PART I  Introduction to Hematology

27 Hemoglobinopathies
(Structural Defects in Hemoglobin)
Tim R. Randolph

OUTLINE OBJECTIVES
Structure of Globin Genes After completion of this chapter, the reader will be able to:
Hemoglobin Development
Genetic Mutations 1. Explain the difference between structural hemoglobin 8. Describe the clinical and laboratory findings for the
Zygosity disorders and thalassemias, and describe the types of compound heterozygous disorders of Hb S with Hb C,
Pathophysiology mutations found in the structural disorders. b-thalassemia, Hb D, Hb O-Arab, Hb Korle Bu, and
Nomenclature 2. Describe globin gene structure and the development Hb C-Harlem.
Hemoglobin S of normal human hemoglobins throughout prenatal 9. Describe the electrophoretic mobility of Hb A, Hb F,
Sickle Cell Anemia and postnatal life. Hb S, and Hb C at an alkaline pH, and explain how
Sickle Cell Trait 3. Differentiate between homozygous and heterozygous other methods (including the Hb S solubility test,
Hemoglobin C states and the terms “disease” and “trait” as they re- citrate agar electrophoresis at acid pH, and high-
Prevalence, Etiology, and late to the hemoglobinopathies. performance liquid chromatography) are used to
Pathophysiology
4. Given the hemoglobin genotypes of parents involving distinguish Hb S and Hb C from other hemoglobins
Hemoglobin C-Harlem
common b chain variants, determine the possible with the same mobility.
(Hemoglobin C-George-
town) genotypes of their children using a Punnett square. 10. Describe the genetic mutations, inheritance pat-
Hemoglobin E 5. Describe the general geographic distribution of com- terns, pathophysiology, and clinical and labora-
Prevalence, Etiology, and mon hemoglobin variants and the relationship of that tory findings in hemoglobin variants that result in
Pathophysiology distribution with the prevalence of malaria and glu- methemoglobinemia.
Clinical Features cose-6-phosphate dehydrogenase deficiency. 11. Describe the inheritance patterns, causes, and clin-
Hemoglobin O-Arab 6. For disorders involving Hb S and Hb C, describe the ical and laboratory findings of unstable hemoglobin
Hemoglobin D and Hemo- genetic mutation, the effect of the mutation on the variants.
globin G hemoglobin molecule, the inheritance pattern, patho- 12. Discuss the pathophysiology of hemoglobin variants
Compound Heterozygosity physiology, symptoms, clinical findings, peripheral with increased and decreased oxygen affinities, and
with Hemoglobin S and
blood findings, laboratory diagnosis, and genetic explain how they differ from unstable hemoglobins.
Another b-Globin Gene
counseling and treatment considerations. 13. Given a case history and clinical and laboratory find-
Mutation
Hemoglobin SC 7. Describe the genetic mutation, clinical findings, ings, interpret test results to identify the hemoglobin
Hemoglobin S b-Thalasse- and laboratory diagnosis for disorders involving Hb variants present in the patient.
mia – C-Harlem, Hb E, Hb O-Arab, Hb D, and Hb G.
Hemoglobin SD and Hemo-
globin SG-Philadelphia CASE STUDY
Hemoglobin S/O-Arab and
After studying the material in this chapter, the reader should be able to respond to the
HbS/D-Punjab
following case study:
Hemoglobin S-Korle Bu
Concomitant Cis Muta-
An 18-year-old African-American woman was
tions with Hemoglobin S Patient Results Reference Interval
seen in the emergency department for fever
Hemoglobin C-Harlem
and abdominal pain. The following results HCT (%) 32.5 35–49
Hemoglobin S-Antilles and
were obtained on a complete blood count: RDW (%) 19.5 11.5–14.5
Hemoglobin S-Oman
Hemoglobin M Platelets (3109/L) 410 150–450
Patient Results Reference Interval Segmented 75 50–70
Unstable Hemoglobin
Variants WBCs (310 /L)9
11.9 3.6–10.6 neutrophils (%)
Clinical Features RBCs (31012/L) 3.67 4.00–5.40 Lymphocytes (%) 18 18–42
HGB (g/dL) 10.9 12.0–15.0 Monocytes (%) 3 2–11

