Professional Documents
Culture Documents
Second Edition
August 2015
Editorial Board
Diane M. Maennle, MD
Chairperson
Kenneth F. Tucker, MD
Member
Member
Member
Member
II
Angie Duong, MD
Assistant Professor, Hematopathology
Department of Pathology and
Laboratory Medicine
Medical University-South Carolina
Charleston, South Carolina
USA
Rita Ellerbrook, PhD
Technical Director Emeritus
Helena Laboratories, USA
1530 Lindberg Drive
Beaumont, TX 77707
USA
Eitan Fibach, MD
Professor, Department of Hematology
Hadassah-Hebrew University Medical Center
Ein-Kerem, Jerusalem
Israel
Bernard G. Forget, MD
Professor Emeritus of Internal Medicine
Yale School of Medicine
New Haven, CT 06520
USA
Piero C. Giordano, PhD
Hemoglobinopathies Laboratory
Human and Clinical Genetics Department
Leiden University Medical Center
The Netherlands
Dina N. Greene, PhD
Scientific Director, Chemistry
Regional Laboratories, Northern California
The Permanente Medical Group
Berkeley, CA 94710
USA
III
IV
Herbert L. Muncie, MD
Professor, Department of Family Medicine
School of Medicine, Louisiana State University
1542 Tulane Ave
New Orleans, LA 70112
USA
Gul M. Mustafa, PhD
Post-Doctorate Fellow
Department of Pathology
The University of Texas Medical Branch
Galveston, TX 77555
USA
Diane M. Maennle, MD
Associate Pathologist
Department of Pathology
St. John Macomb-Oakland Hospital
Warren, MI 48093
USA
Jayson Miedema, MD
Post-Doctorate Fellow
Department of Pathology and Laboratory Medicine
University of North Carolina
Chapel Hill, North Carolina
USA
Christopher R. McCudden, PhD
Assistant Professor, Department of Pathology
and Laboratory Medicine, University of Ottawa
Ottawa, Ontario
Canada
Michael A. Nardi, MS
Associate Professor
Department of Pediatrics and Pathology
New York University School of Medicine
New York, NY 100016
USA
VII
VIII
Financial Disclosure
I neither had nor will have financial relationship
with any of the manufacturers or any other
organization mentioned in the book.
Similarly all the contributors and reviewers
of the book have worked with gratis to further
the cause of education.
This book and its translations into several
languages are provided at no charge.
August 2015
IX
Dedication
This book is dedicated with heartfelt thanks to my
professors responsible for my PhD level education in
Chemistry at the Illinois Institute of Technology, Chicago,
Illinois, and post-doctoral education and training in Clinical
Chemistry at the University of Illinois Medical Center,
Chicago, Illinois.
Illinois Institute of Technology, Chicago, Illinois
Professor Kenneth D. Kopple, PhD
Professor Paul E. Fanta, PhD
Professor Robert Filler, PhD
Professor Sidney I. Miller, PhD
University of Illinois Medical Center, Chicago, Illinois
Professor Newton Ressler, PhD
August 2015
Preface
Higher level education is one of the blessings of God.
Unfortunately, primarily due to economic and logistic reasons
a vast majority of the qualified candidates are denied this
opportunity.
Internet has the potential of mass education at an
infinitesimal cost. This is the 3rd book launched via Internet
by me at no charge.
All the MD/PhD degree holders are most respectfully
requested to utilize the Internet as a means of communication
to launch books at no charge in their areas of expertise.
Love
God
Love
People
Serve
The World
August 2015
XI
Acknowlegement
During the past three years I contacted worldwide >200 family physicians,
clinical chemists, pathologists, hematologists, public health officials and experts in
diagnostic hemoglobinopathy for formatting this book. The contribution of all of these
individuals is heartfelt and very much appreciated.
I am highly indebted to the following persons for their technical support:
Diane M. Maennle, MD
Rita Ellerbrook, PhD
Kimberly R. Russell, MT (ASCP), MBA
Jennifer Randazzo, MS (Information Technology)
The following manufacturers and organizations provided technical support,
and facilities for the collection of data for the book:
Helena Laboratories, USA
Sebia, France
PerkinElmer Corporation, USA
Bio-Rad, USA
ARUP Laboratories, USA
Quest Diagnostics, USA
College of American Pathologists, USA
Seven Universities and four Newborn Screening Laboratories, USA
(names are with held as per their request)
Mr. Mathew Garrin, Biomedical Communications and Graphic Arts Department,
Wayne State University, School of Medicine, Detroit has worked on the figures, scans,
and layout of the book. I am very grateful to him for his contribution.
Finally, I would like to thank the following persons for facilitating my work:
Adrian J. Christie, MD, Medical Director of Laboratories
St. John Macomb-Oakland Hospital, Warren, Michigan, USA
Anoop Patel, MD, Assistant Systems Medical Director
St John Providence Health System Laboratories, Warren, Michigan, USA
Mr. Tipton Golias, President & CEO
Helena Laboratories, Beaumont, Texas, USA
August 2015
XII
Table of Contents
Chapter 1
Hemoglobin
Chapter 2
Hemoglobin Structure
Hemoglobin Function
Hemoglobin Synthesis
Hemoglobin Variants
10
John Lazarchick, MD
Angie Duong, MD
Chapter 3
Basic Concepts
44
Jayson Miedema, MD
and Christopher R. McCudden, PhD
3.1.1
3.1.2
3.1.3
3.1.4
3.1.5
3.1.6
3.2
Unstable Hemoglobins
Altered Affinity Hemoglobins
Sickle Solubility Test
Serum Iron, TIBC, Transferrin, Ferritin
Soluble Transferrin Receptor
Hepcidin
Microcytosis
55
Diane Maennle, MD
and Kimberly Russell, MT (ASCP), MBA
3.3
Introduction
Deletions Associated with the HPFH Phenotype
Non-Deletion Forms of HPFH
HPFH Unlinked to the -Globin Gene Cluster
Conclusion
XIII
62
3.4
3.5
95
Introduction
Cellulose Acetate Electrophoresis (alkaline pH)
Agarose Gel Electrophoresis (alkaline pH)
Agar Electrophoresis (acid pH)
Interpretation of Hemoglobin Agarose Gel (pH 8.6)
and Agar Gel (pH 6.2) Electrophoresis
3.5.6 Requirements for the Identification of Complex
Hemoglobinopathies
3.6
Introduction
Basic Principle
Application of CZE in Diagnostic
Hemoglobinopathies
Interpretation of CZE Results
XIV
3.7
Isoelectric Focusing
117
107
3.8
129
Chapter 4
136
4.2
140
XV
4.4
4.5
181
Chapter 5
Epidemiology
Pathophysiology
Alpha Thalassemia
Beta Thalassemia
Diagnosis
Treatment
Complications
Other Treatment Issues
5.8.1
5.8.2
5.8.3
5.8.4
5.8.5
5.8.6
5.8.7
5.8.8
Hypersplenism
Endocrinopathies
Pregnancy
Cardiac
Hypercoagulopathy
Psychosocial
Vitamin Deficiencies
Prognosis
XVI
Chapter 6
191
166
Hemoglobinopathies
212
Introduction
Methodologies
Laboratory Reports Format & Interpretation
Examples of Neonatal Screening
6.4.1.
6.4.2
6.4.3
6.4.4
6.5
Chapter 7
Chapter 8
Hemoglobin A1c
266
Introduction
HbA1c Diagnostic Role in Diabetes Mellitus, and
Glycemic Control in Adults
Measurement of HbA1c
8.4
XVII
Case Studies
278
Introduction
Case # 1
Normal Adult
281
Case # 2
Hemoglobin S trait
Case # 3
Hemoglobin S homozygous
Case # 4
Case # 5
Case # 6
313
Case # 7
321
Case # 8
Hemoglobin C trait
Case # 9
Hemoglobin C homozygous
Case # 10
Case # 11
Case # 12
Case # 13
286
292
306
327
333
346
353
360
XVIII
Case # 14
367
373
Case # 16
Case # 17
-Thalassemia trait
Case # 18
Case # 19
Case # 20
Case # 21
Case # 22
Case # 23
Case # 24
Case # 25
Case # 26
Case # 27
475
Case # 28
488
XIX
390
396
402
408
415
421
428
434
442
455
466
449
378
384
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.
pocket protects the heme from oxidation and facilitates oxygen transfer to the
tissues of the body. The previously mentioned and chain-containing hemoglobins
have very high oxygen affinities, a factor very important in the early embryonic life of
the fetus.
The hemoglobin molecule can be looked at in four different ways; primary,
secondary, tertiary and quaternary structural views. While outside of the scope of
this volume, each of these structures contributes definitive unique properties to the
various hemoglobin molecules from normal hemoglobins to the very rare and
functionally diverse molecules. The primary structure of all hemoglobins is the order
of amino acids found in the globin chains of the molecule. It is this unique sequence
that is the major differentiator of hemoglobin from each other. The secondary
structure of hemoglobin is the arrangement of these amino acid chains into alpha
2
1.2
Hemoglobin Function
As mentioned above, the primary function of hemoglobin is to reversibly
transport oxygen to the tissues of the body. In addition, however, this flexible
molecule can also transport carbon dioxide (CO2) and nitrous oxide (NO). The
transport of CO2 is facilitated by reversible carbamoylation (formation of carbamoyl
moiety, i.e., H2NCO-) of the N-terminal amino acids of the globin chains and can,
4
via proton scavenging, keep CO2 in the soluble bicarbonate form . Nitrous oxide is
handled in two different ways by hemoglobin: one as a transporter and the other as
a scavenger. Blood levels of NO are therefore, by definition, a balance between NO
production and NO removal by binding to oxyhemoglobin. Since NO is an extremely
potent vasodilator, hypoxic patients will have lower oxyhemoglobin and therefore
higher amounts of free NO. This free NO can cause significant vasodilation, a
physiological effect that is very desirable in hypoxia.
All hemoglobin molecules, either normal or variant, share the same
functionality in the human body. Therefore, the primary structural differences
mentioned above and in more complete treatises (i.e., amino acid
substitutions/deletions) will be the prime reason for functional differences. It is these
amino acid variances that, along with the secondary, tertiary and quaternary
structural differences, will determine if the variant hemoglobin is either benign or
clinically important.
The bottom line is this whether the hemoglobin is normal or variant in
nature, the prime reason for determining the hemoglobin phenotype of the patient is
to assess the functionality of the hemoglobin. If the variant is normally functioning in
both the heterozygous and homozygous states, the clinical picture is benign. If,
however, the variant has normal properties in the heterozygous state (i.e., trait) but
clinical issues in the homozygous state (i.e., disease), the phenotypic analysis and
subsequent interpretation becomes ultimately important to the patient.
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.
Chapter 2
Red Blood Cell Morphology
9
John Lazarchick, MD
Angie Duong, MD
Knowledge of red blood cell (RBC) morphology is essential for the clinical diagnosis of
hemoglobinopathy. The diameter of RBC, when mature under normal circumstances
is approximately 7-8 microns, and RBC is round, anuclear and biconcave disc-shaped.
A study of RBC morphology includes size, shape, color, inclusions and arrangement. In
this chapter we have presented with pictures of the most commonly encountered RBC
morphologies with legends and few examples of the diseases with abnormal RBC
morphology. In the clinical cases of this book, we have mentioned only the main
features of the peripheral blood smear, therefore a review of this chapter is advised
for a nave reader for the proper diagnosis of hemoglobinopathy.
The following RBC morphology cases are presented in this chapter:
Size:
Macrocyte large
Microcyte small
Normocyte normal
Hemoglobin Content:
Hypochromic low
Normochromic normal
Polychromatic high
Shape and Inclusions:
Anisocytosis
Poikiocytosis
Acanthocyte
Basophilic Stippling
Bite Cell
Blister Cell
Burr Cell (Ecchinocyte)
Heinz Body
Howell-Jolly Body
Pappenheimer Body
Schistocyte
Sickle Cell
Spherocyte
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig.10
Fig.11
Fig.12
Fig.13
Fig.14
Fig 15
Fig.16
Fig.17
Fig.18
Fig.19
10
Stomatocyte
Fig. 20
Target Cell
Fig. 21
Teardrop Cell
Fig. 22
RBC Agglutination
Fig. 23
Rouleaux Formation
Fig. 24
Diseases :
Erythroblastosis Fetalis
Fig. 25
Hemoglobin C Disease
Fig. 26
Hemoglobin C/beta Thalassemia Fig. 27
Hemoglobin S/beta Thalassemia Fig. 28
Hemoglobin SC Disease
Fig. 29
Sickle Cell Disease
Fig. 30
Fetal-maternal Hemorrhage:
Fig. 31
Kleinhauer-Betke Stain
11
Fig. 1 Macrocyte-large
The diameter of RBC >9-14 microns (1.5 to 2 times larger than
normal RBC) and the MCV >100 fL is characteristic of macrocyte.
Macrocytes are mostly oval in shape.
12
Fig. 2 Microcyte-small
RBC, when abnormally smaller (< 5 micron) than normacytic
RBC (7-8 micron) is defined as microcyte (also called
microerythrocyte). The MCV of the microcyte RBC is < 80 fL.
13
Fig. 3 Normocyte-normal
The diameter of RBC, when mature under circumstances is
approximately 7-8 microns, and are round, anuclear, biconcave
disc-shaped with an internal volume of 80-100 fL.
The term normocyte is used when the size of the RBC is normal.
14
Fig. 4 Hypochromasia
Hypochromasia is a descriptive term for red blood cells where the
central pallor is greater than one third the diameter of the red
blood cell (black arrows). This is due to a decrease in the amount
of hemoglobin in the cells. Diseases with prominent
hypochromasia are iron deficiency anemia, anemia of chronic
disease, and sideroblastic anemia. Some cases of
myelodysplastic syndrome can also have hypochromatic red
blood cells. Hypochromasia is reflected in the complete blood
count (CBC) by a decreased mean corpuscular hemo-globin
concentration (MCHC).
Also present are: target cells/codocytes (red arrow),
polychromatic forms (blue arrow), fragmented red blood
cells/schistocytes (green arrows), and tear drop
forms/dacryocytes (yellow arrows). Overall, this smear shows
moderate anisopoikilocytosis.
15
Fig. 5 Normochromic-normal
This descriptive term is applied to a red blood cell with a normal
concentration of hemoglobin. The above figure is a peripheral
blood cell smear of a patient treated for iron deficiency anemia.
Blue arrow shows normochromic-normal RBC. Black arrow shows
hypochromic-microcytic RBC.
16
Fig. 6 Polychromatic-high
This smear demonstrates polychromasia. Numerous
polychromatic forms (black arrows), which are young slightly
larger red blood cells with a purple-tinge due to retained RNA, are
present. Polychromasia is the bone marrows response to anemia,
where the bone marrow releases younger red blood cells.
Sometimes, nucleated red blood cells are also released into the
peripheral blood. Due to their larger size, when many
polychromatic forms are present, the CBC values of mean
corpuscular volume (MCV) as well as RDW (red blood cell
distribution width) will be increased.
In a supravital stains, such as cresyl violet, the retained RNA in
the polychromatic forms precipitate out and these cells are called
reticulocytes. Thus, sometimes the terms polychromatic form is
used interchangeably with reticulocytes.
17
Fig. 7 Anisocytosis
The term anisocytosis refers to size variation seen among red
blood cells. As demonstrated above, there are small red blood
cells as well as large red blood cells, some approaching the size
of a neutrophil (green arrow). Ansiocytosis is a reactive process
where the bone marrow is releasing younger red blood cells,
therefore an increased number of polychromatic forms can also
be seen (black arrow). In the complete blood count (CBC),
anisocytosis is reflected by having an increased red cell
distribution width (RDW).
18
Fig. 8 Poikilocytosis
Poikilocytosis refers to shape variation. In poikilocytosis, the red
blood cells have lost their normal discoid appearance. The
example shown here has a predominance of
ovalocytes/elliptocytes, which are red blood cells that have a
length twice their diameter (a few are indicated by blue arrows).
Also seen are schistocytes (red arrows), which are fragmented
red blood cells. Ovalocytes/Elliptocytes are seen in peripheral
blood smear in some conditions, e.g., thalassemia, iron
deficiency, etc.
Note: When both shape and size variation is seen in the red blood
cells, the term anisopoikilocytosis can be used.
19
20
21
22
23
24
25
26
27
28
29
Fig. 19 Spherocytes
This peripheral blood smear is from a patient with autoimmune
hemolytic anemia (AIHA) and is characterized by many
spherocytes (blue arrows) and microspherocytes (black arrows).
Spherocytes are red blood cells that have no central pallor. As the
name implies, microspherocytes are small spherocytes. If the
majority of the cells in a peripheral smear are spherocytes, the
possibility of hereditary spherocytosis arises. Hereditary
spherocytosis is an autosomal dominant disease where one of the
genes that code for red blood cell proteins (such as spectrin and
ankyrin) become mutated.
30
Fig. 20 Stomatocyte
Red blood cells with slit-like central pallor (arrow) caused by a
decrease in surface area to volume ratio associated with a
membrane permeability disorder. Hereditary stomatocytosis is
associated with hemolysis which can be severe. Acquired
stomatocytosis can be seen in acute alcohol intoxication, chronic
liver disease and as drying artifact in peripheral smear
preparation.
31
32
33
34
35
36
37
38
39
41
42
Chapter 3
Diagnostic Laboratory Methods
3.1
Basic Concepts
Jayson Miedema, MD, and Christopher R. McCudden, PhD
43
44
as
pH,
temperature,
pCO2,
and
2,3-diphosphoglycerate
(2,3-DPG).
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
monoclonal proteins (dysproteinemia). Both positive and negative controls should be
47
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
48
presence
proteins,
of
non-transferrin
iron
binding
particularly
in
cases
of
In cases of iron
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
Disorder
Serum
Chronic
Anemia
of Disease
Iron Deficiency
Thalassemia
Hemochromatosi
TIBC
Transferrin
Ferritin
Iron
Saturation
or
or
or
or
or
or
or
s
decreased; within reference interval; increased
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.
3.1.6 Hepcidin
Discovered in 2000, hepcidin is a hormone involved in iron homeostasis.
Hepcidin is produced by the liver and negatively regulates iron balance by inhibiting
52
macrophage recycling and decreasing intestinal absorption. Thus, when iron stores are
replete, hepcidin levels are increased and when iron stores are low, hepcidin is
elevated. Similar to ferritin, hepcidin is an acute phase reactant, making interpretation
of circulating levels in patients with inflammation more challenging.
At the time of
writing, hepcidin testing was not available commercially. The hepcidin in human iron
stores and its diagnostic implications has been recently reviewed (Kroot JJC, Tjalsma
H, Fleming RE, Swinkels DW. Hepcidin in Human Iron Disorders: Diagnostic
Implications: Clin Chem 2011; 57(12): 1650-1669).
Additional Readings
Fairbanks VF, Klee GG. Biochemical aspects of hematology. In Fundamentals of
Clinical Chemistry. Edited by Tietz N. Saunders,1987,789-818.
Guarnone R, Centenara E, Barosi G. Performance characteristics of hemox-analyzer for
assessment of the hemoglobin dissociation curve. Haematologica 1995;80:426-430.
Pincus MR and Abraham NZ. Interpreting laboratory results. In: Henry's Clinical
Diagnosis and Management by Laboratory Methods (Clinical Diagnosis & Management
by Laboratory Methods) Edited by McPherson RA and Pincus MR. 21 st Edition.
Higgins T, Beutler E, Doumas BT. Hemoglobin, Iron and Bilirubin. In Tietz textbook of
clinical chemistry and molecular diagnostics. Edited by Burtis CA, Ashwood ER, Bruns
DE. Elsevier Saunders, 2006,1165-1208.
Marengo-Rowe AJ. Structure-function relations of human hemoglobins. Proc (Bayl Univ
Med Cent) 2006;19:239-245.
Mayomedicallaboratories.com/test-catalog. Accessed April 20, 2011.
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.
53
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.
54
Chapter 3
Diagnostic Laboratory Methods
3.2 Microcytosis
Diane Maennle, MD, and Kimberly Russell, MT (ASCP), MBA
Smaller-than-normal size of Red Blood Cells (RBCs) is defined as microcytosis.
This is quantified by calculating the mean corpuscular volume (MCV) using the following
formula employing the values of hematocrit and RBC count:
MCV = Hematocrit (HCT) X 10 / RBC Count (Million)
In adults, a MCV value of less than 80fL is defined as microcytosis. In pediatric
subjects, the MCV and hemoglobin range distinctly vary with age (Table I).
Table I Age Dependent Mean Hemoglobin and MCV Values 1,2,3,4
Age
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
14.0
90
15.75
90
Iron deficiency anemia, -thalassemia trait, and -thalassemia trait are the most common
causes of microcytosis. However, other clinical conditions are also associated with microcytosis
(Table II).1,3,5,6 In addition to decreased MCV, the patients with -thalassemia trait usually have
55
increased hemoglobin A2. It is pointed out that lower hemoglobin A2 is also observed in patients
with concurrent deficiency of serum iron. Therefore, serum iron deficiency anemia must be ruled
out in order to correctly make the diagnosis of -thalassemia trait in such patients. Conversely,
patients with -thalassemia trait may acquire megaloblastic anemia or liver disease, and may
exhibit a normal range for MCV.7
Thalassemia trait
Thalassemia trait
Other hemoglobinopathies
Pregnancy
Unexplained anemia
Lead toxicity
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).3,8
56
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
Increased
Normal
Slightly increased
Normal
Transferrin
saturation
Decreased
Normal to
increased
Normal to slightly
decreased
Normal to
increased
Van Vranken3 has recently suggested a protocol for diagnosing the cause of microcytosis
(Figure 1). If the cause remains unclear, the diagnosis of hemoglobinopathy by methods besides
electrophoresis alone is imperative. Note: There is a type-setting error in the presentation of
the protocol suggested by Van Vranken.3 We have corrected this error in the figure 1, and
the journal (American Family Physician) editor was also informed.
57
58
Clinical observations of Kenneth F. Tucker, MD, FACP, a practicing hematologist for the
last forty years:
Ordinary hemoglobin electrophoresis (cellulose acetate or agarose gel
electrophoresis) was only able to detect the more common types of thalassemias. Although
there were several other types, many of them did not have microcytosis. I had a large
number of patients, who had -thalassemia minor and a few with probably -thalassemia,
in which the hemoglobin and hematocrit values were relatively normal. Microcytes may or
may not be present. This diagnosis was suggested by the peripheral smear, and proven by
additional laboratory tests (IFE, globin chain analysis, etc.).
I believe that RDW, which is the average red cell width and reflects standard
deviation of red cell volumes, is a very important test. RDW normal deviation is a bellshaped curve. When this value is 2-3% higher, it represents red cells which have varying
widths. This certainly can be seen in patients who are iron deficient with microcytosis, but
have normal or large cells in addition to megaloblastic or dysplastic marrows, elevated
reticulocytes, vitamin B12 or folic acid deficiency, and other conditions. Despite the
availability of automated cell counters, review of the peripheral film is one of the most
informative and rewarding tests that should be done (by pathologist or hematologist) in any
case in which the cause of anemia is not obvious, e.g., bleeding, pure iron deficiency, pure
vitamin B12 deficiency, etc. It is also emphasized that the RDW test is not sensitive or
specific enough to differentiate iron deficiency and -thalassemia trait. 9
A fairly low to extremely low ferritin is an excellent measure of iron deficiency
anemia. In my practice, regardless of what else is going on, any ferritin level of <10 ng/mL,
means there is iron deficiency. As mentioned above (Table III), elevated ferritin levels are
seen in refractory anemias, all types of chronic inflammatory conditions, etc. Since this test
59
is an acute phase reactant (similar to haptoglobin), it must not be used alone, as the ferritin
level may be normal in these clinical conditions.
Women in the second or third trimester are always anemic. This is similar to patients
who are hypervolemic because of renal or cardiac problems. Red cells in these cases are
not microcytes and when the hypervolemia is corrected, the hemoglobin and hematocrit
rises.
Severe anemia in childhood is usually due to the lack of iron in food, since cows
milk does not contain iron.
A nave reader is advised to also review the Full Color pdf of Complete Blood Count
in Primary Care, Best Practice Journal, June 2008 (www.bpac.org.nz),
especially the section on Hemoglobin and Red Cell Indices (page 15).
References
1. Richardson M. Microcytic anemia [published correction appear in Pediatr Rev. 2007;
28(7): 275, Pediatric Rev. 2009; 30(5): 181, and Pediatr Rev. 2007; 28(4):151]. Pediatr
Rev. 2007; 28(1): 5-14.
2. Beutler E, Waalen J. The definition of anemia: what is the lower limit of normal of the
blood hemoglobin concentration? Blood. 2006; 107(5): 1747-1750.
3. Van Vranken ML. Evaluation of Microcytosis. Am Fam Physician. 2010; 80(9): 1117-1122.
4. RBC indices calculation and laboratory procedure (2006). St. John Health Laboratories,
Warren, MI 48093.
5. Moreno Chulila JA, Romero Colas MS, Gutierrez Martin M. Classification of anemia for
gastroenterologist. World J Gastroenterol. 2009: 15(37):4627-4737.
6. Guralnik JM, Eisenstaedt RS, Ferrucci L, Klein HG, Woodman, RC. Prevalence of anemia
in persons 65 years and older in the United States: evidence for a high rate of
unexplained anemia. Blood. 2004; 104(8): 2263-2268.
7. Bain BJ. Hemoglobinopathy Diagnosis. 2nd ed. Malden, Mass.: Blackwell
Publishing; 2006: 94-106.
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.
60
Chapter 3
Diagnostic Laboratory Methods
1
3.3
3.3.1 Introduction
Hereditary persistence of fetal hemoglobin or HPFH consists of a group of
genetic disorders characterized by the presence of a substantial elevation of fetal
hemoglobin (Hb F) in RBCs of heterozygotes, as well as of homozygotes and
compound heterozygotes for HPFH and other hemoglobinopathies. Increased levels of
Hb F can ameliorate the clinical course of hemoglobinopathies such as thalassemia
and sickle cell anemia. HPFH is usually due to deletions of different sizes involving the
-globin gene cluster, but nondeletion types of disorders have also been identified,
usually due to point mutations in the -globin gene promoters (reviewed in refs. 1-3).
Figure 1 diagrammatically illustrates the spatial organization of the -like globin genes in
the -gene cluster on chromosome 11. However, as discussed later in this chapter,
certain forms of nondeletion HPFH are clearly not linked to the -globin gene cluster.
61
Figure 1. Deletions of the -globin gene cluster associated with fusion proteins and
HPFH. The circle 3 to the -globin gene indicates the 3 -globin gene enhancer. The
filled vertical boxes at the 3 breakpoints of the HPFH-1 and HPFH-6 deletions indicate
the locations of DNA sequences with homology to olfactory receptor genes (adopted
from reference 2). The references for the individual mutations are cited in references 1,
3 and 6.
HPFH is frequently contrasted with thalassemia, which is another genetic
disorder associated with elevated Hb F levels. However, one should probably not
consider the two disorders as being unambiguously separate entities but rather as a
group of disorders with a variety of partially overlapping phenotypes that sometimes
defy classification as one syndrome or the other. The following is a working definition
that is generally applied to the classification of these disorders: thalassemia usually
refers to a group of disorders associated with a mild but definite thalassemia phenotype
of hypochromia and microcytosis together with a modest elevation of Hb F that, in
heterozygotes, is heterogeneously distributed among red cells.
In contrast, HPFH
refers to a group of disorders with substantially higher levels of Hb F, and in which there
is usually no associated phenotype of hypochromia and microcytosis. In addition, the
increased Hb F in heterozygotes with the typical forms of HPFH is distributed in a
relatively uniform (pancellular) fashion among all of the red cells rather than being
distributed in a heterogeneous (heterocellular) fashion among a subpopulation of socalled F cells, as in thalassemia. Homozygotes for both conditions totally lack Hb A
and Hb A2, indicating absence of - and -globin gene expression in cis to the
thalassemia and HPFH determinants.
Nevertheless,
-globin genes, the Kenya gene resulted from crossover between the A- and -globin
genes (Fig. 1). The crossover occurred in the second exons of the A and genes,
between the codons for amino acids 80 to 87, and resulted in deletion of ~24 kb of DNA
between the A to the gene. Unlike Hb Lepore, that is associated with a thalassemic phenotype, Hb Kenya is associated with a phenotype of pancellular G
HPFH: erythrocytes of affected heterozygotes have normal red cell indices and contain
7-23% Hb Kenya as well as approximately 10% Hb F, all of which is of the G type and is
relatively uniformly distributed among the red cells. A proposed explanation for the
HPFH phenotype associated with Hb Kenya is the influence on the G- and Kenya gene
promoters of a well characterized enhancer element located in the 3' flanking DNA of
the -globin gene, shown by the filled circle in Fig. 1, that becomes translocated into
close proximity of the -globin gene promoters by the crossover/deletion event, resulting
in enhanced activity of these promoters.
Among the HPFH deletions, there is a relatively short deletion, called HPFH-5 or
Italian HPFH, that extends from a point ~3 kb 5' to the gene to a point 0.7 kb 3' to the
gene, deleting the gene but not its 3' enhancer. The molecular basis of the HPFH
phenotype associated with this deletion is presumably the influence of the translocated
3' -gene enhancer on the -gene promoters, in a manner analogous to that proposed
for the basis of the HPFH phenotype of the Hb Kenya syndrome. In the case of some of
the other larger HPFH deletions, the DNA preserved at or near the 3 breakpoint of the
deletions has been shown in various types of assays to have enhancer-like activity on
gene expression (2, 5-7). Thus, it has been proposed that the DNA sequences at the
HPFH 3' deletion breakpoints, that become juxtaposed to the genes as a result of the
64
The most conclusive evidence for a functional role of the A-intergene DNA in
the regulation of gene expression consists of the observations by Sankaran et al. who
have extensively characterized a negative regulatory transcription factor, called
BCL11A, that down-regulates -gene expression in adult erythroid cells and that binds
to the A-intergene DNA (11-13). BCL11A, originally identified as an important factor in
B-lymphoid cell development, is a component of a multi-protein complex that plays a
negative regulatory role in -gene expression. This complex has been shown to contain
GATA1 as well as all components of the nucleosome and histone deacetylase ( NuRD)repressive complex (14). Additional studies have shown that this complex physically
interacts with another transcription factor called SOX6 that is thought to be a repressor
of embryonic and fetal globin gene expression (15). Chromatin immunoprecipitation
(ChIP) studies have shown that BCL11A binds to a number of regions in the -cluster,
including the upstream locus control region (LCR) and the intergenic region, but does
not bind to the - or -gene promoters (4, 14, 15). Sankaran et al. (4) have
characterized two important deletion mutants with nearly identical distal breakpoints but
different upstream breakpoints around the -gene and its flanking DNA. One mutant
with a more proximal breakpoint has a -thalassemia phenotype, whereas the longer
deletion removing 3.5 kb of additional upstream DNA is associated with a HPFH
phenotype. The authors propose that this 3.5 kb region of DNA contains a silencer
element, deletion of which can cause HPFH. This hypothesis is strengthened by the fact
that the deleted region contains one of the prominent binding sites of BCL11A detected
in their ChIP experiments. These findings provide very strong evidence for a -gene
silencer element in the -gene cluster that associates with a BCL11A-containing
66
repressor complex and that this interaction is an important factor in the suppression of
-gene expression during the perinatal switch from expression of Hb F to Hb A.
