron which exceeds the binding capacity of transferrin is present in the form of non-transferrin bound iron (38). This iron can be taken up through a transferrin-independent pathway, to form the cellular unbound "labile iron pool" (LIP) (16). The small fraction of LIP was suggested as a low molecular weight intermediate or transitory pool between extracellular iron and cellular firmly-bound iron (39). LIP is redox active and it participates in generation of free radicals by the Fenton and Haber-Weiss reactions and consequently in cell and tissue damage (40). Since iron overload plays an important role in the pathology of transfused patients with β-hemoglobinopathies, the patients are commonly treated with iron chelators. The three chelators currently in clinical use are deferioxamine, deferiprone and deferasirox (41). Evaluation of iron overload is important for assessing its severity and for determining the efficacy of iron chelation therapy. The parameters usually tested are serum ferritin protein level and transferrin iron saturation. However, serum ferritin is an acute phase reactant that may increase by iron-independent factors such as infection, inflammation and liver disease (42). In addition, serum ferritin levels often fail to predict impending cardiac iron overload and ensuing cardio-myopathies (43). The advent of non-invasive proton relaxation assays (by NMR R2* or T2*) of organs has 55
  76. 76. provided a significant advance in monitoring iron overload, although, similarly to serum ferritin, substantial changes in these parameters are seen only weeks to months after the initiation of chelator treatment. In addition, these techniques require expensive instrumentation that is not always available.FC quantification of the LIP content in various blood cell types overcomes many of these problems. 2.4.8 Staining Protocol for LIP Cells are washed twice with saline and incubated at a density of 1x10 6 per ml for 15 min at 37oC with 0.25 μM Calcein Acetoxymethyl Ester (CA-AM). After wash, the cells are treated with or without Deferiprone (L1, 100 μM). Fig. 4 shows the results of LIP measurements in RBC. LIP is defined as the difference between histograms of cells treated or untreated with L1. 56
  77. 77. Fig. 4. Flow cytometry of labile iron pool (LIP) in RBC. Blood cells were loaded with calcein, then washed and treated with or without the iron chelator Deferiprone (L1). Distribution fluorescence (FL1-H) histograms are shown. LIP is defined as the difference between the mean fluorescence channels of histograms of cells treated or untreated with L1. References 1. 2. 3. 4. Virgo PF, Gibbs GJ. Flow cytometry in clinical pathology. Ann Clin Biochem 2012; 49(Pt 1): 17-28. Sutherland DR, Keeney M, Illingworth A. Practical guidelines for the high-sensitivity detection and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry. Cytometry B Clin Cytom 2012; 82(4): 195-208. Kedar PS, Colah RB, Kulkarni S, Ghosh K, Mohanty D. Experience with eosin-5'maleimide as a diagnostic tool for red cell membrane cytoskeleton disorders. Clin Lab Haematol 2003; 25(6): 373-6. Krishan A. Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. J Cell Biol 1975; 66(1): 188-93. 57
  78. 78. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. Peterson KR. Hemoglobin switching: new insights. Curr Opin Hematol 2003; 10(2): 1239. Boyer SH, Belding TK, Margolet L, Noyes AN. Fetal hemoglobin restriction to a few erythrocytes (F cells) in normal human adults. Science 1975; 188(4186): 361-3. Bunn H, Forget B. Hemoglobins: Molecular, Genetic and Clinical Aspects. Philadelphia: WB Saunders Co.; 1986. Fibach E. Measurement of total and fetal hemoglobin in cultured human erythroid cells by a novel micromethod. Hemoglobin 1993; 17(1): 41-53. Huisman TH. Separation of hemoglobins and hemoglobin chains by high-performance liquid chromatography. J Chromatogr 1987; 418: 277-304. Epstein N, Epstein M, Boulet A, Fibach E, Rodgers GP. Monoclonal antibody-based methods for quantitation of hemoglobins: application to evaluating patients with sickle cell anemia treated with hydroxyurea. Eur J Haematol 1996; 57(1): 17-24. Leers MP, Pelikan HM, Salemans TH, Giordano PC, Scharnhorst V. Discriminating fetomaternal hemorrhage from maternal HbF-containing erythrocytes by dual-parameter flow cytometry. Eur J Obstet Gynecol Reprod Biol 2007; 134(1): 127-9. Benesch RE, Edalji R, Benesch R, Kwong S. Solubilization of hemoglobin S by other hemoglobins. Proc Natl Acad Sci U S A 1980; 77(9): 5130-4. Gambari R, Fibach E. Medicinal chemistry of fetal hemoglobin inducers for treatment of beta-thalassemia. Curr Med Chem 2007; 14(2): 199-212. Steinberg MH. Determinants of fetal hemoglobin response to hydroxyurea. Semin Hematol 1997; 34(3 Suppl 3): 8-14. Amoyal I, Fibach E. Flow cytometric analysis of fetal hemoglobin in erythroid precursors of beta-thalassemia. Clin Lab Haematol 2004; 26(3): 187-93. Prus E, Fibach E. Heterogeneity of F-cells in β -thalassemia. Transfusion 2012, in press. Sebring ES, Polesky HF. Fetomaternal hemorrhage: incidence, risk factors, time of occurrence, and clinical effects. Transfusion 1990; 30(4): 344-57. Porra V, Bernaud J, Gueret P, Bricca P, Rigal D, Follea G, Blanchard D. Identification and quantification of fetal red blood cells in maternal blood by a dual-color flow cytometric method: evaluation of the Fetal Cell Count kit. Transfusion 2007; 47(7): 1281-9. Tashian RE. The carbonic anhydrases: widening perspectives on their evolution, expression and function. Bioessays 1989; 10(6): 186-92. Brady HJ, Edwards M, Linch DC, Knott L, Barlow JH, Butterworth PH. Expression of the human carbonic anhydrase I gene is activated late in fetal erythroid development and regulated by stage-specific trans-acting factors. Br J Haematol 1990; 76(1): 135-42. Aliakbar S, Brown PR. Measurement of human erythrocyte CAI and CAII in adult, newborn, and fetal blood. Clin Biochem 1996; 29(2): 157-64. Fibach E, Rachmilewitz EA. The role of antioxidants and iron chelators in the treatment of oxidative stress in thalassemia. Ann N Y Acad Sci 2010; 1202: 10-6. Rachmilewitz EA. Formation of hemichromes from oxidized hemoglobin subunits. Ann N Y Acad Sci 1969; 165(1): 171-84. Advani R, Sorenson S, Shinar E, Lande W, Rachmilewitz E, Schrier SL. Characterization and comparison of the red blood cell membrane damage in severe human alpha- and beta-thalassemia. Blood 1992; 79(4): 1058-63. 58

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