426
 CHAPTER 27  Hemoglobinopathies (Structural Defects in Hemoglobin) 427

OUTLINE—cont’d CASE STUDY—cont’d

Treatment and Prognosis After studying the material in this chapter, the reader should be able to respond to the
Hemoglobins with In- following case study:
creased and Decreased
Oxygen Affinity Patient Results Reference Interval
Hemoglobins with Increased
Oxygen Affinity Eosinophils (%) 3 1–3
Hemoglobins with De- Basophils (%) 1 0–2
creased Oxygen Affinity Reticulocytes (%) 3.1 0.5–2.5
Global Burden of Hemo- A typical field in the patient’s peripheral
blood film is shown in Figure 27-1. Electropho-
resis on cellulose acetate at alkaline pH showed
50.9% Hb S and 49.1% Hb C.
1. Select confirmatory tests that should be per-
formed and describe the expected results.
2. Describe the characteristic red blood cell
morphology on the peripheral blood film.
3. Based on the electrophoresis and red
blood cell morphology results, what diag-
nosis is suggested? Figure 27-1  ​Peripheral blood film for the patient in
4. If this patient were to marry a person of geno- the case study (31000). (Courtesy Ann Bell, University
type Hb AS, what would be the expected fre- of Tennessee, Memphis.)
quency of genotypes for each of four children?

H
emoglobinopathy refers to a disease state (opathy) involv-
STRUCTURE OF GLOBIN GENES
ing the hemoglobin (Hb) molecule. Hemoglobinopa-
thies are the most common genetic diseases, affecting As discussed in Chapter 10, there are six functional human globin
approximately 7% of the world’s population.1 Approximately genes located on two different chromosomes. Two of the globin
300,000 children are born each year with some form of inherited genes, a and z, are located on chromosome 16 and are referred to
hemoglobin disorder.2 All hemoglobinopathies result from a as a-like genes. The remaining four globin genes, b, g, d, and e, are
genetic mutation in one or more genes that affect hemoglobin located on chromosome 11 and are referred to as b-like genes. In
synthesis. The genes that are mutated can code for either the the human genome, there is one copy of each globin gene per
proteins that make up the hemoglobin molecule (globin or poly- chromatid, for a total of two genes per diploid nucleus, with the
peptide chains) or the proteins involved in synthesizing or regu- exception of a and g. There are two copies of the a and g genes per
lating synthesis of the globin chains. Regardless of the mutation chromatid, for a total of four genes per diploid nucleus. Each glo-
encountered, all hemoglobinopathies affect hemoglobin synthe- bin gene codes for the corresponding globin chain: the a-globin
sis in one of two ways: qualitatively or quantitatively. In qualita- genes (HBA1 and HBA2) are used as the template to synthesize the
tive hemoglobinopathies, hemoglobin synthesis occurs at a nor- a-globin chains, the b-globin gene (HBB) codes for the b-globin
mal or near-normal rate, but the hemoglobin molecule has an chain, the g-globin genes (HBG1 and HBG2) code for the g-globin
altered amino acid sequence within the globin chains. This chains, and the d-globin gene (HBD) codes for the d-globin chain.
change in amino acid sequence alters the structure of the hemo-
globin molecule (structural defect) and its function (qualitative
HEMOGLOBIN DEVELOPMENT
defect). In contrast, thalassemias result in a reduced rate of he-
moglobin synthesis (quantitative) but do not affect the amino Each human hemoglobin molecule is composed of four globin
acid sequence of the globin chains. A reduction in the amount of chains: a pair of a-like chains and a pair of b-like chains. Dur-
hemoglobin synthesized produces an anemia and stimulates the ing the first 3 months of embryonic life, only one a-like gene
production of other hemoglobins not affected by the mutation in (z) and one b-like gene (e) are activated, which results in the
an attempt to compensate for the anemia. Based on this distinc- production of z and e globin chains that pair to form hemoglo-
tion, hematologists divide hemoglobinopathies into two catego- bin Gower-1 (z2e2). Shortly thereafter, a and g chain synthesis
ries: structural defects (qualitative) and thalassemias (quantita- begins, which leads to the production of Hb Gower-2 (a2e2)
tive). To add confusion to the classification scheme, many and Hb Portland (z2g2). Later in fetal development, z and
hematologists also refer to only the structural defects as hemoglo- e synthesis ceases; this leaves a and g chains, which pair to
binopathies. This chapter describes the structural or qualitative produce Hb F (a2g2), also known as fetal hemoglobin. During
defects that are referred to as hemoglobinopathies; the quantitative the 6 months after birth, g chain synthesis gradually decreases
defects (thalassemias) are described in Chapter 28. and is replaced by b chain synthesis so that Hb A (a2b2), also
428 PART IV  Erythrocyte Disorders