3.3.3 Nondeletion Forms of HPFH
In contrast to the deletional types of HPFH syndromes, where both linked G and
A
genes are over expressed, only one or the other gene is usually over expressed in
the best characterized nondeletional types of HPFH associated with high levels of
pancellular Hb F expression. However, less well characterized nondeletion forms of GA
HPFH have been described that are associated with relatively low levels of
heterocellular expression of both genes. Because of the restricted pattern of -globin
gene expression in the G and A forms of nondeletion HPFH, it was assumed that the
mutations in these syndromes must be located near the affected gene and molecular
studies focused initially on the DNA sequence analysis of the over expressed genes in
these disorders.
67
Table 1 adopted from reference 2. The one patient studied was doubly
heterozygous for Hb A and Hb C. About 20% of Hb F (or 8% of the total Hb) was of the
G
G
A
type, and the gene in cis to the -175 mutation carried the -158 C T change.
The references for the individual mutations are cited in references 1 and 3.
The results of these structural analyses revealed a number of different point
mutations in the promoter region of the over expressed gene in individuals with
different types of nondeletion HPFH, as listed in Table 1 (reviewed in refs. 1-3). These
point mutations have clustered primarily in three distinct regions of the 5'-flanking DNA
of the affected genes. The first region is located approximately 200 base pairs from
the "cap site" or site of transcription initiation of the genes (at least five different point
68
mutations involving single nucleotides between residues -195 to -202 from the cap site).
This region of DNA, which had not previously been suspected of playing a role in the
regulation of -gene expression, is very G+C rich and its sequence bears homology to
that of known control elements of other genes that contain the binding site for the
ubiquitous trans-acting protein factor called Sp1. Subsequent studies of the -gene
promoters have demonstrated that the -200 region is also a binding site for Sp1 and for
at least one other ubiquitous DNA binding protein.
The second region containing a mutation associated with nondeletion HPFH is
located at position -175. A point mutation (T->C) at this position of either the G or A
gene is associated with a phenotype of pancellular HPFH with high levels of Hb F (1525%). This region of DNA is noteworthy because it contains an octanucleotide
sequence that is present in the promoter region of a number of genes and is the binding
site of another ubiquitous trans-acting factor called OCT-1. In addition, the octamer
consensus sequence of the -gene promoters is flanked on either side by a consensus
sequence for the hematopoietic-specific transcription factor GATA-1. The point mutation
at position -175 affects the one nucleotide that is present in the partially overlapping
binding sites of both OCT-1 and GATA-1.
The third region affected by a point mutation in nondeletion HPFH is in the area
of a well known regulatory element of globin and other genes: the CCAAT box
sequence. In the genes, the CCAAT box is duplicated and the mutation associated
with the Greek A type of nondeletion HPFH is a G->A substitution at position -117, 2
bases upstream of the distal CCAAT box of the A-globin gene promoter. The base
change disrupts a pentanucleotide sequence, YYTTGA (Y = pyrimidine), that is highly
69
conserved immediately upstream of the CCAAT sequence in all animal fetal and
embryonic genes. At least two other mutations involving the CCAAT box of one or the
other gene have been reported in other cases of HPFH not associated with large
deletions. The CCAAT box region is known to be the binding site of a number of transacting factors, including the ubiquitous factors CCAAT binding protein (CP1) and
CCAAT displacement factor (CDP) as well as the erythroid-specific factor NF-E3.
The unifying model by which these various mutations are thought to affect
hemoglobin switching proposes that these base changes alter the binding of a number
of different trans- acting factors to critical regions of the -gene promoters and thereby
prevent the normal postnatal suppression of -gene expression (reviewed in refs. 1,2).
The mutations could prevent the binding of negative regulatory factors or enhance the
binding of positive regulatory factors. Either mechanism could be operative with one
mutation or the other.
The locus on chromosome 2 corresponds to the site of the gene encoding BCL11A and
its identification led to the elegant studies of Sankaran and co-workers demonstrating
the role of BCL11A in the regulation of -gene expression. The locus on chromosome 6
is located between the genes encoding HBS1L and MYB. The mechanism by which this
locus causes elevation of Hb F is thus far poorly understood. Finally, mutations in the
gene on chromosome 19 encoding the erythroid-specific transcription factor EKLF1
have been shown to be associated with a form of HPFH (18, 19). The involved
mechanism is probably through the regulation of BCL11A levels, because it has been
demonstrated that EKLF1 binds to the promoter of the BCL11A gene and regulates the
expression of the gene (20).
3.3.5 Conclusion
Significant insights into the normal regulation of expression of the human globin gene cluster have been obtained by a detailed analysis of a group of disorders
called HPFH. On the basis of this information, several important regulatory elements
have been identified for the normal functioning of the individual genes in the cluster
during the developmental switch from fetal to adult hemoglobin gene expression, as well
as for the abnormal persistent expression of the -globin genes in adults with HPFH.
These results provide a more sophisticated understanding of the molecular basis of
these syndromes and point to certain strategies for potential future molecular and
cellular therapies for globin gene disorders.
71
References
1. Bollekens JA, Forget BG. Delta beta thalassemia and hereditary persistence of fetal
hemoglobin. Hematol Oncol Clin North Am 1991;5(3):399-422.
2. Forget BG. Molecular basis of hereditary persistence of fetal hemoglobin. Ann N Y
Acad Sci 1998; 850:38-44.
3 Weatherall DJ, Clegg JB. The Thalassaemia Syndromes. 4th ed. Oxford ; Malden,
MA: Blackwell Science; 2001.
4. Sankaran VG, Xu J, Byron R, et al. Functional element necessary for fetal
hemoglobin silencing. N Engl J Med 2011; 365(9):807-14.
5. Feingold, EA, Forget BG. The breakpoint of a large deletion causing hereditary
persistence of fetal hemoglobin occurs within an erythroid DNA domain remote from the
-globin gene cluster. Blood 1989; 74: 21782186.
6. Kosteas T, Palena A, Anagnou NP. Molecular cloning of the breakpoints of the
hereditary persistence of fetal hemoglobin type-6 (HPFH-6) deletion and sequence
analysis of the novel juxtaposed region from the 3' end of the beta-globin gene cluster.
Hum Genet. 1997;100: 441-5.
7. Anagnou NP, Perez-Stable C, Gelinas R, et al. Sequences located 3' to the
breakpoint of the hereditary persistence of fetal hemoglobin-3 deletion exhibit enhancer
activity and can modify the developmental expression of the human fetal A gammaglobin gene in transgenic mice. J. Biol Chem 1995; 270: 10256-63.
8. Huisman TH, Schroeder WA, Efremov GD, et al. The present status of the
heterogeneity of fetal hemoglobin in beta-thalassemia: an attempt to unify some
observations in thalassemia and related conditions. Ann N Y Acad Sci 1974;232(0):10724.
9. Bank A, O'Neill D, Lopez R, et al. Role of intergenic human - -globin sequences in
human hemoglobin switching and reactivation of fetal hemoglobin in adult erythroid
cells. Ann N Y Acad Sci 2005;1054:48-54.
10. Chakalova L, Osborne CS, Dai YF, et al. The Corfu thalassemia deletion disrupts
-globin gene silencing and reveals post-transcriptional regulation of HbF expression.
Blood 2005;105:2154-60.
11. Sankaran VG, Xu J, Orkin SH. Transcriptional silencing of fetal hemoglobin by
BCL11A. Ann N Y Acad Sci. 2010;1202:64-8.
72
73
Chapter 3
Diagnostic Laboratory Methods
3.4 Flow Cytometry Measurements of Cellular Fetal Hemoglobin, Oxidative
Stress and Free Iron in Hemoglobinopathies
Eitan Fibach, MD
3.4.1 Flow Cytometry of Blood Cells
Flow cytometry (FC) is a common methodology in clinical diagnostic and
research laboratories. In hematology, it is mainly used for diagnosis, prognosis,
determining therapy efficacy and follow up of patients with leukemia or lymphoma
(1). It is also used for diagnosis of red blood cell (RBC) abnormalities such as in
Paroxysmal Nocturnal Hemoglobinuria (2) and hereditary spherocytosis (3). In
this review, I will summarize FC methodologies for analysis of RBC (and other
blood cells) from patients with hemoglobinopathies with respect to their fetal
hemoglobin (HbF) and free iron (labile iron pool, LIP) contents and parameters of
the oxidative state.
FC analyzes individual cells in a liquid medium. Most techniques use antibodies
directed against internal (following fixation and premeabilization of the
membrane) or surface antigens. The antibodies are labeled with fluorescence
probes (fluochromes) either directly or indirectly (by a secondary antibody). In
addition to antibodies, other fluorescent compounds can be used. For example,
propidium iodide, which binds stochiometrically to nucleic acids, is commonly
used for determining cell viability and their distribution in the cell cycle (4).
Following staining, the cells are analyzed by a flow cytometer; they are first
hydro-dynamically focused in a narrow sheath of physiological solution before
74
being intercepted by one or more laser beams resulting in light scatter and
fluorescence emission. Depending on the number of laser sources and
fluorescence detectors, several parameters (commonly 6, but up to 18) can be
simultaneously detected on each cell: Forward light scattering and side light
scattering provide correlates with regards to size and granularity of the cells,
respectively, and fluorescence light emission by the fluorochromes correlates
with the expression of different antigens as well as other cellular parameters (see
below).
FC is superior to other techniques in several aspects: (I) Technology is widely available
as mentioned above, most hematology and immunology laboratories use FC for both diagnosis
and research purposes. (II) Only cell-associated fluorescence is measured, excluding soluble or
particulate fluorescence. (III) Each cell is analyzed individually, but since measurement is rapid
(msec), a large number of cells can be analyzed (ranging from 0.1-10 x10 5 cells) within a few
minutes. The results are therefore statistically sound even for very small sub-populations. (IV)
Various sub-populations can be identified and measured simultaneously. (V) The method
produces mean values for each sub-population, and therefore avoids the inaccuracy of
biochemical methods that produce mean value for the whole population. This is of crucial
importance when mixed populations are studied. (VI) The procedure can be automated to permit
high throughput analysis (e.g., for screening of large libraries of compounds for inducers of
HbF). Although the FC data are expressed in arbitrary fluorescence units rather than weight or
molar concentrations, they are useful for comparative purposes.
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,
75
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).
76
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).
blood to a quantity that allows differences before and after treatment to become
apparent. Measuring differences in F-RBC by FC may be more sensitive, and
measuring F-reticulocytes (retics) may provide early indication of treatment efficacy
(15): Retics have a very short life-span (1-2 days) compared to mature RBC (120 days
in normal subjects) and therefore measuring peripheral blood F-retics more closely
characterizes the current status of HbF production in the bone marrow. Measuring Fretics can indicate the efficacy of the drug and/or the patients compliance several days
after treatment initiation. Such follow up is very important since about 30% of the
patients are non-responders. It is imperative that such patients be identified as early as
possible and the treatment (that is not without potential risks) be discontinued and
replaced by treatment with another drug (e.g., butyroids).
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).
80
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.
another marker was introduced carbonic anhydrase (CA) (18). The CA isoenzymes
that are mainly represented by CAI and CAII (19) are fully expressed in the RBC only
after birth (20,21). The "Fetal Cell Count kit" manufactured by IQ Products (Groningen,
the Netherlands), which uses a combination of a murine monoclonal antibody directed
to HbF and a polyclonal antibody to the CAII isoform, has significantly improved this
assay (11,18). Most of the RBC of fetal origin do not express CA but highly express HbF
(CA-HbF++), while RBC in adult blood express CA but do not express HbF (CA +HbF-).
Some adult F-cells which express CA and HbF (CA+HbF+) can be differentiated from
fetal F-cells (CA-HbF++) present in FMH based on the extent of HbF and CA expression.
Until recently, -thalassemia major was lethal. Improvements in treatment, such as the
introduction of blood transfusions and iron chelation, have considerably improved the life
expectancy as well as the quality of the patients life, including the ability of thalassemic women
to give birth. Recently, we were confronted with a case of a possible FMH in a -thalassemic
woman. To establish the usefulness of the CA/HbF procedure, i.e. differentiating between fetal
RBC and the maternal RBC, we screened non-pregnant -thalassemic patients (men and
women). The results demonstrated, in addition to adult non-F RBC (CA +HbF-) and adult F-RBC
(CA+HbF+), two other sub-populations, CA+HbF++ and CA-HbF++. The presence in these patients
of the latter RBC phenotype, which characterizes fetal cells, precludes the use of the CA/HbF
method for the detection of FMH in thalassemia.
3.4.5 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
generated in most cells mainly during energy production. Although important for various
82
aspects of normal physiology (e.g., signal transduction), ROS interact with and damage
various cell components when they are in excess. To protect against the deleterious
effects of ROS, cells maintain an effective antioxidant system consisting of water- or
lipid-soluble antioxidants and enzymes that remove ROS by metabolic conversion.
When the oxidant/anti-oxidant balance is tilted in favor of the oxidants, oxidative stress
ensues (22). Although oxidative stress is not the primary etiology of
hemoglobinopathies, it mediates several of their pathologies, including hemolysis which
results in chronic anemia. Hemolysis occurs both in the bone marrow, where developing
erythroid precursors undergo enhanced apoptosis (ineffective erythropoiesis) and in the
peripheral blood, where mature RBC undergo lysis in the blood vessels (intra-vascular
hemolysis). Destruction also occurs in reticuloendothelial tissues, such as the spleen,
where mature RBC undergo phagocytosis by resident macrophages (extra-vascular
hemolysis) (22).
Various factors are responsible for oxidative stress in RBC of patients with hemoglobinopathies. In -thalassemia, excess -globin chains form unstable tetramers that
dissociate into monomers and then are oxidized, first to met-Hb and then to
hemichromes which precipitate intracellularly with time (23). Following the release of
heme and iron, there is deposition of the protein moiety on the plasma membrane. The
outcome of this chain of events is enhanced formation of ROS, catalyzed by free iron,
with a variety of deleterious effects on the membrane lipids and proteins, including
oxidation of the membrane protein band 4.1 and a decrease in spectrin/band3 ratio (24).
In -thalassemia, the - and -globins, which are produced in excess, do not precipitate
right away, but form the soluble tetramers 4 (Hb Barts) and later the 4 (HbH), which
83
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
and t-butylhydroxyperoxide and with the catalase inhibitor sodium azide, while treatment
84
with ROS scavengers such as N-acetyl cysteine decreased the fluorescence. ROS can
also be measured by dihydrorhodamine 123, which freely enters into cells, and after
oxidation by ROS to rhodamine 123 emits a bright red fluorescence (29).
Reduced glutathione (GSH), the main cellular antioxidant, can be measured
using mercury orange (26), which forms fluorescent adducts with GSH via the
sulphydryl group, producing an S-glutathionyl derivative that emits red-orange
fluorescence (30). The probe reacts more rapidly with non-protein thiols, such as GSH,
compared with thiol-containing proteins, allowing specificity under controlled staining
conditions (31). The validity of this method was confirmed by demonstrating that Nethylmaleimide, which totally blocks thiol groups, decreased the fluorescence in a dosedependent manner. To ascertain that non-protein thiols are being stained, cells were
incubated with diethylmaleate, a specific non-protein thiol-depleting agent. This weak
electrophil of the ,-unsaturated carbonyl group, which reacts with GSH only in the
presence of glutathione transferase, markedly suppressed the mercury orange
fluorescence, suggesting that GSH was the principle thiol which was stained by the dye
(32). Although there is no direct proof that the probe is specific for GSH, the assay
measures predominantly GSH, since it is the main non-protein thiol constituent of the
cellular thiol pool (33).
Other parameters of oxidative stress measured by FC are membrane lipid
peroxidation by staining with fluor-DHPE (26), and externalization of
phosphatidylserine (PS) moieties, a marker of damage to the membrane lipid, by
fluorochrome-conjugated annexin-V (34).
85
86
Fig. 3. Flow cytometry of ROS and GSH in normal and thalassemic RBC. Blood cells derived
from a normal donor (A,C) and a thalassemic patient (B,D) were stained for ROS (A,B) and GSH
(C,D) following 1-h pre-incubation with (white) or without (pink) 2 mM H 2O2. Histograms of RBC
are shown.
hemin) that are released during hemolysis can add to the iron load and further
aggravate the hemolysis.
Normally, iron is transported in the circulation bound to transferrin and is
transferred into cells through the surface transferrin-receptor (36). Most of the
intracellular iron is firmly bound to various components such as Hb, heme and
cytochrome C; excess is stored in ferritin (37). In iron overload, serum iron which
exceeds the binding capacity of transferrin is present in the form of non-transferrin
bound iron (38). This iron can be taken up through a transferrin-independent pathway,
to form the cellular unbound "labile iron pool" (LIP) (16). The small fraction of LIP was
suggested as a low molecular weight intermediate or transitory pool between
extracellular iron and cellular firmly-bound iron (39). LIP is redox active and it
participates in generation of free radicals by the Fenton and Haber-Weiss reactions and
consequently in cell and tissue damage (40).
Since iron overload plays an important role in the pathology of transfused
patients with -hemoglobinopathies, the patients are commonly treated with iron
chelators. The three chelators currently in clinical use are deferioxamine, deferiprone
and deferasirox (41). Evaluation of iron overload is important for assessing its severity
and for determining the efficacy of iron chelation therapy. The parameters usually tested
are serum ferritin protein level and transferrin iron saturation. However, serum ferritin is
an acute phase reactant that may increase by iron-independent factors such as
infection, inflammation and liver disease (42). In addition, serum ferritin levels often fail
to predict impending cardiac iron overload and ensuing cardio-myopathies (43). The
advent of non-invasive proton relaxation assays (by NMR R2* or T2*) of organs has
88
89
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.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
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.
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.
90
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
92
Chapter 3
Diagnostic Laboratory Methods
3.5
3.5.4 Introduction
electrophoretic methods are accomplished at alkaline pH (8.6) and acid pH (5.6), and
the commonly used solid phases for this purpose are described here.
94
plate form is used in the semi-automated instruments (SPIFE 2000 and SPIFE 3000) for
handling a larger volume of testing. Helenas fully automated instrument (SPIFE 4000)
utilizes a different plate form than QuickGel. Detailed information about AGE procedures
of these two manufacturers can be obtained from their web site (www.sebia.com and
www.helenalaboratories.com).
In Fig 2 we have presented the computer simulation of the electrophoretic
mobilities of the commonly used AFSC control and few hemoglobin bands obtained
from AGE at alkaline pH.
used for the electrophoretic purpose (Beckman-Coulter uses maleate buffer in their
Paragon kit), therefore it is also called citrate agar electrophoresis. Commercially the AE
kits (plates, reagents, consumables, etc.) are also available from Sebia, France
(HYDRAGEL ACID HEMOGLOBIN) and Helena Laboratories, USA (Titan Gel and
QuickGel). In both cases, hemoglobin AFSC control is used to confirm the
electrophoretic mobility of the unknown (i.e. Hb S, Hb C, Hb E, etc.). Quantification of
the bands is not required and the electrophoregrams are evaluated visually. Laboratory
procedures for AE by Sebia and Helena Laboratories can be obtained from their web
sites (www.sebia.com and www.helenalaboratories.com). In Fig 3, we have presented a
computer simulation of an electrophoregram of the AE.
visual one, therefore a quicker way to examine these relative mobilities was to convert
them into a chart as depicted in Appendix I.
It will not be out of place to mention here that for >30 years in the Hemoglobin
Laboratories of Henri Mondor Hospital (Creteil, France), Professor Henri Wajcman and
associates have organized a database of the electrophoretic mobility of > 400 Hb
variants, using a similar format to that proposed by Schneider and Barwick. The
Wajcman group included in their database the results of a) IEF on polyacrylamide gel,
b) electrophoresis on cellulose acetate at alkaline pH, c) citrate agar electrophoresis, d)
electrophoresis of dissociated globin chains in 6M urea at pH 6.0 and 9.0 or in the
presence of Triton X-1004.
An excerpt of eight hemoglobins from the chart developed by Ellerbrook and
Mathews3 is shown in Fig 3 for instructional purposes.
Fig 3. Combined agarose gel (pH 8.6), and citrate agar (pH 5.6) electrophoretic
pattern presentation for instructional purposes.
The area labeled Alkaline on the left side of this figure depicts the mobility of
the named hemoglobins under alkaline conditions. The perpendicular lines
represent the relative mobility of Hb C (-10), Hb S (-5.2), Hb F (-2.6), Hb A (0),
99
100
Looking at the left side of the chart, Hb J Baltimore migrates slower than Hb J
Toronto because the bulk of the hemoglobin has not moved as far as one might expect
Hb J Toronto to move. Acid electrophoresis is of no assistance in this case because
these fast hemoglobins do not migrate differently, and thus all end up lined with Hb A.
The mobility of Hb C Siriraj is not different from Hb C under acidic conditions, but can be
differentiated under alkaline pH. In this case since the alkaline separation would have
been done first the only apparent observation would be the presence of an abnormal
hemoglobin band migrating between Hb F and Hb S. Very few hemoglobins migrate like
hemoglobin S so this second test is very useful in narrowing down the possible identity
of this variant. The chart in Appendix I contains the relative mobilities of 165
hemoglobins. The most common variants were discovered first so this chart should
encompass the relative mobilities of most of the hemoglobins found.
101
References
1. Pauling L, Itano HA, Singer SH, Wells IC. Sickle cell anemia, a molecular
basis disease. Science 1949; 110: 53.
2. Barwick RC, Schneider RG. The computer-assisted differentiation of hemoglobin
variants, in Human Hemoglobins and Hemoglobinopathies:
A review to1981. Texas Reports on Biology and Medicine 1980-81; 40:
143-156
3. Helena Laboratories, Beaumont, Texas, USA
4. Wajcman H. Electrophoretic Methods for Study of Hemoglobins. Methods in
Molecular medicine, vol 82: Hemoglobin Disorders: Molecular methods and
Protocols, Edited by: Ronald L. Nagel, Humana Press Inc., Totowa, NJ.
102
Appendix 1
Appendix 1 continued
104
Chapter 3
Diagnostic Laboratory Methods
3.6 Capillary Zone Electrophoresis
Zia Uddin, PhD
3.6.1 Introduction
During the last five decades separation science has witnessed unparallel growth.
Chromatography and electrophoresis are the main techniques that are routinely used
worldwide for the separation, identification, and quantification of analytes in clinical
laboratories. Capillary zone electrophoresis (CZE) played a significant role in the
completion of the human genome project. Introduction of CZE instruments by BeckmanCoulter, Sebia, and Helena Laboratories not only automated but also increased the
sensitivity, specificity and reproducibility of the clinical laboratory procedures (e.g.,
serum protein electrophoresis, immunotyping, hemoglobin variant identification for both
the adult and newborn). Besides references listed at the end of this section, the
interested reader is advised to also review the online literature on CZE (e.g., Righetti,
PG, and Guttman, A. 2012 Capillary Electrophoresis. eLS.)
3.6.2
Basic Principle
In simple terms CZE is a liquid flow electrophoresis in which buffer has replaced
the solid support medium (e.g., agarose gel), and the separation occurs due to the
interaction of the analyte with the pH of the buffer. For this reason initially CZE was also
called Free Solution Capillary Electrophoresis. In Figure 1, a pictorial illustration of
CZE principle is presented.
105
106
Cathode
Anode
Power supply to generate high voltage (10,000 volts)
Catholyte (buffer solution at the cathode end)
Anolyte (buffer solution at the anode end)
Capillary facilitated with a cooling device
Detector (415 nm for hemoglobins)
Computer for data handling and storage
107
Hemoglobin variants can be separated on CZE as is the case with other proteins.
This method is the most advanced and dedicated alternative to the classic alkaline and
acid electrophoresis and the more sophisticated IEF. Chromatography, the separation
alternative on column, has developed from separation on size, charge and hydrophobic
interaction to the modern dedicated high performance liquid chromatography (HPLC),
as we know today. Both of these dedicated methods (CZE and HPLC) have the
advantage of using minimal amounts of material, of providing a separation in a matter of
minutes, with high reproducibility and sensitivity and above all they are able to measure
virtually all fractions including those present at low levels but essential for the diagnosis
or hemoglobinopathy. In addition these two methods may complement each other up to
a certain extent compensating for specific errors.
In Figure 3 we have presented a CZE scan of the most commonly used AFSC
control in the clinical laboratory, which illustrates the position of HbA,
HbF, HbS, and HbC peaks corresponding to their respective zones. Later on in this
book (case studies) we have also presented the CZE scan of the hemoglobin variant for
each case.
One drawback of CZE is the assignment of the migration position of the
hemoglobin bands into Z1-Z15 (Table 1) in cases when the HbA / Hb A2 are absent in
the specimen of interest, e.g. Hb S-C disease. This drawback is due to the shifting of
band positions in the absence of Hb A / Hb A2. This limitation of CZE is avoided by
mixing (1:1 ratio) the specimen devoid of HbA/HbA 2 with a specimen containing Hb A,
and performing the CZE test thus achieving the relevant migration position and zoning
(Z1-Z15).
109
2=
Z(C)
3=
Z(A2)
4=
Z(E)
5=
Z(S)
6=
Z(D)
110
7=
Z(F)
9=
Z(A)
10
11
12
13
14
Hb N-Seattle
15
Hb I (I-Texas)
111
112
References
1
114
Chapter 3
Diagnostic Laboratory Methods
3.7
Isoelectric Focusing
David R. Hocking, PhD
3.7.1 Introduction
Isoelectric focusing (IEF), also known as electrofocusing and isoelectricfocusing
electrophoresis, is a separation method that resolves complex mixtures of proteins by
their isoelectric points (pI). IEF is a type of electrophoresis that forms a pH gradient
during the run. The technique is capable of extremely high resolution. The formation of
a pH gradient is accomplished by blending a mixture of small molecular weight carrier
ampholytes into a support matrix, or gel, usually of purified high-grade agarose. An
anolyte solution (i.e. acetic acid) and a catholyte solution (i.e. ethanolamine) are
saturated onto paper electrode wicks then are placed directly on opposite ends on the
surface of the agarose gel. Proteins (i.e. hemoglobins) that are to be separated are
placed near the cathode wicks using a clear plastic with rectangular wells cut out. The
protein solution (i.e. hemoglobin hemolysate) is then pipetted in the defined wells and
allowed to diffuse into the gel. An electric current is then passed through the medium.
The proteins move through the changing pH gradient until it reaches a point in which the
pH of that molecules pI is reached. At this point the protein no longer has an electric
charge and becomes neutral, or isoelectric (due to the protonation or de-protonation of
the associated functional amino and carboxyl groups) and as such will not proceed any
further within the gel. The proteins become focused into sharp stationary bands with
each protein positioned at a point within the newly formed pH gradient corresponding to
its pI.
115
Note: All the IEF figures in this compendium were obtained after agarose gel
electrophoresis on the Wallac Resolve Hemoglobin System (Perkin Elmer), and
the scans were procured using the Wallac WS-1010 IsoScan Imaging System
(Perkin Elmer).
3.7.2 IEF of Normal Adult Hemoglobins: HbA (Adult), HbF (Fetal), HbA 2
Normal adult hemoglobins are comprised of , , and globin chains paired as
~96% HbA (2 2), ~3% HbA2 (2 2) and <2% HbF (2 2) tetramers (Figure 1). One can
usually find the glycated form of HbA, or HbA 1c , anodal to it as shown in the Figure 2.
Figure 1.
116
Aging bands, HbA3, are also anodal to HbA and are the result of posttranslational modifications such as acetylation and glutathione
attachment.
It should be noted that beta-chain variants such as HbS, HbE, HbD, etc., will also
Figure 2.
display glycated forms anodal to the variant (HbS 1c, HbE1c, HbD1c). This observation is
critical to note should the patient exhibit symptoms of diabetes where their blood
glucose values are documented to be high. The percentage can be upwards of 10-20%
in cases of uncontrolled blood glucose levels.
3.7.3 IEF of Normal Newborn Hemoglobins: HbF (Fetal) and HbA (Adult)
Normal newborn hemoglobins are comprised of ~ 60-85% HbF ( 2 2) and 1540% of HbA (2 2). It is very rare to see HbA2. About 10% of HbF is partially acetylated
HbFac, which results in higher oxygen affinity, an important property needed for
newborns. Aging hemoglobin bands, or HbF3 are always anodal to HbFac, and are the
result of glutathione (an antioxidant, preventing damage to important cellular
components caused by reactive oxygen species) attachment.
Representative patterns of newborn hemoglobin are shown in Figure 3. Fresh
cord blood is shown in channel 3a. A sample that was collected and stored using a filter
paper is shown in channel 3b. Note the increased levels of HbF 3 in the stored blood
collected on filter paper.
117
Figure 3. IEF of normal newborn hemoglobins: HbF (fetal) and HbA (adult)
118
examples show the beta-chain mutation along with the Relative Charge Value (RCV)
119
parent. If one of the alpha genes has a mutation, then one out of the four, or ~25% of
the hemoglobin, will be the variant, not the typical 50% from a beta-chain variant. The
affected alpha globin chain will form dimers with the non-alpha globin chains. HbGPhiladelphia is a common alpha-chain variant that is shown below (Figure 10).
Note that the percentage of HbG-Philadelphia (Figure 10) relative to HbA is less
than what is seen in the beta variant HbD or HbD-Punjab. This is a clue to suspecting
an alpha variant. Additionally you should also observe that there should be another
120
band cathodal to HbA2. This is due to the variant alpha globin chain combining with the
delta chain.
121
The example in Figure 11 is a rare combination of the beta HbS variant and the
alpha HbG-Philadelphia variant. Note the presence of four prominent bands: HbA, HbGPhiladelphia, HbS and the hybrid, HbSG-Philadelphia, the tetramer formed by the
dimers of -GPhiladelphia and S. Also note the HbA 2 variant that resulted from the G
and dimers. It will be seen cathodal (negative electrode) to the hybrid.
3.7.6 IEF of Thalassemias
A
typical +-
thalassemia is shown in Figure 12. Note that the percentage of HbA is reduced (95%)
and the amount of circulating HbA2 is increased (>3.5%). Beta thalassemias occur in
persons of Mediterranean origin, and to a lesser extent, Chinese, other Asians, and
African Americans. +-thalassemia is also known as Thalassemia Minor and occurs if
you receive the defective beta-globin gene from only one parent. Persons with this form
of the disorder are carriers of the disease, Cooleys anemia or beta thalassemia major
(0), if their other partner also passes their defective gene to the baby.
122
The pattern in Figure 13 is typical of those individuals presenting with a severe form of
Sickle Cell disease. In this example, the patient inherited the HbS from one parent and
is missing the beta globin gene from the other parent. The patient, though missing a
beat globin gene, has compensated for the missing beta-globin gene with the
persistence of making HbF from the gamma-globin gene.
Conclusion
IEF can be an important tool in assiting the laboratorian in the dection and
interpretation of hemoglobin variants. The technique offers improved resolution over
traditional electrophoretic methods and is useful for both adult and newborn patients. By
careful observation, one can determine if the variant is either a or variant or
combination. One can also correctly interpret -thalassemias.
References:
1. David R. Hocking. The Separation and Identification of Hemoglobin Variant by
Isoelectric Focusing Electrophoresis (May 2004), Catalog # HC-60, Perkin
123
Elmer Life and Analytical Sciences, Wallac Oy, P.O. Box 10, FIN-20101
Turku, Finland. Tel. 358-2-2678111 Fax. 358-2-2678357
Web site: www.perkinelmer.com
2. Additional information about the IEF procedures and instruments can be
solicited from:
a) Petra Furu, Ph.D., Global Business Manager, Specialty Diagnostics,
Perkin Elmer , Mustionkatu 6 / 20750 Turku / Finland.
e-mail: petra.furu@perkinelmer.com Tel. 358 2 267 8497
b) William R. Fisher, Technical Support Specialist, Specialty Diagnostics,
Perkin Elmer, 520 South Main Street, Akron, OH 44311, USA
e-mail: william.fisher@perkinelmer.com Tel. 330-564-4883
124
Chapter 3
Diagnostic Laboratory Methods
3.8
3.8.1. Introduction
In 1973 I had the privilege of attending a short course on High Performance
Liquid Chromatography (HPLC), sponsored by the American Chemical Society at
Virginia Polytechnic Institute, Blacksburgh, Virginia, USA. The teachers of this course
were Drs. Lloyd R. Snyder and Joseph J. Kirkland. These two scientists are responsible
for several advancements in HPLC, and their most significant contribution in
collaboration with Dr. John W. Dolan is their latest book (Snyder LR, Kirkland JJ, Dolan
JW, Introduction to Modern Liquid Chromatography, 3 rd Edition, John Wiley & Sons,
Hoboken, NJ 20010). Persons interested in HPLC shall find this book very helpful in
understanding the theory and practice of HPLC, and the components of HPLC (solvent
system, pump, injection port, column, stationary phase, detector, computer, etc.).
Additional literature about HPLC can be accessed from the following Internet sites:
http://www.lcresources.com
http://lchromatography.com/hplc find/index.html
http://tech.groups.yaho.com/group/chrom-L/links
http://userpages.umbc.edu/~dfrey1/Freylink
http://www.chromatographyonline.com
Note: The name High Pressure Liquid Chromatography was initially used, however now
the word Pressure is replaced by Performance. In this book we shall therefore use
High Performance Liquid Chromatography nomenclature.
125
127
Figure 2. Peak at 0.81 (Hb A1c) and at 3.1 (Hb A2). Adopted from the Technical Manual
of D-10, Bio-Rad, Hercules, CA.
b) Hb OIndonesia in India: a rare observation
The father is heterozygous for Hb OIndonesia and the mother is normal, however the
1
daughter has an HPLC pattern similar to her father (Figure 3). Although the normal
hemoglobin fractions (Hb F, Hb A, Hb A2)as well as the common variants (Hb S and Hb
C) all have distinct retention times,there are less prevalent variants with similar or
128
129
130
Cited references:
1.
Chapter 4
Globin Chain Analysis
131
Fig 1.
133
Fig 2.
References
134
135
Chapter 4
Globin Chain Analysis
4.2 Reverse-Phase High Performance Liquid Chromatography
Zia Uddin, PhD and Rita Ellerbrook, PhD
Conventional charge based separation techniques (electrophoresis, ion-exchange liquid
chromatography, and isoelectric focusing) are sometimes ineffective in the separation of
hemoglobins, when the amino acid substitution does not cause a net charge differential.
Several hemoglobin variants migrate upon electrophoresis and elute upon ion-exchange liquid
chromatography in the positions of hemoglobin A, S, D, A2 or C. Further clarification is
necessary for newborn screening or in cases of unexplained clinical disorders. Additional
testing is required to resolve this matter, e.g., DNA studies, reverse-phase chromatography
(RPC), liquid chromatography-mass spectrometry (LC-mass) primarily employing the
electrospray ionization (ESI) technique, and Sanger sequencing.
There are three main chromatographic techniques for the separation of peptides and
proteins, e.g., a) size exclusion, b) ion-exchange, and c) hydrophobic interactions. For a
detailed study of the theory and practice of the liquid chromatography of peptides and proteins
in general and reverse phase high pressure liquid chromatography (RP-HPLC) in particular
(Chapter 13, Section 13.4), the interested reader is advised to review the 3 rd edition of
Introduction to Modern Liquid Chromatography, by Lloyd L. Snyder, Joseph J. Kirkland and
John W. Dolan (A John Wiley and Sons, Inc. Publication, 2010). Howard and Martin 1 first
introduced RPC in 1950, and since then, several improvements in the methodology and
advancements in its application in the separation of peptides and proteins were achieved. The
136
recent literature on RPC can be accessed via the Internet (http://www.lcresources.com) and
the specialized journals in the field.
The separation of globin chains by RP-HPLC is based on the hydrophobicity of the
globin chains, which is defined as a tendency of not combining with water or incapable of
dissolving in water. The RP-HPLC consists of a non-polar column in combination with a polar
mixture of water plus an organic solvent as a mobile phase. In this section, we shall
demonstrate the usefulness of RP-HPLC in the separation of globin chains leading to the
identification of hemoglobin variants. Experimental details of RP-HPLC of globin chains
(hemoglobin specimen preparation, selection of column, solvent system, high pressure liquid
chromatography instrumentation, temperature, retention times, detection system, etc.) were
provided by the work of three research groups in this field in Italy, France and USA 2-7.
A few RP-HPLC chromatograms (Fig 1-5) are shown to illustrate the application of this
technique in the separation of globin chains. These chromatograms are either replicated
exactly as cited in the literature (abscissa depicting actual retention times in minutes), or for
comparison, as a normalized scale for the retention times. In the normalized scale, the
retention time for the normal chain is 10, and for the normal chain is 20 (Fig 4). The elution
window is of 0.5 units width. It is emphasized that the retention times of RP-HPLC might vary
depending upon the experimental conditions, but the overall shape of the chromatogram is
highly reproducible.
137
Normal Cord Blood: Fetal blood obtained at 18-20 weeks of gestation age, shows the
preponderance of chains (Fig 1).
138
Fig 2. RP-HPLC chromatogram of a normal adult blood (Kutlar F, Kutlar A, Huisman THJ.
Separation of Normal and Abnormal Hemoglobin Chains by Reverse-Phase High Performance
Chromatography. J. Chromatogr 1986, 357: 147-153) 3.
Adult hemoglobin A-S trait : In hemoglobin S the variation in the chain is due to the
substitution of glutamic acid by valine [6(A3)]. This is shown in Fig 3 by the separation of A
and S chains.
139
electrophoresis and isoelectric focusing, however both the D-Punjab and Korle-Bu chains can be
easily separated by RP-HPLC7.
Several electrophoretic separation techniques did not distinguish 4 Hb Camperdown
[104(G6) ArgSer] from Hb Sherwood Forest [104(G6) ArgThr]. In this example there is
no change on the charge of the two hemoglobin variants as only the hydrogen atom on serine
is replaced by a methyl group of threonine. The substitution of serine by threonine on the same
position of the chain changes the hydrophobicity (presumably by altering the
secondary/tertiary structure of the globin chain), thus resulting in their separation by RP-HPLC
(Fig 4).
Fig. 4. Normalized scale of retention times of globin chains on RP-HPLC, a) retention times of
A (10), (20), G (28) and A (35), b) Hb Campertown (14.1-14.5), and c) Hb Sherwood
Forest (16.1-16.5). Adopted from: Wajcman H, Riou J, Yapo AP. Globin Chain Analysis by
Reversed Phase High Performance Liquid Chromatography: Recent Developments.
Hemoglobin 2002, 26: 272-2844.
141
142
143
Henri Wajcman and associates published the retention times on RP-HPLC of over 200
abnormal globin chains which were also made available on the web 7. Additional
chromatographic and electrophoretic information about hemoglobin variants can be obtained
from the database9-11.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Howard GA, Martin JP. The separation of the C12-C18 Fatty Acids by ReversedPhase Partition Chromatography. Biochem J 1950; 46: 532-538.
Leone L, Monteleone M, Gabutti V, Amione C. Reversed-Phase High
Performance Liquid Chromatography of Human Globin Chains. J Chromatogr
1985; 321: 407-419.
Kutlar F, Kutlar A, Huisman THJ. Separation of Normal and Abnormal Hemoglobin
Chains by Reversed-Phase High-Performance Liquid Chromatography. J
Chromatogr 1986; 357:147-153.
Wajcman H, Riou J, Yapo AP. Globin Chain Analysis by Reversed Phase High
Performance Liquid Chromatography: Recent Developments. Hemoglobin 2002; 26:
271-284.
Yapo PA, Datte JY, Yapo A, Wajcman H. Separation of Adult Chains of Abnormal
Hemoglobin: Identification by Reversed-Phase High-Performance Liquid
Chromatography. J Clin Lab Anal 2004;18: 65-69.
Zanella-Cleon I, Becchi M, Lecan P, Giordano PC, Wajcman H, Francina A.
Detection of a Thalassemic -Chain Variant (Hemoglobin Groene Hart) by
Reversed-Phase Liquid Chromatography. Clin Chem 2008; 54:1053-1059.
Wajcman H, Riou J. Globin chain analysis: An important tool in phenotype
study of hemoglobin disorders. Clinical Biochemistry 2009; 42:1802-1806.
Dainer E, Wenk RE, Luddy R, Elam D, Holley L, Kutlar A, Kutlar F. Two new
hemoglobin variants: Hb Sinai-Greenspring [34 (16) ValIle, GTC>ATC] and Hb
Sinai-Bel Air [53 (D4) AlaAsp, GCT>GAT]. Hemoglobin 2008; 32(6): 588-591
Hardison RC, Chui DHK, Giardine B, et al. HbVar: a relational database of
human hemoglobin variants and thalassemia mutations at the globin gene server.
Hum Mutat 2002; 19: 225-33 (http://globin.bx.psu.edu/hbvar/smenu.html).
Giardine B, van Baal S, Kaimakis P, et al. HbVar database of human
hemoglobin variants and thalassemia mutations: 2007 update. Hum Mutat
2007; 28(2): 206.
Patrinos GP, Giardine B, Riemer C, et al. Improvements in the Hbvar
database of human hemoglobin variants and thalassemia mutations for
population and sequence variation studies. Nucleic Acids Res 2004 Jan 1;
32: D537-41(Database issue).
144
Chapter 4
Globin Chain Analysis
4.3 Globin Chain Gene Mutations: DNA Studies
Joseph M. Quashnock, PhD
2
4.3.1 Introduction
Hemoglobin A is the designation for the normal hemoglobin that exists after birth.
Hemoglobin A is a tetramer with two alpha chains and two beta chains (22).
Hemoglobin A2 is a minor component of the hemoglobin found in red cells after birth
and consists of two alpha chains and two delta chains (22). Hemoglobin A2 generally
comprises less than 3% of the total red cell hemoglobin. Hemoglobin F is the
predominant hemoglobin during fetal development. The molecule is a tetramer of two
alpha chains and two gamma chains (22). Hemoglobinopathies result from amino
acid changes in the alpha or beta globin chains. Most of the mutations are single amino
acid substitutions caused by a single base change, however, other amino acid
mutations can be found due to various base alterations such as:
1.
More than one amino acid change e.g. the alpha chain mutation of Hb J
Singapore with Asn>Asp and Ala>Gly, the beta chain mutation of Hb
Poissy with Gly>Arg and Ala>Pro.
2.
3.
4.
Hybrids such as the Lepore globin gene that is a crossover of beta and
delta globin genes that produces hemoglobin made up of two normal
145
3.
3.
5.
specimen with DNA primers, the substrate nucleic acid bases of adenosine, thymidine,
guanosine, and cytidine, DNA polymerase, and a DNA detection probe. The mixture is
repeatedly heated to ~ 95 C and cooled; each heating and cooling cycle doubles the
amount of PCR product produced; most PCR assays use 25-40 cycles.
Rapid cycle PCR is based upon the low heat capacity of air and the ability to
ramp through temperatures at a far greater rate than instruments using thermocyclers
that rely upon heating and cooling an aluminum block.
LightCycler from Roche also incorporate the improvement of using glass capillary
tubes to serve as both the reaction vessel and optical cuvette. Detection is by the
Fluorescence Resonance Energy Transfer (FRET) method described below, however,
the time required to complete 25-40 cycles is on the order of 30-40 minutes as opposed
to 3-4 hours for aluminum block thermocyclers.
Detection of FRET probes is performed by measuring hybrid stability as modified
by the introduction of base pair mismatch/es. Mismatch destabilization is measured by
148
The subsequent
transfer to occur between the Light-Up and detection fluorophores and produce a
florescent signal, the two fluorophores must be in close proximity. Proximity is achieved
by conjugating the fluorophores to oligonucleotides such that when the oligonucleotides
are hybridized to their target in an amplicon, the fluorophores are held in proximity. The
mixture is then heated and a melting curve is generated by the slow thermal denaturing
of the probe-template hybrid. Melting curves are generated by monitoring the loss of
fluorescence over the course of denaturation. Melting peaks are generated by plotting
the inverse derivative of fluorescence verses temperature (-dF/dT) - the bigger the
mismatch between the amplicon and the probe, the lower the melting temperature.
Because most hemoglobinopathies are single amino acid mutations such as
base substitution or base pair insertion or deletion, the ASP method is the commonly
used technology. In this procedure, allele-specific primers for sequences are designed
to bind to and amplify a small region surrounding the site of the known mutation. A
probe of oligonucleotides, which matches the normal or abnormal sequence, binds to
the PCR products. The probes incorporate a label (fluorophore) that produces a signal
to show that binding has taken place and a specific sequence has been detected.
149
4.3.3 Mutations
Hemoglobin beta is the name of the hemoglobin gene and is abbreviated HBB.
Sickle cell anemia is the most common mutation and primarily affects African-Americans
with a frequency of 1:400.
capillaries. The -globin gene is located on the small (p) arm of chromosome 11 in the
region of 15.5 (HBB; MIM # 141900; 11p15.5). The mutation is the replacement of an
adenosine with a thymidine in the DNA that causes the substitution of valine for glutamic
acid at position 6 in the beta-globin chain. The codon sequence is shown below, GAG,
in the sixth position below, codes for glutamic acid; the replacement of adenosine (A)
with thymidine (T) produces GTG that codes for valine.
1
3
GTG GAC CTG ACT
Val
Asp
Leu
Thr
6
9
CCT GAG GAG AAG TCT
Glu
Pro
Glu Lys Ser
Val
CCT GTG GAG AAG TCT
- - - (wildtype)
- - - (Hb S)
Asp
Leu
Thr
6
9
CCT GAG GAG AAG TCT
Glu
Pro
Glu Lys Ser
Lys
CCT AAG GAG AAG TCT
150
- - - (wildtype)
- - - (Hb C)
Indus River Valley (Punjab) region of Pakistan and Northwestern India but is
widespread, and has been observed in persons from China, England, Holland,
Australia, Greece, Serbia, Bosnia Herzegovina, Macedonia, Montenegro, and Turkey.
It is the fourth most frequently occurring hemoglobin variant.
Heterozygotes for Hb D are normal. Homozygosity for Hb D is associated with
normal hemoglobin levels, decreased osmotic fragility, and some target cells.
Compound heterozygotes for Hb D and -Thalassemia have mild anemia and
microcytosis.
thalassemia, and in the homozygous state. Hemoglobin D has been shown to interact
with the sickle hemoglobin gene S. Individuals who are compound heterozygotes for
Hb S and Hb D-Los Angeles (SD) have moderately severe hemolytic anemia and
151
occasional pain episodes. Populations that have a high frequency of sickle hemoglobin
(SD) disease are those of Asian and Latin American descent.
Hemoglobin O-Arab is an abnormality due to the amino acid substitution of lysine
for glutamic acid at the 121st position in the beta globin gene. The genetic mutation is a
GAA>AAA at this codon (121 Glu>Lys). The mutation is also known as Hb Egypt and
Hb O-Thrace. The mutation is found mainly in African-Americans, Gypsies, in Pomaks
(a population group in the Balkan countries) and in Arabian, Egyptian, and Black
families of the US and western hemisphere.
-------------------------------------------------------------------------- See the DNA codon table for degeneracy (redundant) codons.
tetramers due to the reduced concentration of alpha chain. The tetramers of -globin
do not transport oxygen.
severe -thalassemia.
152
Below are several melting curves representing the various signals obtained
during an analysis. In allele specific binding assays, it is preferred that the primer and
detection probe are the sequences for the mutation and not the wildtype (normal)
sequence. Because the mismatch in base sequences causes the melting temperature
to be lower, the use of the wild type sequence as the detection probe will indeed
demonstrate a lower melting temperature when a mismatch is present, however it will
not be known as to which base/s mismatch (mutation) was present. The use of the
mutation as the template will always result in the specific mutation producing the highest
melting temperature.
The Hemoglobin S templates were used in the analysis of wildtype (normal)
hemoglobin in Figure 1 and shows a melting point of 55.5 oC.
Figure 2 shows a melting curve for a carrier, both hemoglobin sequences were
detected.
wildtype melts at 55.5 C. A homozygous sickle disease individual would show only one
melting point at 62.5 C.
the mutation are very close, e.g. 3 bases, otherwise the energy transfer would not be
very efficient and no fluorescent signal would be detected.
Figure
3.
Hemoglobin C (Mutant) bound to Hemoglobin S probe.
Melting temperatures: Mutant C - 49.8 C and WT - 55.5 C
155
156
157
158
Degeneracy Table
Amino Acid
DNA codons
Alanine
Arginine
Asparagine
AAT, AAC
Aspartic acid
GAT, GAC
Cysteine
TGT, TGC
Glutamic acid
GAA, GAG
Glutamine
CAA, CAG
Glycine
Histidine
CAT, CAC
Isoleucine
Leucine
Lysine
AAA, AAG
Methionine
ATG
Phenylalanine
TTT, TTC
Proline
Serine
Threonine
Tryptophan
TGG
Tyrosine
TAT, TAC
Valine
Stop codons
159
First
T
TTT
TTC
TTA
TTG
C
Phe
Leu
CTT
C
CTC
CTA
Leu
TCT
TCC
TCA
TAT
TAC
Ser
TAA
TCG
TAG
CCT
CAT
CCC
CCA
CAC
Pro
CAA
CTG
CCG
CAG
ATT
ACT
AAT
ATC
Ile
ATA
ATG
GTC
GTA
GTG
ACC
ACA
Met
GTT
G
Val
AAC
Thr
AAA
ACG
AAG
GCT
GAT
GCC
GCA
GCG
Ala
GAC
GAA
GAG
G
Tyr
stop
His
Gln
Asn
Lys
Asp
Glu
TGT
TGC
Third
Cys
T
C
TGA
stop
TGG
Trp
CGT
CGC
CGA
T
Arg
CGG
AGT
AGC
AGA
AGG
GGA
GGG
A
G
Ser
Arg
GGT
GGC
T
C
A
G
T
Gly
C
A
G
References
2
3
4
5
6
2
3
4
5
6
7
8
9
161
Chapter 4
Global Chain Analysis
4.4 Electrospray Ionization-Mass Spectrometry
Gul M. Mustafa, PhD and John R Petersen, PhD
Mass spectrometry (MS) is an analytical technique that identifies the chemical
composition of a sample on the basis of the mass-to-charge ratio (m/z) of charged ions.
The technique has both qualitative (structure) and quantitative (molecular mass or
concentration) uses. Another way of thinking about mass spectrometry is that it can be
considered as the worlds most accurate scale. Mass spectrometers can be divided
into three fundamental parts, namely the ionization source, the analyzer, and the
detector (Figure 1). The molecules of interest are first introduced into the ionization
source of the mass spectrometer, where they are ionized to acquire positive or negative
charges. This is done because ions are far easier to manipulate as compared to
molecules that do not have a charge. The ions then travel through the mass analyzer
and arrive at different parts of the detector according to their mass to charge (m/z) ratio.
After the ions make contact with the detector, useable signals are generated and
recorded via a computer. The computer displays the signals graphically as a mass
spectrum showing the relative abundance of the signals according to their m/z ratio. The
analyzer and detector of the mass spectrometer, and often the ionization source too, are
maintained under high vacuum to allow the ions to travel from one end of the instrument
to the other without colliding with air molecules which decreases the signal. The entire
operation and often the sample introduction process are under complete data system
162
163
Figure: 1
The method of sample introduction to the ionization source often depends on the
ionization method being used, as well as the type and complexity of the sample. Many
ionization methods are available and each has its own advantages and disadvantages.
The ionization method used depends on the nature and type of sample under
investigation and the mass spectrometer available. Figure 2 shows various ionization
methods of ionization such as Atmospheric Pressure Chemical Ionization (APCI),
Atmospheric Pressure Photo-Ionization (APPI), Electron Impact (EI), and Electrospray
Ionization (ESI). The ionization methods used for the majority of biochemical analyses
are Electrospray Ionization (ESI) and Matrix Assisted Laser Desorption Ionization
(MALDI)
Figure: 2
164
The ionization mechanism first involves the liquid containing the analyte(s) of
interest to be dispersed by electrospray into a fine aerosol. Because the ion formation
involves extensive solvent evaporation, the typical solvents for electrospray ionization
are prepared by mixing water with volatile organic compounds (e.g. methanol,
acetonitrile). To decrease the initial droplet size, compounds that increase the
conductivity (e.g. acetic acid) are customarily added to the solution. Large-flow
electrosprays can benefit from additional nebulization by an inert gas such as nitrogen.
Figure: 3
masses but also has the ability to analyze biological samples that are defined by noncovalent interactions. Since the m/z ratio range of a quadrupole instrument is fairly
small, the mass of the sample can be determined with a high amount of
accuracy. Sensitivity of the instrument is also impressive making it useful in both
quantitative and qualitative measurements. The major disadvantage of ESI-MS is that in
the analysis of mixtures the results are unreliable. In addition to the difficulty in handling
mixtures the multiple charges that are attached to the molecular ions can make for
confusing spectral data. The apparatus is also very difficult to clean and has a tendency
to become contaminated with residues from previous experiments.
In recent years, electrospray ionization (ESI) mass spectrometry has become an
increasingly important method in proteomics not only to analyze peptides but also to
study proteins and protein complexes of increasing size and complexity in structural
biology. The analysis of proteins and protein complexes by mass spectrometry
(macromolecular mass spectrometry) has become possible because of the
development of the relatively gentle ionization procedure related to ESI, which retains
non-covalent interactions. The mass-to-charge (m/z) ratios of these proteins can well be
over 10,000 daltons, and therefore, time-of-flight (TOF) analyzers with orthogonal
injection are the most commonly used analyzers in the field of macromolecules. The
m/z analysis of larger proteins and protein complexes is not a routine technique, since a
careful optimization of the operating conditions is always required. Despite the
theoretically unlimited mass range of TOF analyzers, most instruments have detection
problems when the m/z values exceed 4,000 daltons. It has been shown that a pressure
increase in the first and second vacuum chamber of the mass spectrometer is an
167
absolute requirement for the analysis of large proteins (2-6). The increased pressure
leads to collisional cooling and focusing of large ions in the ion guides and, therefore,
improved transmission through the ion guides and the TOF (5). In ESI-MS, the ion
signal is proportional to analyte concentration and largely independent of flow rate and
injection volume used for sample introduction. The signal is linear from the limit of
detection (usually pmol/L) to around 10 mol/L of analyte concentration. For quantitative
measurements, it is important to incorporate an internal standard in the procedure to
compensate for losses during sample preparation and variable detection sensitivity of
the MS system. The internal standard should have a structure similar to that of the
analyte and the ideal practice is to synthesize an internal standard by incorporating
stable isotopes on the molecules of interest. When an ideal internal standard is not
available, molecules with similar structure can also be used. Another critical issue in
quantitative ESI-MS is suppression of ionization due to matrix interference. A biological
sample can give significantly lower ionization signals compared to pure standard
solutions with similar analyte concentrations. This phenomenon is the result of high
concentrations of non-volatile materials, such as salts and lipids, present in the spray
with the analyte. To overcome the matrix interference, extensive sample purification
processes are required. However, these elaborate procedures are time-consuming and
can cause poor recovery. A recent development is to use short Liquid Chromatography
(LC) columns (or guard columns) and apply a fast High Pressure Liquid
Chromatography (HPLC) purification (e.g. for 25 minutes) prior to MS analysis. The
HPLC serves to separate the non-volatile compounds from the analyte. For HPLC
systems with column-switching capability, the analyte in the biological sample can be
168
purified and concentrated on separate columns before MS analysis. Unlike many other
techniques which measure one analyte at a time, these techniques can measure
multiple analytes (>40) at one time. In recent years the scope of testing using these
techniques has expanded from toxicological purposes to newborn screening to
hormones, proteins, and enzymes.
In recent years a change in the way MS is being used in clinical laboratories has
occurred. In the past MS was commonly used in conjunction with gas chromatograph
(GC). Today it is not uncommon to see MS being coupled to LC in the routine clinical
laboratories. Once considered too expensive and cumbersome to use except in forensic
and reference settings, such systems are now used routinely to generate data for
patient care. Although mass spectrometry has long been recognized as an important
and powerful analytical tool, there were a number of challenges that had to be
overcome to be used in the clinical setting for more than a few special applications. GCMS was introduced into the clinical laboratory more than two decades prior to LC-MS.
With the advent of relatively small, inexpensive, and user-friendly LC/MS and LC
tandem MS (LC/MS-MS) systems along with advances in column chemistries the door
has been opened to many analyses not possible with GC/MS (7). Although the initial
capital investment for LC ESI-MS equipment is substantial compared to other routine
clinical laboratory analyzers, its operational costs are low. The cost-effectiveness of this
technique comes from the fact that it can measure multiple analytes at the same time.
This technology can be expected to exert an important influence in how analytes, both
large and small, are detected and quantified in the clinical laboratory service.
169
141 amino acids in the -chain and 38 of 146 residues in the -chain are charged
residues and the rest are neutral so they cannot be detected by these traditional
analytical techniques, such as ion-exchange HPLC and isoelectric focusing (IEF) on
polyacrylamide gel, as these techniques depend on the presence of charge differences
induced by the mutation. Also in the past, definitive characterization of Hb variants
involved tedious and time-consuming analytical procedures requiring days and even
months for completion. Recently, a strategy for rapid definitive characterization of Hb
variants to identify a single mutated; inserted or deleted amino acid residue was
reported using ESI-MS. In case of Hb San Martin [b6(A3)GluVal;b85(F1) PheLeu],
the second mutation leads to an unstable protein causing chronic hemolytic anemia in
the heterozygous carrier (13). Molecular diagnosis, achieved by DNA analyses, shows
the presence of two mutations, but protein or familial studies was required to prove that
the two mutations are carried by the same allele and not interacting in trans. The
identification by MS methods of a new Hb variant: Hb S-Clichy [b6(A3)GluVal;b8(A5)
LysThr], which presents a double mutation located on the same bT-1 tryptic peptide.
This new variant adds the amino acid substitution of Hb Rio Grande[b8(A5) LysThr]
(14) on the same b-globin chain, to that of Hb S. Difficulties encountered in structural
determinations are caused by the presence of two abnormalities in the same
polypeptide chain. Variants with two amino acid substitutions on the same globin chain
as in Hb S-Clichy, demonstrated the importance of including MS studies.
The procedure comprises the following steps:
I.
Overnight trypsin digestion for investigation of the amino acid substitution on the
Hb variants. ESI-MS on the tryptic digest can identify the specific peptide
harboring the substituted amino acid.
III.
ESI-MS/MS of the target peptide can provide the amino acid sequence of the
peptide and thus the position of the substituted amino acid.
These performances can be applied at different steps of the globin variant
distinct conformational change can diverge from the predicted behavior. Furthermore,
the model cannot predict reliably unstable variants. Nevertheless, pI calculations and
the evaluation of the method-specific detect ability allow the prediction of the number of
the currently undetected, silent variants. So it is now recommended that other methods
that are not based on electrophoretic or chromatographic mobility should be applied in
Hb variant analysis. In this regard ESI and MALDI-TOF MS are the suggested methods
that enable the detection of variants when the mass difference between the abnormal
and the wild-type globin chains exceeds 6 Da. This limitation in MS determination is due
to the complexity between the normal and mutated globin chains which can be
overcome by using high resolution instruments (FT-ICR, Orbitrap) or by special
precautions on low resolution instruments. For these low mass differences between
normal and variant globin chains, MS analysis of digested peptides is required.