known as adult hemoglobin, is produced. Recent evidence sug- Deletions involve the removal of one or more nucleotides,
gests BCL11A and KLF1, zinc-finger transcriptional repressors, whereas insertions result in the addition of one or more nu­
are necessary to silence the g-globin gene and mutations in the cleotides. Usually deletions and insertions are not divisible by
gene that codes for either factor results in elevated HbF levels.3 three and disrupt the reading frame, which leads to the nullifi-
The remaining globin gene, d, becomes activated around birth, cation of synthesis of the corresponding globin chain. This is
producing d chains at low levels that pair with a chains to the case for the quantitative thalassemias (Chapter 28). In he-
produce the second adult hemoglobin, Hb A2 (a2d2). Normal moglobinopathies, the reading frame usually remains intact,
adults produce Hb A (95%), Hb A2 (less than 3.5%), and Hb F however; the result is the addition or deletion of one or more
(less than 1% to 2%). amino acids in the globin chain product, which sometimes af-
fects the structure and function of the hemoglobin molecule.
Of the 1181 variants described in Table 27-1, 72 variants result
GENETIC MUTATIONS
from deletions or insertions, or both.5
More than 1000 structural hemoglobin variants (hemoglobin- Chain extensions occur when the stop codon is mutated
opathies) are known to exist throughout the world, and more so that translation continues beyond the typical last codon.
are being discovered regularly (Table 27-1).4,5 Each of these Amino acids continue to be added until a stop codon is
hemoglobin variants results from one or more genetic muta- reached by chance. This process produces globin chains that
tions that alter the amino acid sequence. Some of these are longer than normal. Significant globin chain extensions
changes alter the molecular structure of the hemoglobin mol- usually result in degradation of the globin chain and a quanti-
ecule, ultimately affecting hemoglobin function. The types of tative defect. If the extension of the globin chain is insufficient
genetic mutations that occur in the hemoglobinopathies in- to produce significant degradation, however, the defect is
clude point mutations, deletions, insertions, and fusions in- qualitative and is classified as a hemoglobinopathy. Hemoglo-
volving one or more of the adult globin genes—a, b, g, and d.5 bin molecules with extended globin chains fold inappropri-
Point mutation is the most common type of genetic muta- ately, which affects hemoglobin structure and function.
tion occurring in the hemoglobinopathies. Point mutation is Gene fusions occur when two normal genes break between
the replacement of one original nucleotide in the normal gene nucleotides, switch positions, and anneal to the opposite gene.
with a different nucleotide. Because one nucleotide is replaced For example, if a b-globin gene and a d-globin gene break in
by one nucleotide, the codon triplet remains intact, and the similar locations, switch positions, and reanneal, the resultant
reading frame is unaltered. This results in the substitution of genes would be bd and db fusion genes in which the head of
one amino acid in the globin chain product at the position the fusion gene is from one original gene and the tail is from
corresponding to the location of the original point mutation. the other. As long as the reading frames are not disrupted and
As can be seen in Table 27-1, 1109 of the 1181 known hemo- the globin chain lengths are similar, the genes are transcribed
globin variants result from a point mutation that causes an and translated into hybrid globin chains. The fusion chains
amino acid substitution. It also is possible to have two point fold differently, however, and affect the corresponding hemo-
mutations occurring in the same globin gene, which results in globin function. Nine fusion globin chains have been identi-
two amino acid substitutions within the same globin chain. fied (see Table 27-1).
Over 35 mutations occur by this mechanism.5