As calculated by various studies, MS method is able to detect 92% of the
undetected variants. Among MS techniques for studying Hb variants, ESI-MS is the
most frequently used and can be associated with peptide sequencing using tandem MS,
but it often gives multiple charged fragment ions. On the other hand, MALDI-TOF MS
gives single-charge peptide ions and has been used for identification of some single
mutation Hb variants. Indeed, with additional MS analysis of lysate samples 3 new
variants, Hb Zurich-Hottingen, Hb Zurich-Langstrasse and Hb Riccarton were detected
by using ESI-MS. Neither variant had a clinical impact. These neutral variants are
exclusively found by MS and are chromatographically silent. Also in an Hb Malay
sample, only the MS analysis revealed the variant chain, as opposed to cationexchange HPLC which identified it as a thalassaemia. Recapitulating, 4 out of 2105
174
samples (0.2%) or 1% of the abnormal samples would be missed without the use of MS
analysis. In ESI-MS, the sample preparation is very simple and requires only the dilution
of the lysate sample. Two important drawbacks of the MS methods are worth
mentioning. First, its insufficient resolution prevents the detection of Hb mutations with
small mass differences of the globin chains. The precision of normal low-resolution
mass measurements is insufficient to distinguish the wild-type chain from several chain
variants, such as Hb C, D, or E. Owing to the isotopic pattern, even high-resolution MS
did not separate globin chains that differed only in 1 or 2 Da from the normal chains
(17). Two intact globin chains are not observed as separate entities in MS unless their
masses differ from one another by more than 6 Da. Second, MS as described here is
only a qualitative technique, and in particular, minor Hb fractions such as HbA1C or
HbA2, which are important for diagnosis of diabetes mellitus or thalassemia,
respectively, cannot be quantified. However high resolution MS enables detection of
variants with low mass difference (<2 Da). Also different signals in the spectrum shows
isotopic pattern.
References
1. Fenn, JB. Electrospray Ionization Mass Spectrometry: How It All Began. J.
Biomol. Techl. 13:101118; 2002.
2. Sanglier S, Leize E, Van Dorsselaer A, Zal F. Comparative ESI-MS study of
approximately 2.2 M Da native hemocyanins from deep-sea and shore crabs:
from protein oligomeric state to biotope. J. Am. Soc. Mass Spectrom.14:419-429;
2003.
3. Schmidt A, Bahr U, Karas M. Influence of pressure in the first pumping stage on
analyte desolvation and fragmentation in nano-ESI MS. Anal. Chem.73:60406046; 2001.
4. Tahallah N, Pinkse M, Maier CS, Heck A. The effect of the source pressure on
the abundance of ions of noncovalent protein assemblies in an electrospray
175
176
Chapter 4
Globin Chain Analysis
4.5 PCR and Sanger Sequencing
Elaine Lyon, PhD
Molecular methods are commonly employed to identify hemoglobin variants.
Polymerase chain reaction (PCR) exponentially amplifies regions of DNA allowing direct
genotyping or targeted mutation analysis. Detecting common alpha globin deletions is
accomplished by amplifying over deletion breakpoints or using quantitative methods to detect
copy number changes. Sanger sequencing is considered the gold standard for mutation
detection, and can confirm abnormal hemoglobinopathy and thalassemia variants. Molecular
analysis confirms a diagnosis, detects carrier status, and predicts disease prenatally in highrisk pregnancies. This section will describe general methods, applications and challenges in
PCR and Sanger sequencing for alpha and beta globin molecular analysis.
deletion which eliminate a single gene, while a 20.5kB deletion and the SEA, MED, FIL and
THAI gene rearrangements delete portions of or completely HBA1 and HBA2. In one assay
commonly used in clinical laboratories, PCR primers are designed to flank the breakpoints,
amplifying a product only when the deletion is present. By multiplexing primers, any of the
common deletions can be detected in a single reaction (1). Amplification products are
visualized by gel electrophoresis (figure 1). Other methods to identify deletions include
quantitative PCR analyses such as multiplexed-ligation probe amplification (MLPA) or high
resolution (exonic level) microarray. These methods are capable of detecting known and
previously unknown alpha globin deletions and alpha globin triplications (2,3). Given the
frequency of alpha globin deletions, a molecular work-up for alpha thalassemia often begins
with a test for deletions.
Figure 1. Gel electrophoresis for common alpha globin deletions. Each patient is tested with
control primers for HBA2 and LIS genes. In a separate reaction, each patient is tested for a
common deletion with a multiplex of deletion-specific primers. M: size marker, C: control
primers (HBA2 and LIS1), D: deletion primers. Patient 1: no common deletions, patient 2; 3.7
kB heterozygous, patient 3; 3.7kb/SEA compound heterozygous. Note that in patient 2, the
control HBA2 band is not present, as this patient has a deletion of this region on both
chromsomes.
178
The alpha globin genes also harbor sequence variants, and full gene sequencing is also
available, although alpha globin sequencing poses challenges. Sequencing is performed on
both genes (HBA1 and HBA2) that are highly homologous, and primers are designed to be
specific for each gene. Sequencing the entire coding region (3 exons for each gene),
intron/exon boundaries, proximal promoter regions, 5 and 3untranslated regions, and
polyadenylation signals provides a comprehensive sequencing test. Sequencing should be
combined with deletion analysis because deletions are not detected by sequencing, and an
apparent homozygous sequence variant may be one copy of the variant with a deletion on the
opposite chromosome. Samples homozygous for the 3.7kB deletion may not be able to be
amplified for sequencing, resulting in a failed test. However, the common 3.7kB deletion has
a single functional gene, but mutations have also been described in that fusion gene. (4,5) To
be able to identify a mutation in a chromosome with the 3.7kB deletion, primers must be
designed that will amplify over the deletion breakpoints.
(UTR), and known pathogenic deep intronic mutations (e.g. IVS-II-654, IVS-II-705 and IVS-II745). Large beta globin deletions or delta-beta fusion genes (e.g. Lepore,) will not be detected
by a sequencing assay designed only for HBB, and require a different analysis. Similar to
methods to detect alpha globin deletions, primers can be designed to amplify over the
breakpoints. One example of this is the 619 base pair (bp) deletion found in Indian and other
Asian populations (6). Recently, novel beta globin deletions have been detected by other
methods, such as exonic-level microarrays or MLPA (2,7). Specific large deletions in the beta
globin gene cluster is one of two molecular mechanisms that can result in HPFH. The other
mechanism is point mutations in the promoters of the gamma globin genes (HBG1 and HBG2).
4.5.3 Sequencing
Sequencing assays begin with PCR for the regions to be interrogated. Primers are
designed to avoid known variants at their 3 end which would prevent polymerase extension,
resulting in a drop-out of that allele (8). PCR products are treated with ExoSAP (exonuclease
1 from shrimp alkaline phosphatase) to remove excess primers.
PCR primers may be tagged with a M13 sequence which allows sequencing of all amplicons
from the same M13 primers. Alternatively, a second set of sequencing primers internal to the
PCR primers may be used. The sequencing reaction utilizes fluorescently labeled dideoxynucleotides (ddATP, ddTTP, ddCTP and ddGTP, collectively refered to as ddNTPs) which
lack a 3 hydroxyl group on the sugar residue and prevent the newly synthesized product from
extending to the next base when incorporated into the product. Sanger sequencing is
performed as a linear rather than exponential amplification, with separate sequence results
180
each from the 5 and 3 directions (bi-directionally). After the sequencing reactions, the
products are again processed to remove excess primers. Sephadex columns are often used
to bind the sequencing products, which are eluted as purified products.
Sequencing products are separated by capillary electrophoresis using a polymer with a singlebase resolution. The last base of each fragment is the ddNTPs with a fluorescent dye which is
incorporated into the product. The sequence is visualized as an electropherogram and aligned
to a reference sequence. The difference between the reference and the patient sequence are
examined to determine the type of mutation present. To accurately identify the mutation, it
should be identified in both the 5 and 3 direction.
thalassemia major. On occasion, two mutations may occur on the same chromosome.
Sequence analysis cant determine the phase of two mutations (whether on the same or
opposite chromosomes). HPLC or parental studies may be necessary to evaluate
phase. Over ten complex variants with the Glu6Val variant are listed in the globin gene
server (6). One example is Hb S-Oman, with Glu6Val and Glu121Lys variants on the
same chromosome. The standard nomenclature for the nucleotide changes is HBB:c.
[20A>T;364G>A] (6). These two variants are also seen alone as in Hb S and Hb 0Arab. The combination of these two mutations on opposite chromosomes is consistent
with severe sickle cell disease, whereas if they are on the same chromosome, the
individual is a carrier of an HBB mutation.
182
183
Figure 3.Beta globin sequencing. A compound heterozygous genotype is detected. The first
mutation is Hb S (c.20A>T, Glu6Val), while the second affects a splice site resulting in a
beta(0) thalassemia mutation. (c.92+1G>A). The yellow arrow in the electropherogram
indicates the exonic region. The sequence from the patient (Forward and Reverse) are
compared against a reference sequence. Differences between the patient and reference
sequences are shown in the middle panes.
4.5.6 Conclusion
Molecular analysis confirms the familial variant in individuals who are carriers of or
affected with globin gene variants. In prenatal analysis, molecular studies are often the most
direct method to predict the status of a fetus. If molecular testing is used prenatally, the
parents should first be tested to identify the familial mutations. In addition, amniotic fluid,
amniocyte or chorionic villi cell cultures should be tested for contamination from the mother. If
the samples show maternal cell contamination, the results may not accurately reflect the fetus
genotype and a second sample should be obtained.
184
The alpha and beta loci have complex structures that lead to a variety of molecular
anomalies, such as sequence variants, and large gene rearrangements resulting in deletions
or duplications.
Because many mutations in HBA1, HBA2 and HBB are well understood, the
interpretations are typically straight forward. However, because these loci have complex
structures that lead to a variety of molecular anomalies, molecular results should be combined
with clinical, family and other laboratory findings.
185
References
1 Tan AS, Quah TC, Low PS, Chong SS. A rapid and reliable 7-deletion
multiplex polymerase chain reaction assay for -thalassemia. Blood. 2001;
98(1):250251.
2 Phylipsen M, Chaibunruang A, Vogelaar IP, Balak JR, Schaap RA, Ariyurek Y,
Fucharoen S, den Dunnen JT, Giordano PC, Bakker E, Harteveld CL. Finetiling array CGH to improve diagnostics for - and -thalassemia
rearrangements. Hum Mutat. 2012 Jan; 33(1):272-80.
3 Galanello R, Cao A. Alpha-Thalassemia. 2005 Nov 1 [Updated 2011 Jun 7].
In: Pagon RA, Bird TD, Dolan CR, et al., editors. GeneReviews [Internet].
Seattle (WA): University of Washington, Seattle; 1993-. Available from:
http://www.ncbi.nlm.nih.gov/books/NBK1435/accessed 10-04-12
4 Zhao P, Buller-Burckle AM, Peng M, Anderson A, Han ZJ, Gallivan MV.
Secondary mutation (c.94_95delAG) in a -3.7 allele associated with Hb H
disease in two unrelated African American individuals homozygous for the
-(3.7) deletion (-3.7/-3.7T). Hemoglobin. 2012; 36(1):103-7.
5 Brennan SO, Chan T, Duncan J. Novel 2 gene deletion (c.349_359 del
GAGTTCACCCC) identified in association with the -3.7 deletion.
Hemoglobin. 2012; 36(1):93-7.
6 Hardison RC, Chui DHK, Giardine B, et al. HbVar: a relational database of
human hemoglobin variants and thalassemia mutations at the globin gene
server. Hum Mutat 2002; 19: 225-33 http://globin.cse.psu.edu/accessed 1004-2012
7 Mikula M, Buller-Burckle A, Gallivan M, Sun W, Franklin CR, Strom CM.The
importance of globin deletion analysis in the evaluation of patients with
thalassemia.Int J Lab Hematol. 2011 Jun;33(3):310-7
8 Pont-Kingdon G, Gedge F, Wooderchak-Donahue W, Schrijver I, Weck KE,
Kant JA, Oglesbee D, Bayrak-Toydemir P, Lyon E; Biochemical and Molecular
Genetic Resource Committee of the College of American Pathologists.
Design and analytical validation of clinical DNA sequencing assays.Arch
Pathol Lab Med. 2012 Jan;136(1):41-6.
186
Chapter 5
Alpha and Beta Thalassemia
Herbert L. Muncie, Jr., MD
Alpha () and beta () thalassemia are hematological disorders that are the
result of a decreased or absent synthesis of a globin chain. 1 These genetic alterations
may have been the result of selective pressure from Plasmodium falciparum malaria
from which thalassemia carriers are relatively protected from invasion. 2, 3 The altered
globin chain synthesis can be asymptomatic or can cause severe hemolytic anemia and
even death.
5.1
Epidemiology
The thalassemias are prevalent in the tropical and subtropical regions of the
world and affect men and women equally. Alpha thalassemia is found more often in
persons of African or Southeast Asian descent and -thalassemia occurs more often in
persons of Mediterranean, African or Southeast Asian descent. Thalassemia trait can be
found in 5 to 30 percent of these populations. 4 An estimated 1.5% of the global
population are -thalassemia carriers but only approximately 200,000 people are alive
with -thalassemia major.5, 6
5.2
Pathophysiology
Hemoglobin has an iron-containing heme ring and four globin chains: normally
two alpha and two nonalpha. The composition of these four globin chains determines
the hemoglobin type. The predominant in utero hemoglobin, fetal hemoglobin (Hb F),
187
has two and two gamma () chains (2 / 2). Adult hemoglobin A (Hb A) has two and
two chains (2/2) and hemoglobin A2 (HbA2) has two and two delta () chains
(2/2).
The transition from -globin synthesis (Hb F) to -globin synthesis (Hb A) begins
before birth. Therefore, at birth approximately 20% to 30% of hemoglobin is Hb A and
the remainder is HbF.7 This transition continues and is usually completed from 6 to 24
months of age. At that time normal children will have mostly Hb A (>96%), small
amounts of Hb A2 (2.0 3.4%) and very small amounts of Hb F (< 1%). 8
5.3
Alpha Thalassemia
Alpha thalassemia occurs when there is reduced or absent -globin chain
globin chain variant that is longer than normal and produced in only small quantities. It
therefore behaves in a similar manner to an alpha gene deletion.
11
When Hb Constant
Spring is inherited with a 2 alpha gene deletion, the condition may be referred to as Hb
H / Constant Spring. Finally if all four genes are deleted (--/--) the result will be
significant amounts of hemoglobin Barts (Hb Barts) with four gamma chains ( 4). With
increased Hb Barts and total absence of Hb F, -thalassemia major results leading to
hydrops fetalis, which is incompatible with life.
5.4
12
Beta Thalassemia
Beta thalassemia occurs when there is reduced or absent -globin chain
synthesis with subsequent excess -globin chains. 3, 6 Most often a mutation is the
genetic defect, with more than 200 reported; a deletion is quite rare. One gene on each
chromosome 11 controls the production of -globin chains (,), therefore, there are two
phenotypical presentations. If a child inherits one normal gene from one parent (/)
and a defective gene from the other parent (-/), the result is -thalassemia trait (minor)
which causes an asymptomatic mild microcytic anemia. If both genes are defective, the
result depends on the degree they are deficient in -globin chain production. If -globin
chain production is severely reduced, the person will have -thalassemia major (Cooley
anemia). Most individuals with -thalassemia are asymptomatic at birth because of the
presence of significant amounts of Hb F. As the -globin chain synthesis decreases,
infants may experience symptoms starting at six months of age. If the -globin chain
synthesis is only partially reduced, the person will have -thalassemia intermedia with
189
less severe symptoms and survival beyond 20 years of age without life-long blood
transfusions.
5.5
Diagnosis
Except for or thalassemia major, the diagnosis of thalassemia is usually
made incidentally when a patient is found to have microcytosis with or without anemia.
The most common etiologies for microcytic anemia are iron deficiency, thalassemia,
lead toxicity and sideroblastic anemia. The patients medical history, mean corpuscular
volume (MCV), red cell count and the red cell distribution width (RDW) can help exclude
many of these etiologies (Table 1). The MCV in -thalassemia trait is usually lower than
in -thalassemia trait. In Hb H disease the MCV will be as low as 64 fl. 13 Mentzer index
(MCV/red blood cell count) was proposed (which is not true in children) to predict the
likelihood of thalassemia. If the ratio is > 13, iron deficiency is the likely etiology
whereas thalassemia is associated with a value < 13. An exact ratio of 13 would be
uncertain.14
The RDW can be helpful in distinguishing thalassemia from iron deficiency and
sideroblastic anemia. With iron deficiency or sideroblastic anemia the RDW is almost
always elevated while it is elevated in approximately 50% of thalassemia trait patients. 15
Therefore, with a microcytic anemia, if the RDW is normal the diagnosis is usually
thalassemia trait. However, if the RDW is elevated additional tests to evaluate for iron
deficiency and sideroblastic anemia will be needed. 16
A serum ferritin level is the best single test to rule out iron deficiency in the absence of
inflammation.17 Serum iron, total iron binding capacity and transferrin may not be
190
Beta thalassemia major is diagnosed during the infants first year of life. The
infant usually displays growth retardation, pallor, irritability and later jaundice with
abdominal swelling. Children who develop these symptoms after their first birthday will
be diagnosed with -thalassemia intermedia.
5.6
Treatment
Patients with or thalassemia trait (minor) require no treatment or regular long-
term follow-up. While these patients have a microcytic anemia they are not iron deficient
and should not be given iron supplements. If iron deficiency did develop, iron
supplements would be appropriate.4, 21
Beta thalassemia major requires treatment with blood transfusions starting as
early as six months of age. Transfusions correct the anemia, suppress erythropoiesis
and inhibit intestinal iron absorption. Transfusions are initiated either when the
hemoglobin is < 7 g/dl for more than 2 weeks (without another etiology) or if other
factors such as facial changes, poor growth, bony expansion or splenomegaly occur.
Without blood transfusions these patients would not survive into adulthood. They will
need periodic (every 2 - 4 weeks) transfusions (lifelong) to maintain their hemoglobin
higher than 9.5 g/dL.4, 22 The post-transfusion hemoglobin goal is 13 14 g/dl. Beta
thalassemia intermedia patients require transfusions only when their reduced
hemoglobin interferes with their quality of life or it effects their growth and development.
Transfusions will occasionally be needed for Hb H disease depending on the severity of
the condition.
192
5.7
Complications
Patients with or thalassemia trait (minor) have no complications. Patients with
193
Because of the need for multiple blood transfusions in -thalassemia major or in some
cases of Hb H disease and the increased intestinal iron absorption with -thalassemia
intermedia, patients develop iron overload which damages visceral organs (liver, spleen,
endocrine organs) and the heart which is the primary cause of early death. 28
Splenomegaly invariably develops in symptomatic thalassemia and can worsen the
anemia. The risk of hepatocellular carcinoma is increased due to iron overload hepatic
damage, longer survival and viral infection with hepatitis B and/or C. 29 Gallstones are
more prevalent with -thalassemia intermedia than with -thalassemia major.
Beta thalassemia major and intermedia cause a hypercoaguable state. 30 This effect
increases the risk of thromboembolic events especially after splenectomy.31
Osteoporosis was found in 51% of patients over age 12 with -thalassemia major.32
5.8
5.8.1
Hypersplenism
Splenectomy is required for patients whose splenomegaly causes a marked
increase in their need for blood transfusions, i.e. the annual red cell requirement
exceeds 180 200 ml/kg.6 Because of the importance of the spleen in clearing bacteria
and preventing sepsis, the surgery is not done until the patient is at least 4 years old.
One month prior to the surgery the child should be given the pneumococcal
polysaccharide vaccine. They should also receive the pneumococcal conjugate vaccine
series if they had not received it during infancy. For the first two years after the surgery
patients should take penicillin 250 mg twice a day. For children the antibiotic prophylaxis
194
continues until age 16.33 Gallbladder removal should be considered if gallstones are
present34 at the time of splenectomy.
5.8.2 Endocrinopathies
While growth retardation can occur with thalassemia, growth hormone therapy
has limited effectiveness and is not recommended. If hypogonadism develops,
hormonal therapy is effective.35 Bone mineral density has been increased with
alendronate, pamidronate and zolendronate; however, studies evaluating fracture
reduction are needed.36
5.8.3 Pregnancy
Couples from high risk ethnic groups should be encouraged to seek
preconception genetic counseling.37 Individuals with a low MCV (<80fl) and MCH (<27
pg) could be assessed with hemoglobin electrophoresis / HPLC. 12 An efficient way to
identify mutations is to study their parents hematology and screen them for single
mutations.38
For couples, if both partners have -thalassemia trait, their child will have a one
in four chance of having -thalassemia major and a two in four chance of -thalassemia
trait.(Figure 1) With four genes controlling the expression of -globin chains, the
inheritance pattern is more complex. If two genes are defective, the phenotype is
influenced by whether the defective genes are on the same chromosome (cis) or
different chromosomes (trans), e.g. if one parent is an -thalassemia silent carrier (-,
) and one parent has -thalassemia trait [(cis),(--,)], they have a one in four chance
195
their child will have Hb H disease. Whereas, if the -thalassemia trait parents defect is
trans (-, -), their children have no risk of Hb H disease (Figure 2).
Once pregnancy occurs, patients should be counseled regarding prenatal
diagnostic testing options. An amniocentesis at approximately 15 weeks gestation or a
chorionic villus sample (CVS) obtained at 10 11 weeks gestation can detect point
mutations or deletions utilizing polymerase chain reaction (PCR) testing. Other
diagnostic options include DNA analysis of fetal cells obtained by amniocentesis and in
the future analysis of maternal blood fetal cells. 39, 40 If Hb Barts is detected, the mother
has an increased risk of pre-eclampsia and postpartum hemorrhage.
For couples using in vitro fertilization, preimplantation genetic testing is available. 41
5.8.4 Cardiac
When iron overload occurs, cardiac infiltration and death are significant
concerns. Serum ferritin levels have been used to predict cardiac complications with
improved survival if levels are kept below 2,500 ng per ml (2500 mcg per L). 42 Ferritin
levels are unreliable when significant liver disease develops. 43
5.8.5 Hypercoagulopathy
While the risk of thromboembolic events in patients with -thalassemia major or
intermedia is increased, no trials have evaluated prevention of these complications with
anticoagulants. A consensus recommendation for patients with a thrombosis history is
prophylactic treatment with low molecular weight heparin especially before surgery and
during pregnancy. Because estrogen containing contraceptives may increase the risk of
196
5.8.6
Psychosocial
The impact on a patient and their family of a chronic disease such as -
5.8.8 Prognosis
197
Beta thalassemia major patients live an average of 17 years and usually die by
age 30. With regular blood transfusions and compliance with chelation therapy, their life
can extend beyond age 40.6 Their deaths are commonly caused by cardiac
complications of iron overload.28 Thalassemia trait patients have a normal life
expectancy.
198
Iron Deficiency
-thalassemia
-thalassemia
-thalassemia
Low
Low
trait
Low
major
Low
High
Normal
Normal,
Normal,
occasionally
occasionally high
Normal
high
Normal
Normal
> 13
< 13
< 13
< 13
count)
Hb electrophoresis
Normal (may
Adults : normal
Reduced HbA ,
Reduced HbA,
(Adult normals
have reduced
Newborns:
increased
increased HbA2,
HbA2 before
HbA2, and
and increased
iron therapy is
have HbH or
increased HbF
HbF
given)
Hb Bart
199
HbA2 level
2.5 3.5 %
1.6 3.2 %
2.0 3.2 %
1 2.4 %
> 4.0 %
> 4.0 %
Normal
Iron deficiency
-thalassemia silent carrier or trait (minor)
HbH disease
-thalassemia trait (minor)
-thalassemia major
200
Route of
Dosage
agent
Desferoxamine
administration
Subcutaneous
Adults - 30 50 mg/kg
Deferasirox
Deferiprone
infusion over 8 - 12
Children - 20 40
hours
Oral
Oral
mg/kg
20 - 30mg/kg/day
75 100 mg/kg/day
(only available
in the US
through FDA
Treatment Use
Program)
201
Frequency of therapy
5 7 days/week
Once a day
3 times/day
X
Mother
(-,)
-thalassemia trait
Children:
(-,-)
( ,)
-thalassemia major
Father
(-,)
-thalassemia trait
(-,)
-thalassemia trait
(-,)
-thalassemia trait
Normal
or intermedia
Note: Shaded symbols indicate an abnormal -globin gene on chromosome 11.
202
203
204
References
1. Muncie HL Jr, Campbell J. Alpha and beta thalassemia. Am Fam Physician 2009;
80 (4): 339-344.
2. Mantikou E, Arkesteijn SG, Beckhoven JM, Kerkhoffs JL, Harteveld CL, Giordano PC.
A brief review of newborn screening methods for hemoglobinopathies and preliminary
results selecting beta thalassemia carriers at birth by quantitative estimation of the
HbA fraction. Clinical Biochemistry 2009; 42(18): 1780-1785.
3. Cao A, Galanello R. Beta-thalassemia. Genetics in Medicine 2010; 12(2): 61-76.
4. Rund D, Rachmilewitz E. Beta-thalassemia. N Engl J Med. 2005; 353(11): 1135-1144.
5. Thalassemia International Federation. Thalassemia. Available at:
http://www.thalassaemia.org.cy/index.html. Accessed 04/10, 2011.
6. Galanello R, Origa R. Beta-thalassemia. Orphanet J Rare Dis 2010; 5:11.
7. Richardson M. Microcytic anemia. Pediatr Rev 2007; 28 (1): 5-14.
8. Mosca A, Paleari R, Ivaldi G, Galanello R, Giordano PC. The role of haemoglobin
A(2) testing in the diagnosis of thalassaemias and related haemoglobinopathies. J
Clin Pathol 2009; 62:13-17.
9. Harteveld CL, Higgs DR. Alpha-thalassaemia. Orphanet J Rare Dis 2010; 5:13.
10. Galanello R, Cao A. Alpha-thalassemia. Genetics in Medicine 2011; 13 (2): 83-88.
11. Chen FE, Ooi C, Ha SY, et al. Genetic and clinical features of hemoglobin H disease
in Chinese patients. N Engl J Med. 2000; 343(8): 544-550.
12. Leung TN, Lau TK, Chung TKH. Thalassaemia screening in pregnany. Curr Opin
Obstet Gynecol 2005; 17 (2): 129-134.
13. Origa R, Sollaino MC, Giagu N, et al. Clinical and molecular analysis of
haemoglobin H disease in Sardinia: Haematological, obstetric and cardiac aspects
in patients with different genotypes. Br J Haematol 2007; 136(2): 326-332.
14. Mentzer WC,Jr. Differentiation of iron deficiency from thalassaemia trait. Lancet
1973; 1(7808): 882.
15. Flynn MM, Reppun TS, Bhagavan NV. Limitations of red blood cell distribution width
(RDW) in evaluation of microcytosis. Am J Clin Pathol 1986; 85(4): 445-449.
16. Marsh WL Jr, Bishop JW, Darcy TP. Evaluation of red cell volume distribution width
(RDW). Hematol Pathol 1987; 1(2): 117-123.
17. Guyatt GH, Oxman AD, Ali M, Willan A, McIlroy W, Patterson C. Laboratory
diagnosis of iron-deficiency anemia: An overview. Journal of General Internal
Medicine 1992; 7(2) : 145-153.
18. Kattamis C, Lagos P, Metaxotou-Mavromati A, Matsaniotis N. Serum iron and
unsaturated iron-binding capacity in the -thalassaemia trait: their relation to the
levels of haemoglobins A, A 2 , and F. J Med Genet 1972; 9(2): 154-159.
19. Van Delft P, Lenters E, Bakker-Verweij M, et al. Evaluating five dedicated automatic
devices for haemoglobinopathy diagnostics in multi-ethnic populations. Int J Lab
Hematol 2009; 31(5): 484-495.
20. Mosca A, Paleari R, Leone D, Ivaldi G. The relevance of hemoglobin F
measurement in the diagnosis of thalassemias and related hemoglobinopathies.
Clin Biochem 2009; 42(18): 1797-1801.
21. Olivieri NF. The beta-thalassemias. N Engl J Med 1999; 341(2): 99-109.
205
207
Chapter 6
Neonatal Screening for Hemoglobinopathies
Zia Uddin, PhD
With the technical support of Patrick Hopkins, Joseph Quashnock,
Aigars Brants, Christine Moore, DAndra Morin, Rachel Lee, Mahin Azimi,
and Bonifacio Dy
6.1 Introduction
A gratifying achievement of my professional career was the organization of an
interdisciplinary conference on Perinatal Care and Neonatal Screening in 1978 at
South Macomb Hospital (now a part of St. John Providence Health System), Warren,
Michigan.
After this conference I started neonatal T4 screening for the four major hospitals
in South Eastern Michigan. Subsequently, with the vision of the then Governor of
Michigan (Honorable Mr. William Milliken), a law was passed for the establishment of a
state of the art laboratory in Lansing, Michigan for the screening of inborn errors of
metabolism and hemoglobinopathies. By statute, each state in the USA performs
newborn screening (NBS), however, the number of tests/neonate and the
methodologies utilized vary from state to state. In 2006, Honorable Senators Edward M.
Kennedy, Barack H. Obama, and Hillary R. Clinton proposed a uniform standard and
protocol of NBS in USA. An integral part of this proposal was to extend this program to
resource-poor countries under the auspices of the United States Agency for
International Development. Unfortunately, due to political events in USA and the death
of Senator Edward M. Kennedy, nothing materialized in this direction.
NBS in America always includes analysis for hemoglobinopathies as described
by the Health Resources and Services Administration (HRSA) Maternal and Child
1
Bioscience), high performance liquid chromatography (Bio-Rad and Trinity Biotech), and
isoelectric focusing instruments (PerkinElmers Resolve, and Helenas SPIFE) for NBS.
6.2 Methodologies
Isoelectric focusing (IEF) and high performance liquid chromatography (HPLC) are
the two most commonly used screening methods for hemoglobinopathies in the
neonate. Recently Sebia (Evry, France) has introduced the capillary zone
electrophoresis (CZE) method (Neonate Hb Fast System) for the newborn screening of
hemoglobinopathies. In order to resolve abnormal results of NBS,the blood of the
biological parents (and sometimes of the siblings) are analyzed by IEF, HPLC, agarose
gel electrophoresis (pH 8.6 and 6.2) and capillary zone electrophoresis to ascertain
genetic inheritance of the abnormality in the neonate. Finally, the diagnosis is confirmed
by means of DNA mutation studies,but for Hb S the final diagnosis can be confirmed by
a Sickle test, and complete blood count (CBC) with manual differential. Further
confirmation, if desired, for Hb S in a newborn can be achieved by testing the blood of
the parents. Electrospray ionization-mass spectrometry (Chapter 3.4), PCR and Sanger
sequencing (Chapter 3.5) are also used to confirm DNA mutation. A table of screening
2
The principle of the assay by IEF (Chapter 2.7) and HPLC (Chapter 2.8) for the
screening of hemoglobinopathies in the neonate and the adult are similar. However
certain adjustments are required for the neonate specimen (dried blood spot on filter
paper) handling and processing. The Resolve kit and instrumentation of the
210
PerkinElmer and SPIFE 2000 or 3000 instrument of the Helena Laboratories are the
most commonly used methods for the screening of hemoglobinopathies by IEF. HPLC is
most commonly performed employing ion-exchange chromatography by the NBS
instruments manufactured by BioRad, USA., or Trinity Biotechs Ultra Resolution
System.
The principle of the CZE for the screening of hemoglobinopathies (Chapter 2.6)
in the adults and neonates is identical, except that modifications in the automated
instrument are necessary for the handling of a dried blood sample on filter paper from
the neonate (Figure 1). Sebia is the main supplier of capillary zone electrophoresis
instrument and reagents for the NBS of hemoglobinopathies.
3-5
IEF and HPLC methodologies for NBS, results have to be considered provisional and
confirmatory procedures are always required because many rare hemoglobin variants
migrate or elute on the same position of the common one and because different
homozygous or hemizygous genotypes look identical with these methods.
FA
212
AF
FAS
FSS
FAC
FAE
FEE
FSE
FAD
213
positive results , therefore the blood is retested when the adjusted gestational age is 40
weeks and two months after transfusion if executed.
214
In this section, selected cases are presented to illustrate the laboratory data
obtained from NBS from commonly used methods.
215
216
217
218
219
Figure 10. IEF results of newborn (Hb SC disease), father (Hb AS trait), and mother (Hb
AC trait).
220
221
Isoelectric
focusing
Figure 11 Normal: FA
IEF of normal phenotype displays three prominent bands, Hb F, Hb A, and acetylated
Hb F. Hb F is the prominent band in newborns. Hb A (the middle band) in the IEF
pattern often appeared weaker in premature babies compared to full term babies. In all
the HPLC separations the prominent Hb peaks (i.e. with significant concentration)
eluted at specific retention times.
Isoelectric
focusing
Isoelectric
focusing
222
Figure
12 Hb
AS trait
FAS
Isoelectric
focusing
Figure 16 Hb S disease FS
223
Isoelectric
focusing
224
225
226
elutes on HPLC at the position of the common Hb E, many other variants elute at the
position of Hb S, Hb D, or Hb C and therefore molecular confirmation is always needed.
2.
3.
4.
5.
6.
newborn bloodspot screening for sickle cell disease and other clinically
significant haemoglobinopathies in England: screening results for 2005-7. J Clin
Pathol 2009; 62: 26-30.
Adorno EV, Couto FD, de Moura Neto JP, Menezes JF, Rego M, dos Reis
MG, Goncalves MS. Hemoglobinopathies in newborns from Salvador, Bahia,
Northeast Brazil. Cad. Saude Publica, Ruio de Janeiro 2005; 21(1): 292-298.
229
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Chapter 7
231
period, in the eighties, the diagnosis of thalassemia was obtained either indirectly by
linkage analysis using RFLP at the -globin cluster [7] or directly by oligonucleotide
hybridization on electro-phoretically separated DNA fragments [8] or by enzymatic
digestion of mutated sites. A major impulse has been given by the PCR technology that
allowed the development of a number of procedures, for easier mutation detection, as
well as the development of both PGD and non-invasive prenatal diagnosis procedures.
Nowadays thalassemias are detected directly by the analysis of amplified DNA from
fetal trophoblast or, more rarely, from amniotic fluid cells.
In this review we will delineate current procedures for prenatal and
preimplantation diagnosis of thalassemias as well as the most promising approaches for
non-invasive prenatal diagnosis.
Prenatal Diagnosis
Detection Methods:
Detection of molecular defect in both parents is a prerequisite for prenatal
diagnosis of the disease. The majority of defects affecting the -globin gene are point
mutations that occur in critical areas for its function, or single/few base addition/deletion
that change the frame in which triplets are translated into protein. Very rarely thalassemia results from gross rearrangement in the -globin gene cluster. In spite of
the marked molecular heterogeneity, a limited number of molecular defects are
prevalent in every at risk population. This may be very useful in practice, because a
panel of most frequent mutations to be searched for can be designed according the
carrier's ethnic origin [9]. Known mutation detection is caried out by a number of PCR234
based techniques. (ARMS, Amplification Refractory Mutation System) and the reverse
oligonucleotide hybridization with specific oligonucleotide probes (RDB, Reverse
Oligonucleotide-probe analysis).
Primer-specific Amplification:
The method is based on the principle that a primer carrying a mismatch in its 3'
region cannot anneal on its template. With this method, the target DNA fragment is
amplified in two separate PCR reactions using a common primer and either of the two
following primers: one complimentary to the mutation to be detected (-thalassemia
primer) and one complementary, at the same position, to the normal DNA (normal
primer). Normal DNA is amplified only by the normal primer while the DNA from
homozygotes only by the -thalassemia primer and DNA from heterozygotes by both
primers. A different sized fragment of the -globin gene is simultaneously co-amplified
as an internal control of the PCR reaction [10]. The method is very simple as it
requires, for each mutation to be searched, only two PCR reactions followed by agarose
gel electrophoresis. A further improvement of the methodology can be obtained by
multiplexing the primers for more than one mutation. In good hands the method is very
safe and particularity useful in fetal DNA analysis to search for mutations previously
detected in the parents.
237
seems to be the faster and most useful approach to detect unknown thalassemia
mutations.
If a mutation is not detected by sequence analysis, we search for the presence of
small deletions by polyacrylamide gel electrophoresis of amplicons designed for the
most frequent small deletional defects of the -globin gene (gap PCR). Furthermore,
the presence of larger deletions of the cluster may be identified by Southern-blotting or
more recently by Multiple Ligation-Dependent Probe Amplification (MLPA) for which a
commercial kit is available (SALSA MLPA KIT P102 HBB-MRC Holland).
In a very limited number of cases, direct sequencing from position -600 to 60 bp
downstream from the -globin gene and methods for deletion detection, failed to detect
the disease causing defect. In these cases, the molecular defect may reside either in
the locus control region of the -globin gene cluster, or in one of the genes, outside the
-globin gene region, encoding for regulatory proteins acting in trans on the function of
the -globin gene. Very recently it has been proved that the -thalassemia-like
phenotype could be caused by the coinheritance of a -globin gene defect and a
duplication of the -globin gene cluster, which results in an excess of chain. In these
selected cases, the characterization of these -globin gene rearrangements (SALSA
MLPA KIT P140-B2 HBA-MRC Holland) can be routinely carried out with success by
MLPA analysis.
Both members of the couple at risk are counseled in a non-directive way. The
nature of the disease, the implications of being carriers and reproductive choices are
analyzed, specifically those concerning birth control, including prenatal or
preimplantation diagnosis and the possibility, in case of affected fetus HLA compatible to
not interrupt pregnancy. As for fetal testing, detailed information is offered regarding the
risk of fetal mortality, the risk of misdiagnosis, and the mortality and morbidity of an
abortion in case of affected fetus.
Fetal DNA is analyzed using the same methods described above for detection of
known mutations during carrier molecular screening. To limit the possibility of
misdiagnosis, we analyze chorionic villous DNA with two different procedures: i.e. RDB
hybridization and primer-specific amplification, using distinct couple primers.
Misdiagnosis may occur for several reasons: failure to amplify one copy of the target
DNA fragment, mispaternity, maternal contamination, and sample exchange.
Misdiagnosis for failure of DNA amplification is obviously limited by the double approach
described above. To avoid misdiagnosis due to maternal contamination as well as
mispaternity and/or sample exchange, a fetal DNA microsatellite analysis is usually
performed to verify the presence of one allele from each parent [9]. In our hands, by the
above mentioned PCR-based procedures, no misdiagnoses have occurred in more than
5000 cases. Figure 1 shows the overall results of the Sardinia prenatal diagnosis
program since the beginning of 1976 up to the end of the past year.
240
241
242
Cell Biopsy:
Preimplantation may be carried out by either cleavage-stage biopsy of 1-2
blastomeres, from an eight-cell embryo three days after in vitro fertilization carried out
by ICSI (Intracytoplasmatic Sperm Injection), or by the biopsy of polar bodies.
For cleavage-stage biopsy the embryo is grown in vitro until it reaches a six-eight cell
stage which usually occurs on the third day after insemination. Polar bodies diagnosis,
pioneered by Verlinsky and his group in 2006 is based on the analysis of the first polar
body of unfertilized eggs [27], and may lead to distinguish between unfertilized eggs
that carry the defective gene and those without the defect. The successive sampling
and analysis of the second polar body that is extruded from the oocyte after fertilization
and completion of the second meiotic division, is carried out in order to avoid
misdiagnosis due to the high rate of recombination that happens during the first meiosis.
By fertilizing in vitro only the eggs without the defect and replacing them in the mother, a
successful pregnancy with a normal fetus can be obtained. Recently a preconceptional
genetic diagnosis based on the analysis of only the first polar body has been proposed
for countries in which the use of PGD and manipulation of embryos is prohibited [28].
This approach although permitting to avoid the manipulation, cryopreservation and/or
discard of sovranumerary and/or affected embryos, shows several problems: the need
to obtain more than 10-12 oocytes, the increased risk of diagnostic error and the
increased risk of the technical difficulties. Blastocyst biopsy, even if it has the advantage
to provide a higher number of cells, is at present more rarely used because of the
difficulties of the embryos to reach this stage in IVF programs. The cleavage-stage
243
biopsy of blastomeres from an eight-cell embryo is the most frequently used PGD
procedure all over the world.
Detection Methods:
Methods for mutation detection in OGD are always based on multiple steps of
PCR. Mutations are detected in PCR products by various methods that combine speed,
analytical sensitivity and specificity. In particular, a first round of multiplex PCR is
performed to amplify both the -globin gene region including the mutation and one or
more polymorphic loci. Secondly, two separated nested PCR reactions are performed
to amplify the two or more selected genomic regions. Finally, the polymorphic alleles
are directly detected by capillary electrophoresis of the amplified fragment, while the
presence of -globin gene mutations are identified by the subsequent mini sequencing
reaction [29]. This approach is expressly designed to detect the presence of the globin gene mutations and to monitor, in the same sample, the presence of
contamination as well as the eventual allele drop-out that represent the most frequent
causes of error in PGD.
Quality Control:
For both techniques a prenatal diagnosis by villocentesis is recommended in
order to avoid diagnostic errors. Successful pregnancies following the transfer of
human embryos in which the -globin gene defect has been excluded, occur only in 2025% of cases and the birth rate of a child is even lower. Due to the low birth rate most
women have to undergo PGD several times in order to give birth to a healthy child [30].
244
PD or PGD?
Among clinical geneticists there has been much discussion about the main goal
of PD. Some have argued that the main aim is to avoid the birth of an affected child.
Others have emphasized the reproductive confidence and the purpose of informing the
couples at risk about the status of the fetus. Several studies indicate that if there is no
PD option, a large proportion (up to 50%) of the couples at high risk of an affected child
refrain from pregnancy despite their wish to reproduce. When PD is possible many
more at-risk couples dare to embark on a pregnancy.
Most experts consider PGD as an additional option for couples at risk and not as
a replacement for conventional prenatal diagnosis. PGD is still considered a highly
specialized experimental procedure with limited results, mainly dedicated to couples
against abortion for ethical and religious reasons and to a small proportion of couples
who have experienced repeated abortion, that ask for referral for this procedure.
At present its use in routine monitoring of pregnancies at risk is precluded by the
technical demand for these procedures, the difficulty in organizing the service, and the
high costs.
245
246
the first evidences that fetal lymphocytes persist in maternal circulation one year after
delivery was published in 1974 by Bianchi et al [40]. Several years later Bianchi et al
described the presence of fetal progenitor cells 27 years after delivery [41]. For these
reasons also lymphocytes, as trophoblasts, became an unattractive candidate for noninvasive prenatal diagnosis.
Erythroblasts:
One of the main advantages to study fetal erythroid cells is that they are
nucleated, terminally differentiated short-lived cells and for this reason they do not
persist in maternal circulation for a long time after delivery. Furthermore, first primitive
erythroblasts appear in the embryonic bloodstream around the four-five week gestations
so they can be detected early during gestation.
Nevertheless, their isolation from maternal peripheral blood is still problematic
because of their rarity and the lack of a fetal specific antibody.
In 1990 Bianchi [41] first described a method for fetal nucleated erythroid cells CD71
transferrin receptor, highly expressed in erythroid cells. Two years later Ganshirt-Ahlert
et al [43] obtained similar results by using a new detection system called MACS
(Magnetic Cell Sorting) which is based on the use of antibodies labeled with magnetic
beads.
Since then, both systems have been extensively improved and used, by several
groups, following different approaches which can consist in the positive selection of
CD71 and/or glycophorin-A fetal cells and/or in the negative depletion of CD45 maternal
cells. Usually, in both cases, a previous density (Ficoll or Histopaque) gradient
centrifugation step is carried out to remove non-nucleated maternal erythrocytes. A
248
schematic workflow resuming one of the strategies used for isolating fetal NRBCs from
maternal peripheral blood is represented in Figure 2. Finally both MACS and FACS
sorted cells are labeled with fluorescent antibodies which recognize embryonic (, ) or
fetal () hemoglobin chains and are eventually subjected to FISH analysis for
chromosome Y detection. An example of positive labeling with the antibody for gammaglobin conjugated with FITC is shown in Figure 3. Molecular characterization can
eventually be carried out in positive fluorescent cells isolated by laser microdissection.
Even with the high progress made in the last twenty years in this field, the methods for
erythroblasts enrichment are still limited as they mostly result in the recovery of fetal
samples with low yield (FACS) and scarce purity (MACS), being variably contaminated
by maternal cells.
For these reasons in recent years several studies have been addressed to the
proteomic field with the attempt to characterize novel fetal erythroblast cell-specific
surface markers. For example, bi-dimensional electrophoresis coupled with mass
spectrometry has allowed the identification of 2 proteins, differentially expressed in
sickle erythrocytes in comparison to healthy erythrocytes, and the detection of proteins
up-or downregulated in fetal erythroid cells in comparison to their adult counterparts.
Some of these results have been published as a full-patent application and the data
concerning the new antibodies developed against these new targets expect to be
validated in large samples of maternal blood [44].
fetal cell recovery are expected to be obtained through the application of micro-fluidic
rare-cell capture technologies [45] which are being developed to detect not only fetal but
also cancer as well as other rare cells in biologic fluids.
249
250
Analysis of Fetal Cells in Maternal Blood and Non Invasive Prenatal Diagnosis
(NIPD) of -Thalassemia:
Despite the difficulties encountered to find the best target cell and the best
method for their enrichment and isolation, several attempts have been made in the last
twenty years, to transfer the results of these researches into clinical practice.
Unfortunately the lack of reproducibility of experiments hardly makes the isolation of
fetal cells from maternal blood as a first choice method of NIPD of monogenic disorders.
Below the most significant results obtained in NIPD of -thalassemia are briefly
summarized. The first example of non invasive prenatal diagnosis of
hemoglobinopathies was described in 1990 by Camaschella et al [46]. The genetic test
was carried out in three selected couples where the mother was a carrier of thalassemia and the father of the Hb Lepore-Boston trait. The absence/presence of the
paternal trait was successfully detected in PCR amplified samples DNA extracted from
T-cell samples were obtained by incubating Ficoll-separated cells of the mother with the
CD 3-specific MoAb Leu 4 and then separating the positive cells with goat-anti-mouse
immunoglobulin G (1gG)-coated immunomagnetic beads.
In those years most of the studies were addressed to couples carrying different
mutations and only aimed to the exclusion of the paternal allele in the enriched fetal
cells, as most of the times they were contaminated from maternal cells.
In subsequent years, even if the fetal cells enrichment and selection methods
have been greatly improved, other IP diagnosis have been carried out but with
fluctuating results. Below are described three significant examples of NIPD realized,
with different levels of success, by using single or pooled erythroid cells.
251
In 1996 the group of Y.W.Kan47 reported the successful identification of two fetal
genotypes by using fetal nucleated erythroid cells selected by MACS, anti- globin
immunostaining and then isolated by microscopy and cell scraping. The
presence/absence of sickle cell and beta thalassemia mutations of both parents were
finally detected by Reverse Dot Blot in PCR amplified samples constituted by pools of
fetal dissected cells.
A few years later the group of Di Naro [48] replicated these results using a
slightly different procedure for erythroblast enrichment which was carried out by Percoll
and Gastrografin multiple gradient centrifugation. Mutation detection was then obtained
by automated sequencing of single cells amplified by PCR. According to authors, even
if the risk of allele drop out is higher when amplifying single cells, however the possibility
to study several individual, instead of pooled, cells guarantees an accurate diagnosis of
the fetal DNA.
More recently the group of Kolialexi [49] has hardly tried to replicate these
results. In this study, NIPD was performed through magnetic cell sorting (MACS) and
microdissection of single NRBCs with a laser micromanipulation system. Single-cell
genotyping was achieved by nested real-time PCR for genotyping -globin gene
mutations; a multiplexed minifingerprinting was used to confirm the origin of the isolated
cells and to exclude their possible contamination. A total of 224 cells were isolated but
only half of them were successfully amplified. In the majority (n=80) of these cells
minifingerprinting was not informative because of allele dropout or homozygosity. In the
rest of the samples, 22 cells resulted to be of fetal origin, 26 maternal while 80 were non
informative.
252
Analysis of Fetal DNA in Maternal Plasma and Non Invasive Prenatal Diagnosis
(NIPD) of Thalassemia:
The existence of cell-free nucleic acids within the human plasma was firstly
reported in 1948 by Mendel and Metais [50] which described their presence both in
normal subjects and in individuals affected by various diseases. Some decades later
other studies have confirmed the presence of circulating DNA as well as of RNA in
several pathological conditions (pancreatitis, inflammatory diseases, cancer, diabetes,
etc) [51].
In 1997 Lo et al discovered for the first time that a fetus may release cell-free
fetal DNA (cffDNA) into maternal plasma, thus providing an alternative to fetal cells for
noninvasive prenatal diagnosis [52].
In recent years more information has been acquired about the concentration, the
origin and the characteristics of the cell-free fetal DNA and several procedures have
been developed in order to use it in prenatal diagnosis.
The cell-free DNA is constantly present in peripheral blood of non pregnant
women and its concentration increases during pregnancy. The cell-free fetal DNA
represents the 3-5% of the DNA present in maternal plasma from which, after delivery, it
is rapidly cleared.
Recent studies carried out by microfluidic digital PCR have revealed that cffDNA
can be present at even higher concentrations which can reach up to 10-20% of total
DNA in maternal plasma [53]. Nevertheless, because of the high background of
maternal DNA, an enrichment step is needed to obtain highly purified fetal DNA
samples suitable for non invasive prenatal diagnosis.
253
but less stable in the presence of single-pair mismatches. In that paper their ability to
clamp wild type -globin sequences was proved in artificial mixture of DNA samples
enriched with increased amounts of wild type alleles, by using a microchip platform to
detect the -thalassemia mutations.
Four years later [56] the efficacy of PNA was evaluated with success in 41 non
invasive prenatal diagnosis of -thalassemia and in combination with three different
techniques (microelectronic chip, pyro sequencing and direct sequencing) to detect fetal
DNA mutations in maternal plasma.
Despite its successful application, this strategy, as the other above described
technologies, is still restricted to couples which carry different mutated alleles and
aimed to the detection of mutated paternal alleles.
Another method recently described for NIPD of -thalassemia is called APEX
namely Arrayed Primer Extension. This is a mutation detection system which is based
on the combined use of the microchip technology and the single nucleotide base
extension method. This system has been recently described by the group of
Papasavva [57] and used to characterize the presence of the paternal -thalassemia
mutations and associated -globin gene SNPs, in cffDNA isolated from maternal
plasma. The possibility to study the polymorphisms associated to the mutated alleles
represent a feature of great value since it would give the possibility to extend NPID to
couples which carry the same mutated allele. Prerequisite for its application is to find
informative SNPs associated with parental mutations which can help to discriminate the
paternal mutated allele and to characterize the haplotype inherited from the fetus. The
authors of the paper described the correct application of this methodology in the NIPD
255
of six out of seven couples at risk for -thalassemia, carried out in the Cypriot
population.
Future Perspective:
As previously reported, one of the major problems which still limits the application
of the described protocols in clinical practice is the impossibility to obtain highly purified
fetal, cellular as well as cffDNA, samples which could allow the detection of parental
alleles, even when they are identical. Few clinical applications of NIPD are actually
restricted to the detection of the Y chromosome, for fetal sex determination, or the
Rhesus D gene, in Rhesus D negative women, or, in general, of genetic loci which are
absent in the maternal genome.
In recent years a great improvement has been obtained in the field of the
technologies which can explore the presence of sequence variations even in single
molecules of DNA. The concept of "Digital PCR" was firstly introduced in 1992 by
Sykes [58] who described a method to determine the number of starting DNA templates
by doing Poisson statistical analysis of PCR results obtained in limiting dilutions. The
more recent development of the emulsion PCR (emPCR) have further enhanced the
possibility to study single molecules of DNA by using a small volume of reactions, wateroil emulsions and microfluidic as well as high-throughput platforms (for a review of both
methods and application to NIPD please see Zimmermann et al [59].
Recent applications of these technologies in the field of NIPD, and in particular in
the diagnosis of aneuploidies and monogenic disorders, have shown that these
methodologies may find useful application in the near future, even if several drawbacks
256
need to be solved and wider validation studies should be carried out before transferring
their use in routine diagnostics.
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54. Li Y, Di Naro E, Vitucci A, Grill S, Zhong XY, Holzgreve W, Hahn S. Size
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261
Chapter 8
Hemoglobin A1c
Zia Uddin, PhD
8.1 Introduction
The term glycated hemoglobin refers to the non-enzymatic, irreversible, covalent
bonding of glucose at one or both N-terminal valine residues of the hemoglobin -chain,
and the N-terminus of the -chains, or the -amino groups of lysine residues (Figure 1).
The term normal hemoglobin phenotype beyond the neonatal period involves a
major fraction due to Hb A (22), and a minor fraction of Hb A2 (22). Occasionally a
very minor fraction of Hb F (22) is also detected. Further chromatographic analysis of
262
263
The fast moving forms of Hb A were separated in the late 1950s and recognized as
being associated with diabetes in the late 1960s
are not elevated in diabetes, the clinical focus has been solely on Hb A1c. For clinical
testing purposes the term Hb A1c analysis is referred to as A1c or the A1c test with the
word hemoglobin omitted as a matter of convenience.
8.2 Hb A1c Diagnostic Role in Diabetes Mellitus, and Glycemic Control in Adults
After three decades of investigation and evaluation of numerous proposals by
various scientific and clinical organizations, Hb A1c found its status as a diagnostic test
4
for diabetes mellitus. One of the four criteria for the diagnosis of diabetes mellitus are:
2-h plasma glucose > or = 200 mg/dL (11.1 mmol/L) during an Oral
Glucose Tolerance Test
Several investigators have recently suggested the combined use of fasting glucose and
Hb A1c for the diagnosis of diabetes mellitus.
5-7
treatment for diabetics is now being performed based on the laboratory test results of Hb A 1c.
One objective would be to get glucose levels to as close to normal as possible with minimal or
264
disease (type I and II). Recently a follow up study of the ACCORD (Action to Control
Cardiovascular Risk in Diabetes) stipulated that the best target of Hb A1c in middle aged or
older patients with cardiovascular risk factors is between 7.0 and 7.9%.
Hb A1c is widely used to judge the treatment of diabetes and adjustment of the
medication dose when necessary. In chronic glycemia the blood glucose is monitored more
frequently (once a day or more). Since Hb A1c is measured less frequently and in percent, and
is a complicated process to explain to the patient, it is convenient for the physician to relate
the result to glucose concentration (in mg/dL or mmol/L) over the preceding 5-12 weeks. This
derived glucose concentration from Hb A1c value is called Estimated Average Value (eAG).
Patients monitor their blood glucose and their physician can relate that performance to the
eAG. This way the patients can see the effect of their behavior over time on the test outcome.
The only way this feat could be accomplished, if the result for Hb A1c be the same no matter
where the result was run. This simple feat required the cooperation of many government
agencies and all Hb A1c laboratory testing manufacturing facilities and was brought about by
the determination of the Diabetes Control and Complication Trial (DCCT) Research Group and
the American Diabetes Association.
The mathematical relationship then developed between HbA1c and eAG is based on
the following linear regression equation.
10
265
eAG (mg/dL)
11
Table 1.
NGSP (Hb A1c%)
eAG(mg/dL)
eAG (mmol/L)
97
5.4
126
7.0
154
8.6
183
10.2
212
11.8
10
240
13.4
11
269
14.9
12
298
16.5
266
Currently over 100 different methods are available for quantification of Hb A1c.
Most available Hb A1c methods are certified by the NGSP
12
Immunoassay
Boronate affinity binding/HPLC
Ion-exchange HPLC
Capillary zone electrophoresis
Enzymatic
Sacks.
8.4
diabetes (type I and II), several factors influence the accuracy of its laboratory results,
e.g., a) hemolytic disease or other conditions with reduced red blood cell survival, b)
recent blood loss, c) iron deficiency anemia, d) patients with renal failure, and e)
hemoglobin variants. All these interferences cannot be easily delineated by the
laboratory personnel and the physician. Due to the diluvial of methods, reagents, and
267
instruments for the assay of Hb A1c , it is impossible for the laboratory to be aware of
the methods limitation with respect to the presumptive interference by >1000
hemoglobin variants reported so far in the literature (http://globin.cse.psu.edu). In the
case of the most common hemoglobinoathies (AS, AE, AC, AD), Hb A1c can be
accurately measured if the correct method is used. The affect of these hemoglobin
variants (AS, AE, AC, AD) and elevated Hb F in HPFH (not pathological) on the results
of Hb A1c by the most often used methods is presented in Table 2.
268
Several studies have shown a correlation of Hb A1c with fructosamine and was thus
recommended in patients with hemoglobinopathies.
14
References
1
2
3
4
5
6
8
9
10.
11.
12.
13.
14.
iv
vi
vii
viii
ix
x
xi
Lee S-T, Weykamp CW, Lee Y-W, Kim J-W, Ki C-S. Effects of 7
Hemoglobin Variants on the Measurement of Glycohemoglobin by 14
Analytical Methods. Clin Chem 2007; 53(12): 2202-2205.
Roberts WL. Hemoglobin Constant Spring can interfere with Glycated
Hemoglobin Measurements by boronate Affinity Chromatography. Clin
Chem 2007; 53(1): 142-43.
272
Case Studies
Introduction
The following case (# 1-28) studies include laboratory data representing results
from five different hemoglobin separation methods commonly used in the clinical
laboratory. Due to the large number of variants possible the mandate is that
more than one separation method be used in identification. The question is which
two methods would provide discriminative information. The results of the lab
tests for each case are presented in a tabular form to assist in these choices.
The alkaline electrophoresis images are of Helena SPIFE Alkaline
Electrophoretic results but identical separation results would have also been
obtained using alkaline cellulose acetate, Helena Biosciences SAS alkaline
hemoglobin gels, Helena Quick Gels or Sebia Hydrasys alkaline hemoglobin
gels.
The acid electrophoretic images are of Helena SPIFE or QUICK Gel Acid
electrophoresis. For Acid electrophoretic separation, two classes of media have
been used with differing separation results. Historically, acid hemoglobin
separation was done on agar using citric acid buffer. Helena SPIFE and Quick
Gels are of this type. Agarose purified from agar has more recently been used by
Beckman, Sebia and Helena BioSciences. The purified nature of the agarose
makes these products easier to produce but historically they lacked easily
available documentation of the differences in mobilities compared to the
273
historically used agar. These differences have been documented in the table
associated with the attached case studies. All acid agarose data were adopted
from (Variant Hemoglobins. a Guide to Identification. 1 st edition, by Barbara J.
Bain , Barbara J. Wild , Adrian D. Stephens, Lorraine A. Phelan . Published
2010 by Wiley-Blackwell Publishing Ltd).
All Capillary Zone Electrophoresis (CZE) data were generated using the Sebia
Capillarys System. This CZE system separates hemoglobins into 15 zones and
provides a list of possible variants that migrate in that zone. The operator then
selects the hemoglobin variant they expect that peak to represent. The peaks in
the CZE reports in the case studies have been labeled in such a fashion but a
different assignment could have been made by the operator had they had
information warranting the choice. Details of other vendor results would require
contact with the vendor but the goal again would be to maintain equality as close
as possible and the assumption would be that the order of separation would not
be different.
All isoelectric focusing images are actual or simulated from actual data obtained
with the Helena Isoelectric Focusing Gels either on the SPIFE or the REP
systems. The Perkin Elmer Resolve (formerly IsoLab) isoelectric focusing
systems would obtain the same results, because the pH range of the ampholytes
are the same. These agarose gels contain acrylamide to sharpen the bands.
Again the end user is the final discriminator. In this case, proper selection of the
274
Case # 1
Normal Adult
Laboratory Data:
Hemoglobin
Hematocrit
RBC
MCV
MCH
MCHC
RDW
Platelet
13.5
39.6
4.7
84.3
28.8
34.1
13.5
200
Hb A
Hb A2
Hb F
98.0
1.8
0.2
94.3 - 98.5%
1.5 - 3.7%
0.0 - 2.0%
276
Isoelectric focusing
277
Reference
Bain BJ. Hemoglobin and the genetics of hemoglobin synthesis: In:
Haemoglobinopathy Diagnosis, Blackwell Publishing, second edition, 2006, pp 12-22.