ZYGOSITY
Zygosity refers to the association between the number of gene
TABLE 27-1  ​Molecular Abnormalities of Hemoglobin mutations and the level of severity of the resultant genetic de-
Variants fect. Generally, there is a level of severity associated with each
NUMBER OF VARIANTS BY GLOBIN CHAIN gene that is normally used to synthesize the globin chain prod-
a b d g Total uct. For the normal adult globin genes, there are four copies
Amino acid 413 535 65 96 1109 of the a and g genes and two copies of the b and d genes.
substitution In theory, this could result in four levels of severity for a and
Deletions or 22 48 1 1 72 g gene mutations and two levels of severity for the b and d gene
insertions mutations. Expressed another way, if all things were equal,
Total 435 583 66 97 1181 it would require twice as many mutations within the a and
Fusions — — — — 9* g genes to produce the same physiologic effect as mutations
within the b and d genes. Because the g and d genes are tran-
*Seven fusions involve the b and d chains; two fusions involve the b and g chains. scribed and translated at such low levels in adults, however,
Data from Patrinos GP, Giardine B, Riemer W, et al: Improvements in the HbVar data- mutations of either gene would have little impact on overall
base of human hemoglobin variants and thalassemia mutations for population and
sequence variation studies, Nucl Acids Res 32(database issue):D537-541, 2004. hemoglobin function. In addition, because the dominant
Available at: http://globin.cse.psu.edu/hbvar/menu.html. Accessed November 29, hemoglobin in adults, Hb A, is composed of a and b chains,
2013. b gene mutations would affect overall hemoglobin function to
The table is designed to provide a relative distribution of mutation types and just in-
cludes structural variants. Fifty-one of the variants are also categorized as thalasse- a greater extent than the same number of a gene mutations.
mias. Mutations are being added regularly. This partially explains the greater number of identified b chain
 CHAPTER 27  Hemoglobinopathies (Structural Defects in Hemoglobin) 429