280
Laboratory Data:
Hemoglobin
Hematocrit
RBC
MCV
MCH
MCHC
RDW
Platelet
Hb A
Hb S
Hb A2
Hb F
13.1
39.6
4.4
82.1
27.3
32.1
12.6
267
59.2
38.4
1.8
0.6
281
Isoelectric focusing
282
Summary of Results
Method
Hb A
area
Hb S
area
Alk Agarose
Major
band
(Hb A)
Major
band
(Hb S)
Acid
Agar/Agaros
e
Major
band
(Hb A+
Hb A2)
Major
peak
(Hb A)
Zone 9
Major
band
(Hb S)
IEF
Major
band
(Hb A)
Major
band
(Hb S)
HPLC
Major
peak
(Hb A)
RT=2.3
4
Major
peak
(Hb S)
RT=4.2
6
CZE
Major
peak
(Hb S)
Zone 5
Hb
A2/C
area
Minor
band
(Hb A2)
Minor
peak
(Hb A2)
Zone 3
Minor
band
(Hb A2)
Minor
peak
(Hb A2)
RT=3.6
5
Since the solubility test was positive and the aberrant band fell between
35 40%, a diagnosis of Hb S trait was made. Concentrations of Hb S other than
35 40% require consideration of the effect of a transfusion, the possibility of
iron deficiency, a concurrent Hb S--thalassemia (Hb S < 33%), a Hb S-thalassemia (Hb S >49%) or the possibility that the fraction may not be Hb S at
all. Mutation at the 6th amino acid position of the chain [6 (A3) GluVal)
causes the substitution of glutamic acid by valine that results in the formation of
Hb S.
284
References
285
1.
2.
3.
Bain BJ. Sickle cell haemoglobin and its interactions with other variant
haemoglobins and with thalassaemias. In: Bain BJ, Ed. Haemoglobinopathy
Diagnosis, 2nd edition, Blackwell Publishing; 2006:141-149.
Steinberg MH. Sickle cell trait. In: Steinberg MH, Forget BG, Higgs DR,
Nagel RC, eds. Disorders of Hemoglobin: Genetics, Pathophysiology and Clinical
Management. Cambridge, England: Cambridge University Press; 2001: 811-830.
Feliu-Torres A, Eberle SE, Bragos IM, Sciuccati G, Ojeda MJ, Calvo KL,
Voss ME, Pratti AF, Milani AC, Bonduel M, Diaz L, Noguera NI. Hb S-San Martin:
A new sickling hemoglobin with two amino acid substitutions
[6(A3)GluVal;105(G7)LeuPro]. Hemoglobin 2010; 34(5): 500-504.
Laboratory Data:
Hemoglobin
7.6
RBC
2.6
MCV
80.0
RDW
21.0
Platelet
201
Hb A
4.2
Hb S
90.0
Hb A2
2.8
Hb F
3.0
(Hemoglobin fractions from HPLC)
Isoelectric focusing
288
Note: The original CZE on this specimen showed no presence of Hb A, therefore the analysis was
repeated after mixing the specimen 1:1 with a normal blood. This is the standard practice in cases
whenever Hb A is not detected.
Summary of Results
Method
Hb A
area
Alk
Agarose
Acid Agar
/ Agarose
CZE
Hb S
area
Major
band
(Hb S)
Major
band
(Hb S)
Major
peak
(Hb S)
Zone 5
IEF
Major
band
(Hb S)
HPLC
Major
peak
(Hb S)
RT=4.38
Hb A2/C
area
Minor
band
(Hb A2)
Minor
peak
(Hb A2)
Zone 3
Minor
band
(Hb A2)
Minor
peak
(Hb A2)
RT=3.6
Minor
peak
(Hb F)
RT=1.04
References
1.
2.
3.
4.
5.
Case # 4
African-American adult male, apparently healthy and without any previously known major
clinical condition visited his family physician for his annual check-up.
292
Laboratory Data:
Hemoglobin
13.4
RBC
4.78
MCV
80.9
MCH
28.0
Hb A
5.7
Hb S
56.0
Hb A2
3.3
Hb F
35.0
(Hemoglobin fractions from HPLC)
Peripheral Blood Smear: No abnormality was noticed.
Sickle cell solubility test for Hb S: Positive.
293
Isoelectric focusing
294
Summary of Results
Metho
d
Alk
Agaros
e
Hb F
area
Major
band
(Hb F)
Acid
Agar /
Agarose
Major
band
(Hb F)
Major
peak
(Hb F)
Zone 7
Major
band
(Hb S)
Major
peak
(Hb S )
Zone 5
IEF
Major
band
(Hb F)
Major
band
(Hb S)
HPLC
Major
peak
(Hb F)
RT=1.16
Major
peak
(Hb S)
RT=4.38
CZE
Hb A
area
Hb S
area
Major
band
(Hb S)
Hb A2/C
area
Faint
band
(Hb A2)
Minor
peak
(Hb A2)
Zone 3
Faint
band
(Hb A2)
Minor
peak
(Hb A2)
RT=3.6
296
From all the five laboratory methods, three abnormalities are evident:
a
b
c
Absence of Hb A
Hb S (56%)
Hb F (35%)
Hb S HPFH patients are the result of a point mutation on one beta gene
forming Hb S and a deletion of the delta and beta area on the other gene
permitting the production of Hb F to continue. The Hb F expression will be 25
35%. This is a pancellar condition so every erythrocyte will contain Hb F as well
as Hb S and the damage caused by Hb S is not seen. Generally speaking
patients with Hb S-HPFH are clinically well, with a benign clinical course, little
evidence of hemolysis and without severe anemia. It is prudent to make a
clinical diagnosis based on all available resources. In this case other laboratory
data showed a positive sickle solubility test, a normal CBC, serum iron and
ferritin, and no other abnormalities except for some sickling. Consultation with the
physician indicated the patient was clinically well and certainly had not been
treated with hydroxyurea. This patient is presumed to be Hb S HPFH.
Approximately 1% of Homozygous S patients present with 5% or less Hb
F and these patients clinically do better than those without Hb F. Therefore much
has been done to increase the production of Hb F in homozygous S patients in
general. Degree of success of hydroxyurea treatment has been very variable for
297
References
1. Murray N, Serjeant BE, Serjeant GR. Sickle cell-hereditary persistence of
fetal hemoglobin and its differentiation from other sickle cell syndromes. Br J Haemotol
1988; 6: 89-92. (available online since March 2008).
2. Hoyer JD, Connie SP, Fairbanks VF, Hanson CA, Katzmann JA. Flow cytometric
measurement of hemoglobin F in RBCs: Diagnostic usefulness in the distinction of
hereditary persistence of fetal hemoglobin (HPFH) and hemoglobin S-HPFH from other
conditions with elevated levels of hemoglobin F. Am J Clin Pathol 2002; 117: 857-863.
3. Akinsheye I, Al-Sultan A, Solovieff N, Ngo D, Baldwin CT, Sebastiani P, Chui DH,
Steinberg MH. Fetal hemoglobin in sickle cell anemia.Blood 2011; 118: 19-27.
4. Ngo D, Aygun B, Akinsheye I, Hanjins JS, Bhan I, Luo HY, Steinberg MH, Chui DH.
Fetal haemoglobin levels and haematological characteristics of compound
hegterozygotes for haemoglobin S and deletional hereditary persistence of fetal
hemoglobin. Br J Haematol 2012; 156(2): 259-64.
5. Whyte D, Forget BG, Chui DH, Luo HY, Pashankar F. Massive splenic infarction in
an adolescent with hemoglobin S-HPFH. Pediatr Blood Cancer 2013; 60(7): 49-51.
6. Chapter 2.3 of this book: Bernard G. Forget, MD. Hereditary Persistence of Fetal
Hemoglobin
299
7. Bain BJ. Hereditary persistence of fetal haemoglobin and other inherited causes of
an increased proportion of haemoglobin F. In: Haemoglobinopathy Diagnosis, Blackwell
Publishing, second edition, 2006, pp119-127.
Laboratory Data:
Hemoglobin
RBC
14.5
5.06
MCV
84.0
80 - 100 fL
MCH
28.7
27 - 34 pg
Hb A
77.1
94.3 - 98.5%
Hb A2
0.9
1.5 - 3.7%
Hb F
0.0
0.0 - 2.0%
Hb G
22%
(Hemoglobin fractions from HPLC)
Peripheral Blood Smear: No abnormality was detected.
Sickle cell solubility test: Negative.
301
Isoelectric focusing
Method
Hb A
area
Hb S
area
Alk Agarose
Major
band
(Hb A)
Major
band
(Hb G)
Acid
Agar/Agaro
se
Major
band
(Hb A+
Hb A2 +
Hb G+
Hb G2)
Major
peak
(Hb A)
Zone 9
Major
peak
(Hb G)
Zone 6
CZE
IEF
HPLC
Major
band
(Hb A)
Major
band
anodal
to Hb S
(Hb G)
Major
peak
(Hb A)
RT=2.4
5
Major
peak
(Hb G)
RT=4.0
4
Hb
A2/C
area
Minor
band
(Hb A2)
Minor
band
close to
carbonic
anhydras
e
Minor
peak
(Hb A2)
Zone 3
Minor
band
(Hb A2)
Minor
peak
(Hb G2)
Zone 1
Minor
peak
(Hb A2)
RT=3.6
Minor
peak
RT=4.54.6
Minor band
(Hb G2)
as far
cathodal to
A2 as G is
anodal to it.
* Note: HPLC retention time (RT) varies with the type of the instrument used and
several other factors, e.g. temperature etc.
The sickle cell solubility test was negative, ruling out the possibility of Hb
S. The separation table shows the presence of a non Hb S band migrating in the
Hb S area for all methods except Acid Electrophoresis. Common variants found
anodal to Hb S on alkaline electrophoresis that migrate in Hb A position on acid
electrophoretic conditions are Hb D, Hb G-Philadelphia and Lepore. Of these
options only Hb G- Philadelphia is an -chain variant. If -chain variant is
expressed in a large enough percentage to compete with normal -chains for
combination with chains a new small modified delta band is created. In
individuals with Hb G-Philadelphia [68(E17)AsnLys], a combination of Hb GPhiladelphia -chains with normal -chains leads to the formation of about 1% of
a molecule Hb G2 (2G2). Hb G2 has no clinical significance, but plays an
important role in the distinction between Hb D and Hb G-Philadelphia. Since Hb
G-Philadelphia is entirely innocuous, globin chain electrophoresis and DNA
studies are usually not necessary.
There is a temptation to analyze the available hemoglobin variants by
percentage since Hemoglobin Lepore runs less than 15 % and Hb D runs about
40 while Hb G-Philadelphia trait runs 20-25% in the heterozygote. Differentiation
between Hb D and Hb G-Philadelphia on the basis of the percentage of the
variant is not advised because the percentages of either would be effected by a
concurrent -thalassemia -2 trait or homozygous -thalassemia-2 (see below).
The single alpha gene deletion resulting in -thalassemia-2 trait is found in 1/3 of
African Americans therefore this silent mutation could be likely found in
305
References
1.
2.
3.
4.
5.
6.
7.
Laboratory Data:
307
Hemoglobin
11.7
RBC
4.29
MCV
81.6
MCH
27.4
Hb A
54.0
Hb S
19.9
Hb G
17.8
Hb A2
1.1
Hb F
0.2
Hb S-G Hybrid
7.0
(Hemoglobin fractions from HPLC)
Peripheral Blood Smear: No abnormality.
Sickle cell solubility test for hemoglobin S: Positive.
Unstable hemoglobin (isopropanol) Test: Negative.
No record of blood transfusion during the past six months.
308
1.5-3.7%
0.0-2.0%
Isoelectric focusing
Summary of Results
Method
Alk
Agarose
Acid Agar
/ Agarose
CZE
Hb A
area
Major
band
Hb A
Major
band
(Hb A +
G)
Major
peak
(Hb A)
Zone 9
IEF
HPLC
*Note:
Medium
peak
(Hb A)
RT=2.35
Hb S
area
Major
band
Hb S +
G
Major
band
(Hb S)
Major
peak
Zone 6
Major
peak
poorly
separated
from Zone
6 in Zone
5
Major Hb
G band
Anodal
to Hb S
Major
band
(Hb S)
Minor
peak
(Hb A2)
RT=3.58
Hb A2/C
area
Minor
band
(Hb A2)
Minor
Hb A2
peak
Zone 3
Medium
peak
(Hb G)
RT=4.0
Minor
band
cathodal to
A2
Major Hb
GS
hybrid
peak
Zone 2
Minor
Hb G
A2
peak
Zone 1
Medium
band (Hb
S-G
hybrid +
A2 )
Minor
Hb G2
Band
Medium
peak
(Hb S)
RT=4.24
Medium
peak
(Hb S-G
hybrid)
RT=4.8
HPLC retention time (RT) varies with the type of the instrument used and
several other factors, e.g. temperature etc.
Agarose gel electrophoresis (pH 8.6) showed three major bands in the
familiar positions of Hb A ( 50%), Hb S ( 38%), and Hb A2/C (>10%) and a
barely visible minor band slightly cathodal to the carbonic anhydrase position. It
should be emphasized here that Hb A, Hb S, and Hb C cannot all be
manufactured in any single person because there are only 2 beta genes and
these hemoglobins represent three different beta compositions. Either this patient
311
had a transfusion or one of the hemoglobin variants is not a beta chain variant.
The transfusion could be to a patient with Hb S and C or a transfusion using
blood from an Hb A-S heterozygous donor or an Hb A-C heterozygous donor to a
patient who was a heterozygote of the other type. These unlikely scenarios were
all ruled out as the patient received no blood transfusion.
Beta-thalassemia in conjunction with Hb A-S trait can result in an elevated
Hb A2 which migrates with or near Hb C by most of these methods. In S-thalassemia the Hb A2 is rarely higher than 10% so a >10% band is unlikely
Hb A2.
Secondly in S--thalassemia patients Hb F concentration is often
increased especially if the patient is thalassemic to the point that the Hb A2 is
very elevated but in this patient the Hb F was normal ( 0.2%).
The identity of the small barely visible minor band is the key to the
identification. The most common alpha chain variant is Hb G-Philadelphia which
would present in the Hb S area at 30 to 35%. This alpha chain variant then
competes with the unmodified alpha chains to combine with the beta and delta
chains available. Since the sickle solubility test was positive we know the band in
the position of Hb S is indeed at least partly due to the S beta gene combined
with normal alpha chains. This Hb S beta gene when combined with a modified
Hb G-Philadelphia alpha gene creates a new double hemoglobin variant
combination, Hb S-G Philadelphia hybrid which unfortunately migrates with Hb A2
on alkaline, acid or IEF electrophoresis. This explains the elevated Hb A2.
312
If
half of the alpha chains are modified they would be competing also with the
unmodified alpha chains for delta chains. The unmodified alpha chain delta
combination is Hb A2 seen normally and the modified alpha variant delta
combination is new hemoglobin, Hb G2 which migrates close to the carbonic
anhydrase. The number of different hemoglobin molecules created by a Hb S GPhiladelphia double mutation is 6. The Hb S-G hybdrid migrates with A2 on
acid, alkaline and IEF electrophoresis.
IEF, CZE and HPLC data support the presence of a heterozygous Hb GPhiladelphia [68(E17)AsnLys] and Hb S in that two distinct, approximately
equal bands or peaks were seen in the position of Hb S and Hb G. IEF indicated
that the Hb G band is closer to the Hb A band (more anodal) than the Hb S. Two
additional bands in the position of Hb A2 and Hb G2 were also detected from IEF
although the low intensity of the Hb G2 band made it difficult to see.
CZE showed six distinct peaks in the following zones with alleged
hemoglobins indicated in parenthesis:
i
ii
iii
iv
v
vi
Zone 9 (Hb A)
Zone 6 (Hb G-Philadelphia)
Zone 5 (Hb S)
Zone 3 (Hb A2)
Zone 2 (Hb S/G hybrid)
Zone 1 (Hb G2)
Hb F ( 0.2%)
Hb A (54%; RT = 2.35)
313
c
d
e
f
Hb A (
S
2 2)
G
S
S/G ( 2 2)
G
G2 ( 2 2)
Hb S (
Hb
Hb
Had this patient been a newborn the situation would further have been
complicated by the addition of 2 new gamma chain containing forms of HbF.
Hb S-G Philadelphia double heterozygous hemoglobinopathies are
essentially healthy and without anemia.
References
1
4
5
LeCrone CN, Jones JA, Detter JC. Hemoglobin G Trait and S Trait in the
Same Patient. Hemotology 1983; 49(3): 165-167.
Lawrence C, Hirsch RE, Fataliev NA, Patel S, Fabry ME, Nagel RL. Molecular
interactions between Hb alpha-G Philadelphia, Hb C, Hb S: phenotypic
implications for SC -G Philadelphia disease. Blood 1997; 90: 2819-2825.
Laboratory Data:
Hemoglobin
RBC
MCV
RDW
Platelet
12.7
4.49
81
13.2
243
Hb A
Hb A2
Hb F
Hb variant
56.0
2
1
41.0%
13.5-18.5 g/dL
3
4.6-6.2 Mil/mm
80-100 fL
11.5-14.5%
3
150-400 Th/mm
94.3-98.5%
1.5-3.7%
0.0-2.0%
316
Isoelectric focusing
Summary of Results
Method
Hb A
area
Alk
Agarose
Major
band
(Hb A)
Acid
Agar /
Agarose
Major
band
(Hb A+
Hb A2 +
Hb G)
Major
peak
(Hb A)
Zone 9
CZE
IEF
Major
band
(Hb A)
HPLC
Minor
peak
(Hb F)
RT=1.0
5
Hb S
area
Major
band
Major
peak
( Hb G )
Zone 6
Major
Hb G
band
anodal to S
Major
peak
(Hb A)
RT=2.5
Hb
A2/C
area
Minor
band
(Hb A2)
Minor
peak
(Hb A2)
Zone 3
Minor
band
(Hb A2)
No G2
band
was
detected
Major
peak
(Hb G +
Hb A2)
RT=3.6
*Note: HPLC retention time (RT) varies with the type of the instrument used and several
other factors, e.g. temperature etc.
Agarose gel electrophoresis (pH 8.6) indicated major bands in the position
of Hb A and at the position of Hb S. Besides a minor band at the position of Hb
A2 and carbonic anhydrase band no other band was detected. Citrate agar
electrophoresis (pH 6.2) showed one major band at the position of Hb A, and a
faint band was also detected in the position of Hb F. CZE showed major peaks
in Zone 9 (Hb A), and Zone 6 (Hb variant) and a minor peak in Zone 3 (Hb A2).
IEF indicated that the second major band was in the position of Hb G, or
319
The safest interpretation for this case is that this patient has Hb G trait (-chain
variant ) known as Hb G-Coushatta[ 22 (4) GluAla (GAAGCA)] because of the
320
References
1
2
3
4
5.
6
Laboratory Data:
Hemoglobin
RBC
MCV
RDW
Platelet
14.8
4.91
77
15.1
248
13.5-18.5 g/dL
3
4.6-6.2 Mil/mm
80-100 fL
11.5-14.5%
3
150-400 Th/mm
Hb A
58.0
Hb A2
2
Hb F
1
Hb variant
39.0%
(Hemoglobin fractions from HPLC)
94.3-98.5%
1.5-3.7%
0.0-2.0%
322
Isoelectric focusing
323
Method
Alk
Agarose
Acid Agar
/Agarose
CZE
IEF
HPLC
Hb A
area
Major
band
(Hb A)
Major
band
(Hb A+
Hb A2)
Major
peak
(Hb A)
Zone 9
Hb S
area
Hb A2/C
area
Major
band
Major
band
Minor
peak
(Hb A2)
Zone 3
Major
peak
(Hb C)
Zone 2
Major
band
(Hb A)
Minor
band
(Hb A2)
Major
peak
(Hb A)
RT=2.45
Minor
peak
(Hb A2)
RT=3.6
Major
band
cathodal
to A2
(Hb C)
Major
Peak
(Hb C)
RT=5.l0
* Note: HPLC retention time (RT) varies with the type of the instrument used and
several other factors, e.g. temperature etc.
References
1
2
3
4
5
Case # 9
Hemoglobin C homozygous
Laboratory Data:
327
Hemoglobin
12.1
RBC
4.3
MCV
73
RDW
13.3
Platelet
248
Hb A
Not detected
Hb A2
2.5
Hb F
1.6
Hb variant
95.9%
(Hemoglobin fractions from HPLC)
328
Isoelectric focusing
Summary of Results
Method
Hb A
area
Hb S
area
Alk
Agarose
Acid Agar
/Agarose
CZE
IEF
HPLC
Hb A2/C
area
Major
band
Major
band
Minor
peak
(Hb A2)
Zone 3
Minor
band
Minor
peak
(Hb A2)
RT=3.6
Major
peak
(Hb C)
Zone 2
Major
band
cathodal
to A2
(Hb C)
Major
peak
(Hb C)
RT=5.06
*Note: HPLC retention time (RT) varies with the type of the instrument used and several
other factors, e.g. temperature etc.
Agarose gel electrophoresis (pH 8.6) showed only one intense and major band in
the position of Hb C/E/O, and Hb A was not detected. Citrate agar electrophoresis (pH
6.2) also showed one intense band in the position of Hb C, therefore at the very outset
the presence of Hb E and O were ruled out. It appeared that the solitary band in the Hb C
position is most likely due to the substitution of amino acid lysine with glutamic acid at
the sixth position of -chain [ 6(A3) GluLys]. Hb C has prevalence of 0.017% among
the African-Americans in the United States, but it has been also reported in persons of
Hispanic and Sicilian ancestry.
331
Other laboratory tests (CZE, HPLC, and IEF) also indicated the prominent Hb C
band or peak, however contrary to alkaline and acid electrophoresis (see above) minor
bands or peaks due to Hb F ( 1.6%) and Hb A2 ( 2.5%) were also detected. Absence
of Hb A by all the five methods in this person suggested either homozygous Hb C or Hb
+
Hb C/0-thalassemia
Homozygous Hb C
3.2 3.9%
Hb F
0.8 1.9%
MCV
68 - 76
55 - 70
References
1
2
3
4
5
6.
7.
8.
Bunn HF, Forget BG, Hemoglobin: molecular, genetic and clinical aspects. 1 st
edition, Philadelphia, PA: WB Saunders Co; 1986: 421-425.
Nagel RL, Steinberg MH. Hb S/C disease and Hb C disorders. In: Steinberg
MH, Forget BG, Higgs DR, Nagle RL, eds. Disorders of Hemoglobin:
Genetics, Pathophysiology and Clinical Management. Cambridge, England:
Cambridge University Press; 2001: 756-785.
Fairhurst RM, Casella JF. Homozygous hemoglobin C disease. N Engl J Med
2004; 350: e24 (Web only).
(Available at www.nejm.org/cgi/content/full/350/26/e24).
Schwab JG, Abelson HT. Hemoglobin C. N Engl J Med 2004; 351(15): 1577.
Weatherall DJ, Clegg JB. The thalassemia syndrome, 4th edition, Oxford,
England: Blackwell Science, 2001: 415-419.
Modiano D, Luoni G, Sirima BS, et al. Hemoglobin C protects against
clinical Plasmodium falciparum malaria. Nature 2001; 414 (6861): 305-8.
[Medline].
Rihet P, Flori L, Tall F. Hemoglobin C is associated with reduced
Plasmodium falciparum parasitemia and low risk of mild malaria. Hum
Mol Genet 2004; 13(1): 1-6.
Hoyer JD, Kroft SH. Color Atlas of Hemoglobin Disorders. College of
American Pathology 2003. Case # 8 (pp 45), Case # 15 (pp 75), Case
# 29 (pp 135), Case # 30 (pp 139).
Case # 10
A 23 years old white female presented to the Emergency Department of the hospital
(2011) complaining of pelvic pain. She was found to have a ruptured right hemorrhagic
ovarian cyst which was suspected on CT and ultrasound and then confirmed by
laparoscopy. No blood transfusion was executed.
Laboratory Data:
Hemoglobin
11.9
12.0 -16.0 g/dL
3
RBC
4.8
4.0 - 5.5 Mil/mm
MCV
74
79 - 98 fL
RDW
20.8
11.5 -14.5%
Hb A
Not detected
94.3 - 98.5%
Hb A2
2.2
1.5 - 3.7%
Hb F
29.4
0.0 - 2.0%
Hb variant
68.4%
(Hemoglobin fractions from HPLC)
Peripheral Blood Smear: Abundant target cells
Sickle cell solubility test for hemoglobin S: Negative
Flow cytometry (monoclonal antibody for Hb F) showed a homogeneous distribution of
Hb F.
335
Isoelectric focusing
Note: HPLC and hemoglobin electrophoresis tests were performed at three independent
laboratories, and all the results were concordant.
Method
Alk
Agarose
Acid Agar
/Agarose
CZE
IEF
HPLC
Hb A
area
Hb S
area
Major
band
in Hb F
area
Major
Band in
Hb F
area
Major
peak Hb
F
Zone 7
Hb A2/C
area
Major
band
Major
band
Very
minor
peak
(Hb A2)
Zone 3
Minor
band
(Hb A2)
Major
band
in
Hb F
area
Major
peak
(Hb F)
RT=1.1
5
Very
minor
peak
(Hb A2)
RT=3.6
Major
peak
(Hb C)
Zone 2
Major
band
cathodal
to A2
(Hb C)
Major
peak
(Hb C)
RT=5.14
*Note: HPLC retention time (RT) varies with the type of the instrument and several other
factors, e.g. temperature etc.
References
1
Bain BJ. Hereditary persistence of fetal hemoglobin and other inherited causes
of an increased proportion of hemoglobin F: In: Hemoglobinopathy Diagnosis,
Blackwell Publishing, 2nd edition, 2006, pp 119-127.
2 Bollekens JA, Forget BG. thalassemia and hereditary persistence of fetal
hemoglobin. Hematol Oncol Clin North Am. 1991; 5: 399-422.
3 Hoyer JD, Penz CS, Fairbanks VF, et al. Flow cytometric measurement of
hemoglobin F in RBCs: diagnostic usefulness in the distinction of hereditary
persistence of fetal hemoglobin (HPFH) and hemoglobin S-HPFH from other
conditions with elevated levels of hemoglobin F. Am J Clin Pathol 2002; 117: 857863.
4 Weatherall DJ, Legg JB. Hereditary persistence of fetal hemoglobin. In: The
thalassemia Syndromes. 4th ed. Oxford: Blackwell Science, 2001: 450-484.
5 Wood WB. Hereditary persistence of fetal hemoglobin and thalassemia. In:
Steinberg MH, Forget BG, Higgs DR, Nagel RL. Disorders of Hemoglobin:
339
Case # 11
340
A 22 year old African American male, who was working at the Chrysler Stamping plant,
complained of headache and difficulty in breathing. His supervisor suspected carbon monoxide
poisoning and sent him to the Emergency Department.
Laboratory Data:
Hemoglobin
10.8
RBC
3.6
MCV
90.1
MCH
30.0
Hb A2
2.4
Hb F
1.8
Hb Variant-1
49.0%
Hb Variant-2
46.8%
(Hemoglobin fractions from HPLC)
Isoelectric focusing
Metho
d
Hb A
area
Hb S
area
Alk
Agaros
e
Major
band
Acid
Agar/
Agarose
Major
band
Major
peak
Hb S
(Zone 5)
Major
band
(Hb S)
CZE
IEF
HPLC
Major
peak
(Hb S)
RT=4.37
Hb
A2/C
area
Major
band
Major
band
Minor
peak
(Hb A2)
Zone 3
Very
minor
band
(Hb A2)
Very
minor
peak
(Hb A2)
RT=3.6
Major
peak
(Hb C)
Zone 2
Major band
(Hb C)
slightly
cathodal to
A2
Major
peak
(Hb C)
RT=5.12
*Note: HPLC retention time (RT) varies with the type of the instrument used and several
other factors, e.g. temperature etc.
the shape of the red blood cells (Professor James D. Hoyer, MD, Mayo
Clinic, Rochester, MN).
Hemoglobin C-Harlem (also called Hb C-Georgetown) is a rare double chain mutation hemoglobin (
6(A3) GluVal
73(E73) AspAsn
) and patients
References:
1
2.
3
6
7
8
9
10
Laboratory Data:
347
Hemoglobin
14.7
RBC
4.9
MCV
82
MCH
30.2
Platelet
239
Hb A
58.0
Hb A2
1.5
Hb F
0.3
Hb Variant
40.2
(Hemoglobin fractions from HPLC)
348
Isoelectric focusing
349
Method
Hb A
area
Hb S
area
Alk
Agarose
Major
band
(Hb A)
Major
band
Major
peak
(Hb A)
Zone 9
Major
band
(Hb A)
Major
peak
(Hb A)
RT=2.42
Major
band
Acid Agar
/Agarose
CZE
IEF
HPLC
Major
peak
(Hb D)
Zone 6
Major Hb D
band
slightly
anodic to S
Hb
A2/C
area
Minor
band
Minor
peak
(Hb A2)
Zone 3
Minor
band
(Hb A2)
Minor
peak
(Hb A2)
RT=3.6
Major
peak
(Hb D)
RT=3.99
*Note: HPLC retention time (RT) varies with the type of the instrument and several other
factors, e.g. temperature etc.
(2G2) and the observation that the percentage of the abnormal variant
approaches 50%. -chain variants percentages do not run this high without other
genetic complications (see Case# 5).
The differentiation between Hb D-Los Angeles and other kinds of
heterozygous D hemoglobins (which are also -chain variants) or heterozygous
Hb Korle-Bu (G-Accra) on the basis of electrophoretic tests (alkaline, acid, IEF,
CZE) was not possible with certainty due to identical mobilities. HPLC
differentiated Hb D-Los Angeles from Hb Korle-Bu. We have summarized the
HPLC retention times from three separate studies for Hb D-Los Angeles and Hb
Korle-Bu:
Nardi et al*
Nardi-2013**
Hoyer et al
Hb Korle-Bu
3.92 + 0.050
3.9+ 0.034
3.88+ 0.08
Hb D- Los Angeles
4.18+ 0.007
4.11+ 0.078
4.08+ 0.08
References
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Case # 13
17 years old male patient. No other information provided due to the privacy requested
by the patient. No record of blood transfusion during the past three months.
Laboratory Data:
Hemoglobin
10.2
RBC
MCV
MCH
Platelet
3.2
90.7
30.9
229
Hb A (mostly Hb A1c)
6.0
Hb A2
2.7
Hb F
2.5
Hb Variant-1
49.3
Hb Variant-2
39.5
(Hemoglobin fractions from HPLC)
1.5 - 3.7%
0.0 - 2.0%
Peripheral Blood Smear: Moderate sickle cells. Target cells and polychromasia.
Sickle cell solubility test for Hb S: Positive.
Hemoglobin instability (isopropanol) test: Negative.
355
Isoelectric focusing
356
Summary of Results
Method
Alk
Agarose
Acid Agar
/Agarose
CZE
Hb A
area
Hb S
area
Major
band
(Hb D)
Major
band
Major
band
(Hb S)
Major*
peak
(Hb D)
Zone 6
IEF
HPLC
Minor
peak
(Hb F)
RT=1.0
5
Major
peak
(Hb D)
RT=4.0
Major*
peak
(Hb S)
Zone 5
Major
band
(Hb S)
Major
peak
(Hb S)
RT=4.3
Hb
A2/C
area
Minor
band
Minor
peak
(Hb A2)
Zone 3
Major
band
anodal to
S position
(Hb D)
Minor
band
(Hb A2)
Minor
peak
(Hb A2)
RT=3.6
* Overlap of the two peaks (Zone 5-6) due to approximately equal and higher
concentration of Hb S and Hb D-Los Angeles.
Note: HPLC retention time (RT) varies with the type of instrument used and several
other factors, e.g. temperature etc.
Agarose gel electrophoresis (pH 8.6) showed a major and very intense
band in the position of Hb S. Another band of faint intensity was detected in
the Hb F position. A faint band in the position of Hb C/E/O/A 2 was also noticed.
No band was detected in the position of Hb A. Citrate agar electrophoresis (pH
6.2) presented with two major bands in approximately equal intensity in the
358
In a heterozygous situation upon CZE, Hb S migrates in Zone 5 and Hb DLos Angeles in Zone 6. In this case since the concentration of the two variants is
intense ( 40-49% from HPLC), thus clearly separated peaks were not detected
359
but the scan positively showed two overlapping peaks in the position of Zones 56. Distinct peaks for Hb F and Hb A2 from CZE were noticed in Zone 7 and and
Zone 3 respectively.
The specimens of father and mother of this person were not available for
additional studies. Furthermore globin chain and DNA studies were also not done
on the blood of this person. On the basis of the available laboratory data a
tentative diagnosis of a double heterozygosity of Hb S [6 (A3) GluVal] and
Hb D-Los Angeles [121(GH4)GluGln.GAA>CAA] was advised to the
physician.
Hb D-Los Angeles in both the heterozygous (Case # 12) and homozygous
state is clinically silent and harmless. However patients with homozygous Hb Do
Los Angeles and -thalassemia do have mild anemia and also exhibit mild
hemolysis. Hb D-Los Angeles is not itself a sickling hemoglobin, but compound
heterozygosity (Hb S + Hb D-Los Angeles) produces a severe sickle cell anemia
because Hb D-Los Angeles enhances Hb S polymerization by forming an
additional contact stabilizing the Hb S polymer.
References
1
2
3
4
5
6
7
8
9
10
11
12
361
2.
3.
4.
7.
363
9.
Note: It will not be out of place to mention here that another disorder Hb S-E
heterozygous has been also diagnosed in persons of Southeast Asian
origin (Case # 14 C).
References
1 Fucharoen S, Weatherall DJ. The Hemoglobin E Thalassemias. Cold Spring
Harb Perspect Med 2012; 2: a011734.
2 Sae-ung N, Srivorakun H, Fucharoen G, Yamsri S, Sanchaisuriya K,
Fucharoen S. Phenotypic expression of hemoglobins A2 , E and F in various
hemoglobin E related disorders. Blood Cells, Molecules, and Diseases 2012;
48: 11-16.
3 Tatu T, Kasinrerk W. A novel test tube method of screening for hemoglobin E.
Int. Lab. Hem 2012; 34: 59-64.
4 Moiz B, Hashmi MR, Nasir A, Rashid A, Moatter T. Hemoglobin E syndromes
in Pakistani population. NMC Blood Disorders 2012; 12: 1-6.
5 Khan MR, Aziz MA, Shah MSU, Imam H. Hemoglobin E trait- in Rajshahi,
Bangladesh. Bangladesh Med ResCounc Bull 2012; 38: 72-73.
6 Tamminga RYJ, Doombos ME. Muskiet FAJ, Koetse HA. Rhabdomyolysis in a
child with Hb SE. Pediatric Hematology-Oncology 2012; 29(3): 267-269.
7 Edison ES, Shaji RV, Chandy M, Srivasta A. Interaction of Hemoglobin E with
Other Abnormal Hmoglobins. Acta Haematol 2011; 126: 246-248.
8 Tay SH, Teng GG, Poon M, Lee VKM, Lim AYN. A Case of Hemoglobin SE
Presenting with Sickle Cell Crisis: Case Report and Histological Correlation.
Annl Acad Med 2011; 40 (12): 552-553.
9 Colah R, Gorakshakar A, Nadkarni A. Global burden, distribution and
prevention of -thalassemias and hemoglobin E disorders. Expert Review of
Hematology 2010; 3: 103-117.
10 Patel J, Patel A, Patel J, Kaur A, Patel V. Prevalence of Haemoglobinopathies
in Gujrat, India: A Cross-Sectional Study. The Internet Journal of Hematology
2009; 5 (1): DOI: 10.5589/1764.
365
366
Case # 14 a
Hemoglobin E trait
A 56 year old male of Southeast Asian origin, migrated to America in 1972 with his
parents. Physical examination showed no abnormalities.
Laboratory Data:
Hemoglobin
RBC
MCV
RDW
Platelet
14.8
5.7
74
13.5
240
Hb A
Hb A2 (CZE)
Hb variant
64.0
2.0
34.0
13.5-18.5 g/dL
3
4.6-6.2 Mil/mm
80-100 fL
11.5-14.5%
3
150-400 Th/mm
94.3-98.5%
1.5-3.7%
367
368
Isoelectric focusing
369
Method
Hb A
area
Alk Agarose
Major
band
(Hb A)
Acid
Agar/Agaros
e
Major
band
(Hb A+
Hb E+
Hb A2)
Major
peak
(Hb A)
Zone 9
CZE
IEF
Hb S
area
Major
peak
(Hb E)
Zone 4
Major
band
(Hb E)
Slightly
anodal to
Hb A2
Major
band
(Hb A)
HPLC
Very
faint
peak
(Hb F)
RT=1.0
7
Hb
A2/C
area
Major
band
(Hb E +
Hb A2)
Major
peak
(Hb A)
RT=2.4
2
Minor
peak
(Hb A2)
Zone 3
Very
minor
band
(Hb A2)
Major
peak
(Hb E +
Hb A2)
RT=3.6
3
* Note: HPLC retention time (RT) varies with the type of the instrument used and
several other factors, e.g. temperature etc.
Alkaline agarose electrophoresis (pH 8.6) showed two major bands in the
position of Hb A (65%), and Hb C/E/O/A 2 (35%). Citrate agar electrophoresis
showed only one band in the position of Hb A. This kind of electrophoretic
migration pattern (pH 8.6 and 6.2) ruled out the possibility of Hb C
and Hb O, and suggested the possibility of Hb E, as Hb A2 is never > 10%. IEF
also showed a major band in the position of Hb A and another band slightly
371
anodal to Hb A2 suggesting the presence of Hb E variant. Hb E and Hb A2 coeluted upon HPLC, therefore their quantification was not feasible. However,
CZE presented three distinct peaks in the zones for Hb A, Hb E and Hb A2 and
also provided quantification of the peaks.
Case # 14 b
Hemoglobin E homozygous
A 25 year old female from Laos-Northern Vietnam region, asymptomatic; physical examination
showed no abnormalities. No icterus, no splenomegaly.
372
Laboratory Data:
Hemoglobin
RBC
MCV
MCH
Platelet
14.1
5.7
68
24.9
223
12.0-16.0 g/dL
3
4.0-5.5 Mil/mm
80-100 fL
26-34 pg
3
150-400 Th/mm
Hb A
Hb A2 (CZE)
Hb F (HPLC)
Hb variant
Not Detected
0.5
0.8
98.7%
94.3-98.5%
1.5-3.7%
0.0-2.0%
373
Isoelectric focusing
Summary of Results
Metho
d
Hb A
area
Hb S
area
Hb
A2/C
area
Alk
Agaros
e
Acid
Agar /
Agarose
Major
band
(Hb E +
Hb A2)
Major
band
(Hb E +
Hb A2)
CZE
Major
peak
(Hb E)
Zone 4
Very
minor
peak
(Hb A2)
Zone 3
IEF
Major
band
(Hb E)
slightly
anodal to
Hb A2
HPLC*
Major
peak
(Hb E +
Hb A2)
RT=3.65
In addition to Hb F peak at RT= 1.05 minutes, there are two additional minor
peaks at RT= 1.75 minutes and RT= 2.1 minutes. The peak at RT=2.1 minutes
(A0 window) must not be construed as Hb A. Similar peaks were detected upon
HPLC (Bio-Rad Variant II), and were alleged to post-translationally modified
Hb E (Other Significant Hemoglobinopathies. In: Hemoglobinopathy Diagnosis,
Bain, Barbara J., 2nd edition, pg 206, Blackwell Publishing, 2006).
376
Alkaline agarose electrophoresis (pH 8.6) showed only one band in the
position of Hb C/E/O/Hb A2, therefore Hb A was not present. Citrate agar
electrophoresis (pH 6.2) indicated only one major band in the position of Hb A,
thus the possibility of Hb C and Hb O was ruled out. IEF also indicated the
absence of Hb A and only one major band slightly anodal to Hb A2 was detected.
Absence of Hb A was also shown by HPLC, and only one major peak eluted at
RT = 3.65. CZE clarified the ambiguity about the solitary band /peak in
electrophoretic methods and HPLC, and two peaks were detected at Zone 4
(major peak presumably of Hb E) and Zone 3 (minor peak of Hb A2).
A diagnosis of Hb E homozygous was made in view of the electrophoretic,
and HPLC results and the following characteristics:
prevalent in the population areas (e.g. Cambodia and Northeastern India) that
also have the higher frequency of -thalassemia minor. Therefore genetic
counseling is advised to prevent the occurrence of severe thalassemic
o
377
Case # 14 c
A 29 year old female nurse (Southeast Asian descent) complained of knee joint pain and
weakness of the lower extremities. Hemoglobin electrophoresis was ordered after lower MCV
378
was found from CBC. Clinical and laboratory data of her parents were not available to the
physician.
Laboratory Data:
Hemoglobin
RBC
MCV
MCH
Platelet
12.2
4.6
72
26
212
12.0-16.0 g/dL
3
4.0-5.5 Mil/mm
80-100 fL
26-34 pg
3
150-400 Th/mm
Hb A
Hb A2 (CZE)
Hb F (HPLC)
Hb S
Hb E
Not Detected
0.5
0.5
55%
44%
94.3-98.5%
1.5-3.7%
0.0-2.0%
379
Isoelectric focusing
Summary of Results
Metho
d
Hb A
area
Alk
Agaros
e
Acid
Agar/
Agaros
e
Major
band
(Hb E)
Hb S
area
Hb
A2/C
area
Major
band
(Hb S)
Major
band
(Hb E)
Major
band
(Hb S)
CZE
Major
peak
(Hb S)
Zone 5
Major
peak
(Hb E)
Zone 4
Minor
peak
(Hb A2)
Zone 3
IEF
Major
band
(Hb S)
Major
band
(Hb E)
slightly
anodal to
Hb A2
HPLC*
Major
peak
(Hb S)
RT=4.48
Major
peak
(Hb E +
Hb A2)
RT=3.65
*Note: HPLC retention time (RT) varies with the type of the instrument used and
several other factors, e.g. temperature etc.
Alkaline agarose gel electrophoresis (pH 8.6) showed two major bands in
the position of Hb S (55%) and Hb C/E/O/A 2 (45%). Citrate agar
electrophoresis (pH 6.2) confirmed the presence of a major band due to Hb S and
presence of another band at the position of Hb A ruled out the possibility of Hb C
382
and Hb O. IEF indicated a major band in the position of S and another major
band in the position of Hb A2. These three electrophoretic methods suggested
the presence of double heterozygous Hb S and Hb E. HPLC was not helpful as
both Hb E and Hb A2 co-eluted at RT = 3.65. CZE provided a clear separation of
the three hemoglobin entities, i.e. Hb S (Zone 5), Hb E (Zone 4) and Hb A2 (Zone
3), therefore a presumptive diagnosis of Hb S-E double heterozygous disorder
was made.
Hb S [6 (A3) GluVal) and Hb E [26 (8) GluLys] are the two most
prevalent hemoglobin variants in the world. However, due to their existence in
different ethnic groups and continents, compound heterozygosity for Hb S and
Hb E is extremely rare. As of 2011 only thirty (30) cases were reported, therefore
hematological parameters are too scant to provide a module for diagnostic
purposes.
Majority of Hb S-E subjects have mild or absent anemia, microcytic
indices, and some target cells. Contrary to some earlier reports, a severe sickle
cell crisis was recently reported in a 66-year-old Bangladeshi woman in
Singapore (Ann Acad Med 2011; 40: 552-553).
A recent review of Hb S-E double heterozygosity by Masiello et al (Am J
Hematol 2007; 82: 643-649) mentioned that patients aged 18 and younger are
usually well. Sickling-related complications, including potentially life threatening
acute chest syndrome was developed in a majority of cases. Generally these
383
384
Laboratory Data:
Hemoglobin
RBC
MCV
MCH
MCHC
RDW
Platelet
Hb A
Hb S (HPLC)
Hb Korle-Bu
Hb A2 (CZE)
Hb F
13.1
4.4
82.1
27.3
32.1
12.6
267
Not Detected
53.4 %
45%
1.6
Not Detected
1.5 -3.7%
0.0-2.0%
Isoelectric focusing
386
Hb A
Hb S
387
Hb
area
area
Broad
Major
band
starting
anodic to
S
(Hb
Korle-Bu
+ Hb S)
Alk Agarose
A2/C
area
Very
faint
band
(Hb A2)
Acid Agar
Major
band
slightly
anodic
to Hb A
position
(Hb
KorleBu)
Major
band
(Hb S)
Acid
Agarose
Major
band in Hb
A position
(Hb KorleBu)
Major
band
(Hb S)
Major
peak
Zone 6
(Hb
KorleBu)
Major
peak
Zone 5
(Hb S)
Minor
peak
Zone 3
(Hb A2)
Major
band
(Hb S)
Very
faint
band
(Hb A2)
Major
peak
RT=4.48
(Hb S)
Major
peak
(Hb A2 +
Hb
Korle
Bu)
RT=3.75
CZE
Small
degradatio
n peaks
Zone 7
Major
band
slightly
anodal
to Hb S
band
(HbKorleBu)
IEF
HPLC
area of Hb S and a very faint band in the Hb A2 area, but Hb A was not
detected. Citrate agar electrophoresis (pH 6.2) showed two variants in the
position of Hb S and very slightly anodic to the Hb A band position.
IEF also indicated the presence of two variants, one in the position of Hb S and
another band anodal to Hb S in the migration position of Hb D-Los Angeles/GPhiladelphia and few other variants. The presence of Hb S as one of the variants
was also confirmed by HPLC, CZE and the positive solubility test. It is obvious
that the three electrophoretic methods (i.e. alkaline agarose, acid agar, and IEF)
could not identify the second major band with certainty. Hb G-Philadelphia was
ruled out due to the absence of G2 variant (G22) in both the alkaline agarose
electrophoresis and IEF (Case # 5). In situations like this the patients history and
clinical status may indicate the likelihood of the hemoglobin variant. Both of these
hemoglobin variants (Hb D-Los Angeles and Hb Korle-Bu) are found in the
population sector dominated by Hb S. HPLC is helpful in the separation of Hb DLos Angeles from Hb Korle-Bu, since Hb D-Los Angeles has a longer elution time
(Case # 12) as compared to Hb Korle-Bu. The D-Los Angeles chain can be easily
separated from Korle-Bu chain by reverse phase chromatography, but all these
additional tests are not necessary. Hb S interacts with Hb D-Los Angeles causing
sickle cell disease. Hb S also interacts with Hb Korle-Bu, but in opposite
direction, i.e. inhibiting sickling. This patient does not have an abnormal blood
picture so the second variant in this case is most likely Hb Korle-Bu (G-Accra).
Korle-Bu means valley of the Korle lagoon, and this hemoglobin was
named after its discovery at Korle-Bu Hospital, Accra, Ghana. Initially it was
389
References
1
2
3
4
AKL PS, Kutlar F, Patel N, Salisbury CL, Lane P, Young AN. Compound
Heterozygosity for Hemoglobin S [6(A3)Glu6Val] and Hemoglobin Korle-Bu
[73(E17)ASP73Asn]. Lab Hematol 2009; 15: 19-23.
Chico A, Padros A, Novials A. The Korle-Bu Hb Variant in Caucasian Women
With Type I Diabetes. A pitfall in the assessment of diabetes control. Diabetes
Care 2004; 27(9): 2280-2281.
Vukelja SJ. Hemoglobin Korle-Bu (G-Accra) in Combination with Hemoglobin
C. Am J Hematol 1993; 42(4): 412.
Nagel RL, Lin MJ, Witkowska HE, Fabry ME, Bestak M, Hirsch RE.
Compound Heterozygosity for Hemoglobin and Korle-Bu: Moderate Microcytic
Hemolytic Anemia and Acceleration of Crystal Formation. Blood 1993; 82(6):
1907-1912.
Konotey-Ahulu FID, Gallo E, Lehman H, Ringelhann B. Hemoglobin Korle-Bu
(73 aspartic acidasparagine). J Med Genet 1968; 5: 107-111.
Case # 16
A 23 year old male student of Northern African descent at Michigan State University,
East Lansing, Michigan, USA.
Laboratory Data:
390
Hemoglobin
14.8
RBC
4.9
MCV
86
MCH
29.3
MCHC
32.6
RDW
12.5
Platelet
273
Hb A
56.5%
Hb O-Arab
41%
Hb A2
2%
Hb F
0.5%
(Hemoglobin fractions from HPLC)
391
Isoelectric focusing
392
Metho
d
Hb A
area
Alk
Agaros
e
Major
band
(Hb A)
Acid
Agar
Actually
on Hb S
side of
Hb A
Acid
Agaros
e
CZE
Major
band
Major
band
(Hb A)
Small
peak
(Hb F)
Zone 7
IEF
Hb S
area
(Hb OArab)
May
appear
as a
broader
Hb A
band
Major
band
(Hb OArab)
Actually
migrates
cathodal
to Hb S)
Major
band
Actually
on Hb C
side of
Hb S
(Hb OArab)
Major
peak
(Hb A)
Zone 9
Small
peak
(Hb F)
RT=1.08
Actually on
Hb S side
of Hb C
Minor
peak
(Hb OArab)
Zone 3
Major
band
Major
band
HPLC
Hb
A2/C
area
Major
band
(Hb A)
(Hb OArab)
Major
peak
(Hb A)
RT=2.38
Minor
peak
(Hb A2)
RT=3.64
Major
peak
(Hb OArab)
RT=4.3
Note: Separation under acidic conditions has traditionally been done with agar instead of
agarose because all the early descriptions of hemoglobin variants contained data collected in
this manner. As the separation quality of agar has deteriorated many vendors have chosen to
switch to use of the purified agarose in order to maintain a more constant product. Hemoglobin
O-Arab migrates close to hemoglobin A historically and with well selected current agars. In the
heterozygous state one broad band is seen starting with Hb A but extending on toward Hb S
somewhat. When agarose is substituted Hb O-Arab migrates close but on the Hb C side of the
Hb S location. The user is advised to take note of the separation media used for acid
electrophoresis in the interpretation of the results.
was ruled out because Hb A2 virtually never has such an increase. Citrate agar
References
1
2
3
4
5
7
8
Laboratory Data:
396
Hemoglobin
12.5
RBC
5.13
MCV
63.8
MCH
20.5
RDW
12.1
Platelet
267
Serum Iron
110
Ferritin
75
Hb A
93.5
Hb A2
6.0
Hb F
0.5
(Hemoglobin fractions from HPLC)
397
Isoelectric focusing
398
Metho
d
Hb A
area
Alk
Agaros
e
Major
band
(Hb A)
Acid
Agar/
Agaros
e
Major
band
(Hb A +
Hb A2)
Major
peak
(Hb A)
Zone 9
CZE
Small
peak
(Hb F)
Zone 7
IEF
Hb S
area
Minor
peak
(Hb A2)
Zone 3
Minor
band
(Hb A2)
Minor
peak
(Hb A2)
RT=3.64
Major
band
(Hb A)
HPLC
Small
peak
(Hb F)
RT=1.05
Hb
A2/C
area
Minor
band
(Hb A2)
Major
peak
(Hb A)
RT=2.42
Note: HPLC retention time (RT) varies with the type of instrument used and
several other factors, e.g. temperature etc.
Alkaline agarose electrophoresis (pH 8.6) showed no abnormality except
that the staining of the band at the Hb A2 position was relatively denser than the
normal adult. No abnormal band was detected from citrate agar
electrophoresis (pH 6.2). Two bands in the migration position of Hb A (major
band) and Hb A2 (minor band but more intense than a normal adult) were
indicated by IEF. CZE and HPLC results were concordant suggesting
an increased concentration of Hb A2 and no other abnormal peaks.
Hemoglobinopathies can be classified as a manufacture of a modified
400
References
1
2
3
4
5
6
7
8
Laboratory Data:
Hemoglobin
RBC
10.3
4.28
MCV
74.8
MCH
23.9
RDW
16.2
Platelet
203
Hb A
22.1
Hb A2
6.4
Hb F
3.4
Hb S
68.1
(Hemoglobin fractions from HPLC)
79-98 fL
26-34 pg
11.5-14.5%
3
150-400 Th/mm
94.3-98.5%
1.5 -3.7%
0.0-2.0%
403
Isoelectric focusing
Metho
d
Hb A
area
Hb S
area
Hb
A2/C
area
Minor
band
(Hb A2)
Alk
Agaros
e
Very
weak
band
(Hb F)
Small
band
(Hb A)
Major
band
(Hb S)
Acid
Agar/
Agaros
e
Weak
band
(Hb F)
Small
band
(Hb A+
Hb A2)
Major
band
(Hb S)
CZE
Small
peak
(Hb F)
Zone 7
Small
peak
(Hb A)
Zone 9
Major
peak
(Hb S)
Zone 5
Minor
peak
(Hb A2)
Zone 3
IEF
Very
small
band
(Hb F)
Very
small
band
(Hb A)
Major
band
(Hb S)
Minor
band
(Hb A2)
HPLC
Small
peak
(Hb F)
RT=1.05
Small
peak
(Hb A)
RT=2.40
Major
peak
(Hb S)
RT=4.32
Minor
peak
(Hb A2)
RT=3.6
Agarose gel electrophoresis (pH 8.6) showed one major band in the
position of Hb S, minor band (less in intensity than Hb S band) in the migration
position of Hb A, and another band in the position of Hb C/E/O/A 2 with intensity
greater than a normal adult. Citrate agar electrophoresis (pH 6.2) indicated
increased Hb F than a normal adult, and a major band in the position of Hb S,
major band in the position of Hb A but less in intensity than Hb S. IEF, CZE, and
HPLC also provided concordant results and the evidence for the following four
406
bands:
Hb S
Hb A
Hb A2
Hb F
Quantitatively increased Hb A2 (6.4% by HPLC) suggested the presence of thalassemia in conjunction with Hb S. Two types of Hb S--thalassemia are
found in African Americans:
+
Hb S- -thalassemia:
Hb S- -thalassemia
+
-thalassemia.
Appendix:
0
I looked for a case of Hb S- -thalassemia for > three years but no luck. Just
yesterday a 23 year female came to the Emergency Department with a severe sickle
407
cell crisis. I immediately contacted the attending physician and my associates involved
in this book to include the data of this patient for the benefit of readers for understanding
the distinction between the two types of Hb S--thalassemias.
CBC
Hemoglobin
Hematocrit
RBC
MCV
MCH
RDW
Platelet
Reticulocyte Count
5.3
15.1
2.13
70.7
24.8
24.3
407
13.5
Alkaline agarose (pH 8.6), Citrate agar (pH 6.2), IEF, HPLC and CZE indicated the
absence of Hb A. The concentration of hemoglobin fractions from CZE were:
Hb A
Hb A2
Not Detected
4.5
Hb F
Hb S
8.6
86.9
References
1.
2.
3.
4.
Bain BJ. Sickle cell/ thalassemia, Sickle cell hemoglobin and its interactions
with other variant hemoglobins and with thalassemia. In: Hemoglobinopathy
Diagnosis, 2006, 2nd edition. Blackwell Publishing,
England, pg 170-173.
Steinberg MH. Compound heterozygous and other sickle
hemoglobinopathies. In: Steinberg MH, Forget BG, Higgs DR, Nagle RL.
Disorders of Hemoglobin: Genetics, Pathophsiology and Clinical Management.
Cambridge , England: Cambridge University Press; 2001: 786-792.
Sunna EI, Gharaibeh NS, Knapp DD, Bashir NA. Prevalence of
Hemoglobins S and -Thalassemia in Northern Jordan. J Obstet Gynecol
Res 1996; 22(1): 17-20.
Gonzalez-Redondo JM, Kutlar A, Kutlar F, McKie VC, Mckie KM, Baysai
E, Huisman THJ. Molecular Characterization of Hb S (C) -Thalassemia
408
5.
Laboratory Data:
Hemoglobin
10.3
12.0 16.0 g/dL
3
RBC
5.4
4.0 5.5 Mil/mm
MCV
66.5
79-98 fL
MCH
20.9
26-34 pg
Hb A (all methods)
Not Detected
94.3-98.5%
Hb A2
5.9
1.5 -3.7%
Hb F
1.4
0.0-2.0%
Hb C
87.0
Hb A1c + other fractions
5.7
Peripheral Blood Smear: Hypochomosia, microcytosis, target cells,
anisocytosis and poikilocytosis
Sickle cell solubility test for Hb S: Negative
Hemoglobin instability (isopropanol) test: Negative
410
Isoelectric focusing
Method
Alk
Agarose
Hb A
area
Hb S
area
Faint
band
(Hb F)
Hb A2/C
area
Major
band
(Hb C +
Hb A2)
Acid Agar
/Agarose
Faint
band
(Hb F)
Major
band
(Hb C +
Hb A2)
CZE
Minor
peak
(Hb F)
Zone 7
IEF
HPLC
Minor
peak
(Hb F)
RT=1.0
5
Minor
peak
(Hb A2)
Zone 3
Major
peak
(Hb C)
Zone 2
Minor
band
(Hb A2)
Major
Hb C
band
cathodal
to A2
Minor
peak
(Hb A2)
RT=3.6
Major
peak
(Hb C)
RT=5.09
414
References
1
3
4
5
Case # 20
A 55 year old male computer programmer with age onset diabetes mellitus, was screened for
hemoglobinopathy since one of his family member was anemic, and another having a
hemoglobin variant. His parents (Ashkenazi Jews) migrated from Poland to Detroit, Michigan.
415
Laboratory Data:
Hemoglobin
RBC
MCV
MCH
RDW
Platelet
Hb A
Hb A2 (CZE)
Hb F (CZE)
Hb variant (CZE)
14.8
5.1
88.0
28.3
12.3
203
77.3
1.6
0.8
20.3
416
Isoelectric focusing
Metho
d
Hb A
area
Hb S
area
Alk
Agaros
e
Major
band
Major band
slightly
toward C
Acid
Agar
Major
band
(Hb A)
(Hb A)
Acid
Agaros
e
CZE
Major
band
(Hb A)
Small
peak
Major
peak
(Hb F)
(Hb A)
Zone 7
Zone 9
(Hb
Hasharon)
Major band
slightly
toward A
(Hb
Hasharon)
Major band
directly in S
position
(Hb
Hasharon)
Major
peak
(Hb
Minor
peak
Zone 3
Hasharon)
(Hb A2)
Zone 5
IEF
Major
band
(Hb A)
Hb
A2/C
area
Minor
band
(Hb A2)
Major
band
slightly
cathodal
Minor
band
(Hb A2)
Very
small
peak
(Hb F)
RT=1.05
Major
peak
(Hb A)
RT=2.40
Major
peak
eluted
between S
and C
(Hb
Hasharon)
RT=4.74
(Hb-Hash
aron-A2)
A very
faint band
cathodal to
Hb C
Hb A2
of Hb S.
(Hb
Hasharon)
HPLC
Very
minor
peak
Zone 1
(2 2)
Minor
peak
(Hb A2)
RT=3.58
faint band was present in Hb A2 position. Citrate agar electrophoresis (pH 6.2)
also revealed two major bands with intensities equivocal to that described for
agarose gel electrophoresis (pH 8.6) in the respective Hb A and Hb S positions.
The sickle cell test was negative, ruling out the presence of Hb S.
IEF showed four bands: two intense bands and two faint bands. One
intense band in the position of Hb A, and a second band slightly cathodal to
Hb S. Additionally, there was a very faint band migrating in the Hb A2 position
and a second faint band in the delta chain variant position (cathodal to Hb C).
Hb A2 variants are due to the presence of an abnormal -chain as seen in Hb GPhiladelphia trait (Case # 5) or due to the presence of an abnormal delta chain.
Since this specimen also has an abnormal Hb A, the Hb A2 variant is likely due
to an alpha mutation.
CZE showed a major peak in the Hb A zone (Zone 9), and a lesser
intense peak than Hb A in Zone 5. Two minor peaks in the position of Hb F
(Zone 7) and Hb A2 (Zone 3) were also detected as well as a very small peak
in Zone 1.
HPLC showed a major peak for Hb A and two minor peaks for
Hb A2 and Hb F. Another major peak was detected between the Hb S and Hb
C window.
A narrative report was communicated to the attending physician with a
request for consultation with him to identify the exact hemoglobinopathy. The
420
References
1
2
3
4
5
Case # 21
A 18 year old white female student from Grand Rapids, Michigan. Parents migrated from
Europe, but no information available about their ethnicity and country of origin.
Her physical examination was normal.
Laboratory Data:
422
Hemoglobin
RBC
MCV
MCH
RDW
Platelet
Hb A
Hb A2 (HPLC)
Hb F (HPLC)
Hb variant
11.6
4.4
102.0
28.4
12.3
228
66.0
1.6
0.8
31.6%
Peripheral Blood Smear: Macrocytic red blood cells. Serum iron and ferritin were
normal; very mild anemia with slight reticulocytosis.
Sickle cell solubility test for Hb S: Negative
Hemoglobin instability (isopropanol) test: Positive
No congenital deficiency of glucose-6-phosphate dehydrogenase.
423
Isoelectric focusing
Metho
d
Hb A
area
Hb S
area
Alk
Agaros
e
Acid
Agar
Major
band
Major
band
(Hb A)
Hb
A2/C
area
Minor
band
(Hb A2)
Major
band
(Hb A +
Hb A2+ Hb
variant)
CZE
IEF
HPLC
Very
minor
peak
(Hb F)
RT=1.06
Major
peak
Zone 9
(Hb A)
Major
band
Major
peak
Zone 6
Minor
peak
Zone 3
Major
band
Minor
band
(Hb A)
Slightly
cathodal to
Hb S
(Hb A2)
(Hb A2)
Major
peak
(Hb A)
RT=2.34
Major
peak
RT=3.55
*The hemoglobin variant eluted with Hb A2, and Hb A2 was in the normal range from
CZE.
Note: HPLC retention time (RT) varies with the type of instrument used and several
other factors, e.g. temperature etc.
Please note that we do not have data of acid agarose electrophoresis separation
at this time.
Agarose gel electrophoresis (pH 8.6) showed two major bands; one at
the migration position of Hb A, and one at the Hb S position. In addition,
a very weak band was noticed in the position of Hb C/E/O/Hb A2. Citrate
426
agar electrophoreis (pH 6.2) showed only one band in the position of Hb A and
a very weak, smudged band in the position of Hb F.
In view of the negative sickle cell test and the migration patterns of the
alkaline and acid electrophoresis, the presence of Hb S was ruled out. Similarly,
the possibility of Hb G-Philadelphia and Hb D- Los Angeles was eliminated on
the basis of the positive (isopropanol) instability test, the absence of G 2 band of
Hb G-Philadelphia and a lower percentage of the variant as compared to Hb DLos Angeles. Hemoglobin electrophoresis on this patient was performed about
seven years ago in our laboratory. At that time, neither the HPLC nor the CZE
testing instruments were available in our laboratory. After consultation with the
attending physician, the specimen was sent to a reference laboratory for globin
chain analysis by reverse phase HPLC and DNA studies. Note: The IEF, CZE
and HPLC scans inserted here in this case are adopted from other sources for
educational purposes.
The globin chain analysis and DNA studies provided the correct
identification of the hemoglobin variant (31.6% from alkaline agarose
electrophoresis) as Hb Zurich. Hb Zurich is an unstable hemoglobin and found
only in the heterozygous state. It is caused by the substitution of histidine by
arginine at the 63rd position of the -chain [22
63
was initially found in Europeans of Swiss descent, but later this variant
was reported in Japanese, and Brazilian citizens of non-Swiss ancestry.
427
References
1.
2.
3.
4.
5.
6.
7.
8.
Case # 22
A 41 year old male employee of General Motors, Detroit, Michigan. Parents migrated
from Italy.
Laboratory Data:
Hemoglobin
14.3
RBC
MCV
MCH
RDW
Platelet
5.72
69
22.4
13.2
243
Hb A
Hb A2
Hb F
Hb variant (CZE)
80.7
2.1
5.4
11.8
94.3 - 98.5%
1.5 - 3.7%
0.0 - 2.0%
429
Isoelectric focusing
430
Method
Hb A
area
Hb S
area
Alk Agarose
Major
band
(Hb A)
Major
band
(Hb
Lepore)
Acid
Agar/Agaros
e
Major
band
(Hb A+
Hb
Lepore
+ Hb
A2)
CZE
Small
peak
(Hb F)
Zone 7
IEF
HPLC
Small
peak
(Hb F)
RT=1.0
5
Hb
A2/C
area
Minor
band
(Hb A2)
Major
peak
(Hb A)
Zone 9
Major
peak
(Hb
Lepore)
Zone 6
Minor
peak
(Hb A2)
Zone 3
Major
band
(Hb A)
Major
band in Hb
G position
(Hb
Lepore)
Minor
band
(Hb A2)
Major
peak
Hb A2 &
Hb Lepore
(RT=3.5)
Major
peak
(Hb A)
RT=2.4
5
.
Since the sickle cell test was negative and also no band was observed in
the area of Hb S upon acid electrophoresis, the presence of Hb S was ruled out.
The possibility of other commonly encountered variants (e.g. Hb D, Hb G, Hb
Russ) that exhibit alkaline and acid electrophoresis pattern similar to this case
432
433
Hb Lepore-Boston
[(1-87) (115-146)]
Hb Lepore-Baltimore
[(1-50) (86-146)]
iii)
Hb Lepore-Hollandia
[(1-22) (50-146)]
To the best of our knowledge both CZE and HPLC do not differentiate
among these variants.
434
All the Lepore traits have the same clinical symptoms. Hb Lepore trait is a
stable hemoglobin but exhibits features of thalassemia minor due to the
decreased production of -chains. -thalassemia intermedia or major type
disorder is exhibited by homozygous Hb Lepore. Hb Lepore interacts with Hb S
to give Hb S/Hb Lepore, however very few cases (<18) were reported in the
literature. Hb S/Hb Lepore patients exhibited mild microcytic anemia, and
clinically were either asymptomatic or with complications generally associated
with Hb S disease. A case of Hb E interaction with Hb Lepore-Hollandia was
described in the literature but without any significant clinical condition except
microcytic anemia without the need for transfusion.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Case # 23
A 55 year old male farmer from Saginaw, Michigan. His mother belonged to a French
settlement in Newfoundland, Canada. Ancestors from father side immigrated from
Norway. No abnormality was found from an annual medical examination except a slight
elevation of serum cholesterol.
436
Laboratory Data
Hemoglobin
RBC
MCV
MCH
Platelet
14.8
5.1
90.7
29.9
279
Hb A2
Hb F
Hb A
Hb Variant
2.2
0.8
72.0
25.0 %
1.5 - 3.7%
0.0 - 2.0%
94.3 98.5%
437
Isoelectric focusing
Summary of Results
Metho
Hb A
d
area
Alk
Agaros
e
Major
band as
much
anodal to
Hb A as
Hb S is
cathodal
to Hb A
(Hb J)
Acid
Agar/
Agaros
e
Hb S
area
Major
band
(Hb A)
Hb
A2/C
area
Very
faint
band
(Hb A2)
See note
below
Major
band
(Hb A +
Hb A2+
Hb J)
CZE
Major
peak
(Hb J)
Zone 12
Major
peak
(Hb A)
Zone 9
IEF
Major
band
anodal
to Hb A
(Hb J)
Very
small
peak
(Hb F)
RT=1.05
Major
band
(Hb A)
HPLC
Major
peak
(Hb A)
RT=2.44
Barely
visible
peak
(Hb "JOxfordA2")
Zone 6
Very
minor
peak
(Hb A2)
Zone 3
Very
minor
band
(Hb A2)
Major
peak
(Hb J)
RT=1.62
Barely
visible
peak
(Hb A2)
RT=3.64
case, the alkaline agarose gel electrophoresis (pH 8.6) was repeated with a
heavier application, and a faint Hb "A2-J" band was detected in the area of Hb S.
The reason for this additional test was to rule out a beta chain variant form of Hb
J. Because the intensity of the fast band is far less than that of the Hb A band
this variant is most likely an -chain variant.
During my discussion with the attending physician the following points
were brought to his attention:
i
ii
iii)
a)
b)
iv)
At the time this patient was analyzed, both the CZE and HPLC testing
facilities were not available in our laboratory. For instructional purposes, we have
illustrated the CZE and HPLC scans of another established Hb J-Oxford trait
patient. Both the CZE (major peak in zone 12) and HPLC (minor peak at RT=
1.62) provided concurrent evidence for Hb J trait (-chain variant).
Hb J was first discovered in 1956 (Thorup OA, Itano HH, Wheyby M,
Leavll BS. Hemoglobin J. Science 1956; 123: 889-90), and the 57 variants found
so far are roughly divided equally between -chain and -chain variants. Hb J
variants are rarely found, but have been reported from the USA, northern
European countries, China and Japan. The only Hb J variant which is
encountered with any notable frequency, is Hb J-Baltimore (Case # 24).
view of their faster mobility on agarose gel electrophoresis (pH 8.6). Additional
cases of other "fast hemoglobins" will be included in the 2nd edition of the book.
References
1.
2.
3.
Case # 24
A 33 year old male, residing in Windsor, Canada, whose ancestors migrated from
Europe. While donating blood, his hemoglobin was found to be low, therefore his family
physician ordered hemoglobin electrophoresis.
443
Laboratory Data
Hemoglobin
RBC
MCV
MCH
Platelet
12.8
4.9
86.0
28.3
232
Hb A2
Hb F
Hb A
Hb Variant (HPLC)
2.3
3.8
55.2
38.7 %
1.5 - 3.7%
0.0 - 2.0%
94.3 98.5%
444
Isoelectric focusing
Summary of Results
Metho
d
Alk
Agaros
e
Hb A
area
Major
band as
much
anodal to
Hb A as
Hb S is
cathodal
to Hb A
(Hb J)
Acid
Agar/
Agaros
e
Major
band
(Hb A)
Hb S
area
Note:
Both
major
bands of
equal
intensity
Hb
A2/C
area
Very
faint
band
(Hb A2)
Major
band
(Hb A +
Hb A2+
Hb J)
CZE
Major
peak
(Hb J)
Zone 12
Major
peak
(Hb A)
Zone 9
IEF
Major
band
anodal
to Hb A
(Hb J)
Major
band
(Hb A)
HPLC
Small
peak
(Hb F)
RT=1.05
Major
peak
(Hb A)
RT=2.4
Note:
Minor
peak
(Hb A2)
Zone 3
Minor
band
(Hb A2)
Note:
Both
major
bands of
equal
intensity
Major
peak
(Hb J)
RT=1.8
Very
small
peak
(Hb A2)
RT=3.6
Agarose gel electrophoresis (pH 8.6) showed two major bands. One band
was in the position of Hb A. Another major band was approximately as much
447
CZE showed a major peak (approximately 40%) in zone 12 (Hb JBaltimore), and HPLC also showed a major peak at a retention time of 1.8
448
minutes (Hb J-Baltimore). We suspect that this case was most likely a
representative of Hb J-Baltimore. Confirmation by DNA studies, globin chain
analysis, and LC-Mass spectrometry would be necessary for definitive diagnosis.
However, for financial reasons, all these additional tests are not required in view
of the benign status of the hemoglobinopathy in the patient.
Hb J-Baltimore (also called J-Trinidad, J-Ireland, J-Georgia) is a -chain
variant [ 16(A13) GlyAsp] and is encountered rarely in Afro Americans, and
very rarely in Europeans.
References
1.
2.
3.
4.
Laboratory Data:
449
Hemoglobin
RBC
MCV
MCH
Platelets
Hb A (HPLC)
Hb A2
Hb F
Hb Variant (HPLC)
20.1
6.8
88.0
29.7
270.0
56.0
2.0
4.0
38%
450
Isoelectric focusing
451
Method
Alk
Agarose
Acid Agar
Minor
Hb F
band
detected
CZE
IEF
Faint
band
in
Hb F
area
HPLC
Major
band
slightly
anodal
to Hb A
band
Minor
Major
peak
peak
(Hb F)
(Hb
RT=1.03 Malmo)
RT=1.66
Hb A
area
Major
band
(Hb A +
Hb
Variant)
Major
band
(Hb A +
Hb
Variant)
Major
peak
(Hb A +
Hb
Malmo)
Zone 9
Major
band in
Hb A
position
Hb S
area
Hb A2/C
area
Very
faint
band
(Hb A2)
Minor
peak
(Hb A2)
Zone 3
Faint
band in
Hb A2
area
Major
peak
(Hb A)
RT=2.44
Minor
peak
(Hb A2)
RT=3.6
*Note:
HPLC retention times varies with the type of the instrument used and several
other factors, e.g. temperature, etc.
CZE was not available in our laboratory when we encountered this case,
therefore the CZE scan provided here is of another Hb Malmo patient. However
453
the CZE scan of a Hb Malmo trait was not helpful in the identification of the
variant since both the Hb A and Hb Malmo migrated together in zone 9.
IEF did separate Hb Malmo from Hb A in a pattern identical with a proven
case of Malmo hemoglobinopathy reported in the literature. Hb Malmo migrated
slightly anodal to Hb A, and the migration mobility was much less than some fast
hemoglobins (e.g. Hb J-Oxford, Hb J-Baltimore, Hb N, Hb I, etc).
HPLC provided two clearly separated major peaks and three small peaks
of Hb F, Hb A1c and Hb A2. One major peak was due to Hb A (RT= 2.43
minutes), and another with a faster elution time (RT=1.66), was due to the
hemoglobin variant of this case. In summary, Hb Malmo elutes with Hb A or
before, depending on the chromatographic system used. In our system it eluted
before Hb A.
We were aware that occasionally erythrocytosis has been found to be
associated with high-oxygen affinity hemoglobins, but we did not have the
capability to determine a hemoglobin-oxygen dissociation curve and its p50 ( the
point on the curve where the hemoglobin molecule is half-saturated with oxygen).
a hyperbolic shape. The oxygen delivery to the tissues is impaired whenever the
oxygen affinity is high (low p50). Erythropoietin production is stimulated, which in
turn increases the red cell mass, resulting in erythrocytosis.
After consultation with the attending physician, a narrative report was
submitted stating that a hemoglobin variant is present and in view of marked
erythrocytosis a possibility of a high affinity hemoglobin cannot be ruled out.
Fortunately, the parents of the patient agreed to provide their blood for
analysis. The mother was found to have a normal CBC and hemoglobin pattern.
The father, who had immigrated from Sweden to USA belonged to a family with
known erythrocytosis. Some years ago, when he complained of fatigue,
headaches and lethargy a diagnosis of Hb Malmo was made in Sweden. In order
to relieve his symptoms, phelebotomy was performed. The electrophoretic
(alkaline, acid, IEF) results and HPLC curve were identical for both the father and
son.
In view of the ancestral background and the laboratory results on both the
patient and the parents, a putative diagnosis of Hb Malmo was made. The
attending physician and the family were advised that the hemoglobin disorder
455
was essentially benign. However, the patient should refrain from smoking and
should be followed periodically for any signs of fatigue, headaches or lightheadedness.
More than 100 high oxygen affinity hemoglobin variants are reported in
the literature. Hb Malmo is a member of this class and is the result of the
substitution of glutamine for a histidine amino acid at the 97 th amino acid of the
chain [97(FG4)HisGln]. This mutation is in the area of the peptide chain that
moves during the oxygenation deoxygenation process. The substitution inhibits
movement in such a manner that deoxygenation becomes more difficult and
deters transfer to the tissue to the point that the patient would become anemic if
the body did not compensate by making excess erythrocytes. The amino acids
from position 94 through 103 constitute a nonhelical section of the beta chain and
mutations effecting ionicity of those positions effects the spacing between the
alpha and beta chains near the point of oxygenation. This area of the globin
chain is called the FG segment or FG corner and a list of these variants is
found in Table 1 (courtesy of Hoyer & Kraft, College of American Pathologists).
The fit between the alpha and beta chains is critical because the gap becomes
narrower when oxygen is attached to the ferrous iron and expands as oxygen is
released. A second region of the beta chain (amino acids 143 through 146 on
456
the Carboxy end of the molecule) also effects this spatial control. Several
hemoglobin variants have been identified as possessing mutations in this area
and thus assisting in understanding the synchronous action involved in the
oxygenation / deoxygenation process. Table 2 is a list of these variants
(also courtesy of Hoyer & Kraft, College of American Pathologists).
In all the cases on this list except for Heathrow and Brigham the
mutation effects the shape of the globin chains such that the electrophoretic
mobility is altered so the mutations are not silent (personal communication, Rita
Ellerbrook, PhD, Helena Laboratories, USA).
Hb Malmo only exists in the heterozygous state. The homozygous state
has not been reported and is thus most probably incompatible with life.
Reference
1
2
3
4
5
6
7
Helical #
Substitution
Name
Effect
94
FG1
AspHis
Barcelona
polycythemia
458
AspAsn
Bunbury
normal
95
FG2
LysAsn
LysGlu
Detroit
normal
N-Baltimore normal
97
FG4
HisGln
HisLeu
Malmo
Wood
98
FG5
ValMet
Koln
polycythemia
polycythemia
ValGly
ValAla
hemolysis, O2
Affinity
Nottingham hemolysis
Djelfa
(?)
99
G1
AspAsn
AspHis
AspAla
AspTyr
AspGly
AspVal
Kempsey
Yakima
Radcliffe
Ypsilanti
Hotel Dieu
Chemilly
polycythemia
polycythemia
polycythemia
polycythemia
polycythemia
polycythemia
100
G2
ProLeu
Brigham
polycythemia
101
G3
GluGly
GluGln
GluAsp
GluLys
Alberta
Rush
Potomac
British
Columbia
polycythemia
hemolysis
polycythemia
polycythemia
102
G4
AsnLys
AsnThr
AsnSer
AsnTyr
Richmond
Kansas
Beth Israel
Saint Mande
normal
cyanosis
cyanosis
cyanosis
103
G5
PheLeu
Heathrow
polycythemia
Helical #
Substitution
Name
459
Effect
143
144
H21
HC1
HisArg
HisGln
HisPro
HisAsp
Abbruzzo
Little Rock
Syracuse
Rancho
Mirage
polycythemia
polycythemia
polycythemia
(?)
HisTyr
Old
Dominion
normal CBC
O2 affinity
LysAsn
Andrewpolycythemia
Minneapolis
LysGlu
Mito
polycythemia
145
HC2
TyrHis
TyrCys
TyrAsn
TyrStop
Bethesda
Rainier
Osler
McKees
Rocks
polycythemia
polycythemia
polycythemia
polycythemia
146
HC3
HisAsp
HisPro
HisArg
Hiroshima
York
Cochin-Port
Royal
Cowtown
Kodaira
polycythemia
polycythemia
(?)
HisLeu
HisGln
polycythemia
polycythemia
The contents of these tables are presented from Hoyer JD, Kroft SH, eds. Color Atlas of
Hemoglobin Disorders: A Compendium Based on Proficiency Testing. Northfield, IL:
College of American Pathologists: 2003 (Reproduced with Permission)
Case # 26
A 18 year old female student. No ancestral information was available. Physical examination
revealed scleral icterus and spleen palpable 4 cm below left costal margin.
Laboratory Data:
Hemoglobin
RBC
MCV
RDW
10.7
3.9
106
13.6
Platelet
235
WBC
Reticulocyte
Serum Iron
Total Bilirubin
Indirect Bilirubin
Hb A (HPLC)
Hb A2 (HPLC)
7.1
2.9
39
2.8
2.4
74.8%
2.2
461
Isoelectric focusing
462
Metho
d
Hb A
area
Hb S
area
Alk
Agaros
e
Major
band
Hb A
Broad
smudge
from Hb S
through
Hb C
Acid
Agar /
Agaros
e
CZE
Major
band
(All
Hbs)
Major
peak
(Hb A +
Zone 9
Minor
peak
(Hb
Koln)
Zone 6
Major
band
(Hb A)
Broad
smudge of
Hb Koln
from Hb
Hb Kln)
IEF
A-Hb A2
HPLC
Major
peak
(Hb A)
RT=2.39
Two
barely
visible
minor
peaks (Hb
Koln)
RT=4.54.8
Hb
A2/C
area
No discrete
band
detected
Minor
peak
(Hb
Koln)
Zone 4
Minor
peak
(Hb A2
+ Hb
Koln)
Zone 3
Barely
visible
minor
band
Hb A2
Minor
peak
Hb A2
RT=3.6
Minor
peak
(Hb
Koln)
RT=4.95
# Due to the instability of Hb Koln, several minor bands were noticed in HPLC. A similar
phenomenon was observed in other electrophoretic methods except in the acid
agar/agarose electrophoresis, where only one major band was detected.
Alkaline agarose gel electrophoresis (pH 8.6) showed a major band in the
position of Hb A and a smudged band migrating on both the anodal and cathodal
sides of Hb S extending further towards the area of Hb C. Citrate agar
electrophoresis (pH 6.2) showed only one band in the position of Hb A. IEF
showed an intense band in the position of Hb A, and a broad smear between Hb
464
A and Hb A2 (similar to a fresh painting with a brush). CZE showed a major peak
in zone 9 (Hb A area) and three minor peaks in zones 3, 4 and 6. HPLC showed
a major peak at a retention time (RT) of 2.39 minutes (Hb A), and minor peaks
from a RT of 3.5 5.0 minutes.
The laboratory tests indicated that either the hemoglobin variant did
not separate from Hb A, or it formed discrete peaks, or it formed blurred bands.
Since the hemoglobin instability test was positive, the attending physician was
advised of the possibility of an unstable hemoglobin variant.
In August 2013, Professor Steinberg reviewed (reference # 1) the current
clinical and hematological characteristics of unstable hemoglobins. According to
this recent review, approximately 140 of the1028 known mutations of hemoglobin
were found to be unstable. To date, it is not feasible to identify an unstable
hemoglobin variant (either alpha-, beta-, gamma, and delta-globin chains
abnormalities) by the commonly used laboratory methods.
In most worldwide laboratories, Hb E, Hb H, Hb Hasharon and Hb
Koln are the most frequently reported unstable hemoglobin variants. Three of
these variants (Hb E, Hb H, and Hb Hasharon) were excluded in our patient on
the basis of their electrophoretic mobilities and retention times on HPLC. Hb E
(Case # 14a) migrates in the position of Hb A2/E/O/C. Hb H is a fast migrating
hemoglobin variant (more anodal to Hb A) on alkaline agarose gel
electrophoresis (pH 8.6). Hb Hasharon (Case # 20) migrates in the Hb S area on
465
Unstable hemoglobin
Negative sickle cell solubility test for Hb S
Minimal or no anemia
Splenomegaly and regenerative erythrocyte changes even in the
absence of anemia
Hypochromasia and macrocytosis are usually evident
Increased oxygen affinity
Atypical migration pattern on alkaline agarose gel electrophoresis
(pH 8.6), IEF, CZE and multiple peaks on HPLC.
466
A suspicious peripheral blood smear would lead to a Heinz body test and
hemoglobin instability (isopropanol) test, but the later two tests should not be run
in the presence of Hb F levels >5%.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
468
Laboratory Data
Hemoglobin
RBC
14.8
5.4
MCV
MCH
Platelet
86.0
28.3
232
Hb A2
Hb F
Hb A (HPLC)
Hb Variant (HPLC)
1.1
0.8
78.8
19.3 %
80 - 100 fL
27 - 34 pg
3
150 - 400 Th/mm
1.5 - 3.7%
0.0 - 2.0%
94.3 - 98.5%
470
Isoelectric focusing
Hb A
Hb S
472
Hb
area
area
Alk
Agaros
e
Major
band
(Hb A)
Major
band
slightly
towards
Hb A side
of Hb S
(Hb QIndia)
Acid
Agar
Major
band
between
Hb A and
Hb S (Hb
A + Hb
Q-India)
Hb A and
Hb QIndia
combine
as a
broader
band
Acid
Agaros
e
Major
band
(Hb A +
Hb QIndia)
Exactly
in the
position
of Hb A
CZE
Major
peak
(Hb A)
Zone 9
Major
peak
(QIndia)
Zone 6
IEF
Major
band
(Hb A)
Major
band
(Hb QIndia)
HPLC
Major
peak
(Hb A)
RT=2.39
Major
peak
(Hb QIndia)
RT=4.7
A2/C
area
Faint
band
(Hb A2)
Slightly
anodal
towards
Hb S
Minor
peak
(Hb A2)
Zone 3
Slightly
anodal
towards
Hb A
Minor
peak
(Hb QIndia +
Hb A2 )
Zone 1
Faint
band
(Hb A2)
Faint
peak
(Hb A2)
RT=3.6
Review of Hemoglobin Q
Bushra Moiz, PhD
Introduction
Hb Q was first reported in 1958 in a Chinese patient (1). Since then a number of
cases have been described in Asians. It is a rare hemoglobinopathy resulting from a
single point mutation (GACCAC) of -1 globin gene present on chromosome 16. The
resulting hemoglobin is modified structurally at polypeptide chain replacing aspartic
acid by histidine. Depending on the implicated codon, three variants have been
described namely Hb Q-India ( 64 AspHis), Hb Q-Thailand ( 74 AspHis), and Hb
Q-Iran ( 75 AspHis). Using computerized models for protein structure, it was
observed that there is no difference between the predicted secondary structures of
normal -globin and that of Hb Q-India (2). In contrast, Hb Q-Iran carries an extra helix
while Hb Q-Thailand carries two extra helices. The predicted results of tertiary structure
474
also support these findings (2). Since the residue and hence charge changes involve
the surface of the hemoglobin tetramer, the properties of the hemoglobin molecule are
not affected (3).
Hb Q-Thailand [74(EF3) AspHis] is often found in Thailand, China, and other
Southeast Asian countries (4). It has several synonyms including Hb Mahidol, QChinese, G-Taichung, Kurashiki, and Asabara (5). The alpha-Q-Thailand gene is
strongly linked to gene deletion and has important implications in the identification and
diagnosis of hemoglobinopathies and thalassemias. Subjects with Hb Q-Thailand
invariably show microcytosis as the variant is invariably linked to (-
4.2
). However,
individuals who are doubly mutated for Hb Q-Thailand and thalassemia may be more
severely anemic (6,7). More complex interaction of Hb Q-Thailand with Hb E, Constant
Spring and hereditary persistence of fetal hemoglobin has been described in the
literature (8-10).
Hb Q-Iran was first described in 1970 by Lorkin et al (11) and later by Rahimi in
an Iranian individual (12).
Hb Q-India was first reported in 1972 by Sukumaran in a Sindhi family (13). Later
reports were published by Dash (14), Abraham (3) and Desai (15); their observations
came from Sindhi and Punjabi families. Hb Q-India usually occurs in the heterozygous
state (
Q-India
Q-India
Q-India
Clinical manifestation
The presence of Hb Q does not impart any functional deficit since the
hemoglobin is not altered structurally at its tertiary level (8). Hb Q is a stable
molecule and has normal oxygen affinity. Therefore, Hb Q is clinically silent
in a heterozygous individual. In contrast, subjects who are compound
heterozygotes with other hemoglobinopathies exhibit a thalassemic phenotype.
For example, co-inheritance of Hb Q-India and -thalassemia results in a mild
anemia (17). Similarly, Hb Q-H disease caused by the co-inheritance of Hb Qo
HPLC
.The separation of hemoglobin variants depends on their retention
times (21). Hb Q-India, Iran and Thailand exhibited retention times similar
to that of other -chain variants in the range of 4.76 to 4.78 minutes (3). In
477
ARMS-PCR
This technique can be used for the successful detection of various
hemoglobin variants including Hb Q-India (3). This technique is based on
the amplification of allele specific primers because of 3-terminal matches
and mismatches. The methodology is simple, rapid and inexpensive;
however, it is non-specific since either sub-optimal amplification or
deteriorating primers can lead to false positive results (25).
RFLP-PCR
Recently, a restriction enzyme digestion assay was employed for
the diagnosis of Hb Q-India (22). Restriction enzyme EaeI was utilized in
RFLP-PCR since Hb Q-India abolishes the recognition site of this enzyme.
It can be used as a simple, robust and alternative method to ARMS-PCR
for DNA diagnosis of Hb Q-India. However, any other rare variant that
abolishes the same EaeI restriction site would also be detected. Hence,
RFLP-PCR can be used as an adjuvant test after HPLC and or IEF for
primary diagnosis of Hb Q-India.
Gene sequencing
This is the most definitive technique for identifying a hemoglobin
variant. Recently, Bhat described DNA sequencing in a patient with Hb QIndia (26). This methodology of sequence electrophoretogram clearly
demonstrates the specific location of the mutation of Hb Q-India.
479
Not only did it show that the codon of GAC encoding aspartic acid was
mutated to the codon CAC encoding for histidine, but it also depicted the
zygosity of the patient (26).
References
1
2
3
4
5
6
7
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
481
Case # 28
A 24 year old male, belonging to Qara tribes from the Dhofar region of the Sultanate of
Oman. The patient had not been transfused during the past six months.
Laboratory Data:
Hemoglobin
RBC
MCV
MCH
RDW
Platelet
Hb A
Hb A2 (HPLC)
13.0
5.1
68
22.4
13.2
243
81.5
4.1
Hb F
Hb Dhofar (HPLC)
0.8
13.6
0.0 - 2.0%
483
Isoelectric focusing
484
Summary of Results
Metho
d
Hb A
area
Hb S
area
Alk
Agaros
e
Major
band
(Hb A)
Medium
size
band
(Hb
Dhofar)
Acid
Agar
Major band
( Hb A +
Hb Dhofar)
Hb A and
Hb Dhofar
combine as
a broader
band
Acid
Agaros
e
Major
band (Hb
A + Hb
Dhofar)
Major
band
broadened
by Hb
Dhofar
CZE
Major
peak
(Hb A)
Zone 9
Medium
size peak
(Hb
Dhofar)
Zone 5
Minor
peak (Hb
A2) Zone
3
IEF
Major
band
(Hb A)
Medium
size
band
(Hb
Dhofar)
Minor
band
(Hb A2)
Major
peak
(Hb A)
RT=2.35
Medium
size peak
(Hb
Dhofar)
RT=4.04
Minor
peak (Hb
A2)
RT=3.6
HPLC
*Note:
Hb
A2/C
area
Minor
band
(Hb A2)
HPLC retention time (RT) varies with the type of the instrument used and
several other factors, e.g. temperature etc.
Alkaline agarose gel electrophoresis (pH 8.6) showed a major band in the
486
Hb Dhofar [
29 (GGC-GGT) Gly-Gly
58 (CCT-CGT) ProArg
] exists
References
1.
2.
3.
4.
5.
6.
488