variants compared with a chain variants, because a single b oxygen affinity; and abnormal hemoglobins with no clinical or
gene mutation would be more likely to create a clinical condi- functional effect. Imbalanced chain production also may be
tion than would a single a gene mutation. associated in rare instances with a structurally abnormal chain,
The inheritance pattern of b chain variants is referred to as such as Hb Lepore,4 because of the reduced production of the
heterozygous when only one b gene is mutated and homozygous abnormal chain. The functional classification of selected he-
when both b genes are mutated. The terms disease and trait are moglobin variants is summarized in Box 27-1.
also commonly used to refer to the homozygous (disease) and Many of the variants are clinically insignificant because they
heterozygous (trait) states. do not show any physiologic effect. As discussed previously,
most clinical abnormalities are associated with the b chain fol-
lowed by the a chain. Involvement of the g and d chains does
PATHOPHYSIOLOGY
occur, but because of the small amount of hemoglobin in-
Pathophysiology refers to the manner in which a disorder trans- volved, it is rarely detected and is usually of no consequence.
lates into clinical symptoms. The impact of point mutations on Box 27-2 lists clinically significant abnormal hemoglobins. The
hemoglobin function depends on the chemical nature of the most frequently occurring of the abnormal hemoglobins and
substituted amino acid, where it is located in the globin chain, the most severe is Hb S.
and the number of genes mutated (zygosity). The charge and
size of the substituted amino acid may alter the manner in
NOMENCLATURE
which the globin chain folds. A change in charge affects the
interaction of the substituted amino acid with adjacent amino As hemoglobins were reported in the literature, they were desig-
acids. In addition, the size of the substituted amino acid makes nated by letters of the alphabet. Normal adult hemoglobin and
the globin chain either more or less bulky. Therefore, the fetal hemoglobin were called Hb A and Hb F. By the time the
charge and the size of the substituted amino acid determine its middle of the alphabet was reached, however, it became apparent
impact on hemoglobin structure by potentially altering the that the alphabet would be exhausted before all mutations were
tertiary structure of the globin chain and the quaternary struc- named. Currently, some abnormal hemoglobins are assigned a
ture of the hemoglobin molecule. Changes in hemoglobin common designation and a scientific designation. The common
structure usually affect function. Location of the substitution name is selected by the discoverer and usually represents the
within the globin chain also has an impact on the degree of geographic area where the hemoglobin was identified. A single
structural alteration and hemoglobin function based on its capital letter is used to indicate a special characteristic of the he-
positioning within the molecule and the interactions with the moglobin variants, such as hemoglobins demonstrating identical
surrounding amino acids. In the case of the sickle cell muta- electrophoretic mobility but containing different amino acid
tion, one amino acid substitution results in hemoglobin po- substitutions, as in Hb G-Philadelphia, Hb G-Copenhagen, and
lymerization, leading to the formation of long hemoglobin Hb C-Harlem. The variant description also can involve scientific
crystals that stretch the red blood cell (RBC) membrane and designations that indicate the variant chain, the sequential and
produce the characteristic crescent moon or sickle cell shape. the helical number of the abnormal amino acid, and the nature
Zygosity also affects the pathophysiology of the disease. In of the substitution. The designation [b6 (A3) GlunVal] for the
b-hemoglobinopathies, zygosity predicts two severities of dis- Hb S mutation indicates the substitution of valine for glutamic
ease. In homozygous b-hemoglobinopathies, in which both acid in the A helix in the b chain at position 6.4
b genes are mutated, the variant hemoglobin becomes the
dominant hemoglobin type, and normal hemoglobin (Hb A)
HEMOGLOBIN S
is absent. Examples are sickle cell disease (SCD, Hb SS) and
Hb C disease (Hb CC). In heterozygous b-hemoglobinopathies, Sickle Cell Anemia
one b gene is mutated and the other is normal, which suggests History
a 50/50 distribution. In an attempt to minimize the impact of Although the origin of sickle cell anemia has not been identified,
the abnormal hemoglobin, however, the variant hemoglobin is symptoms of the disease have been traced in one Ghanaian fam-
usually present in lesser amounts than Hb A. Nevertheless, in ily back to 1670.7 Sickle cell anemia was first reported by a Chi-
some cases they may be present in equal amounts. Examples cago cardiologist, Herrick, in 1910 in a West Indian student with
are Hb S trait (Hb AS) and Hb C trait (Hb AC). Patients with severe anemia. In 1917, Emmel recorded that sickling occurred
homozygous sickle cell disease (Hb SS) inherit a severe form of in nonanemic patients and in patients who were severely ane-
the disease that occurs less frequently but requires lifelong mic. In 1927, Hahn and Gillespie described the pathologic basis
medical intervention, which must begin early in life, whereas of the disorder and its relationship to the hemoglobin molecule.
heterozygotes (Hb AS) are much more common but rarely These investigators showed that sickling occurred when a solu-
experience symptoms. tion of RBCs was deficient in oxygen and that the shape of the
Fishleder and Hoffman6 divided the structural hemoglobins RBCs was reversible when that solution was oxygenated again.4,8
into four groups: abnormal hemoglobins that result in hemo- In 1946, Beet reported that malarial parasites were present less
lytic anemia, such as Hb S and the unstable hemoglobins; ab- frequently in blood films from patients with SCD than in indi-
normal hemoglobins that result in methemoglobinemia, such viduals without SCD.9 It was determined that the sickle cell trait
as Hb M; hemoglobins with either increased or decreased confers a resistance against infection with Plasmodium falciparum
430 PART IV  Erythrocyte Disorders

BOX 27-1  ​Functional Classification of Selected Hemoglobin (Hb) Variants

I. Homozygous: Hemoglobin Polymorphisms: c. Hb Providence: a2b282Asn

The Variants That Are Most Common d. Hb Agenogi: a2b290Lys
Hb S: a2b26Val—severe hemolytic anemia; sickling e. Hb Beth Israel: a2b2102Ser
Hb C: a2b26Lys—mild hemolytic anemia f. Hb Yoshizuka: a2b2108Asp
Hb D-Punjab: a2b2121Gln—no anemia
Hb E: a2b226Lys—mild microcytic anemia C.  Unstable Hemoglobins
1. Hemoglobin may precipitate as Heinz bodies after splenectomy
II. Heterozygous: Hemoglobin Variants Causing (congenital Heinz body anemia)
Functional Aberrations or Hemolytic Anemia in a. Severe hemolysis: no improvement after splenectomy
the Heterozygous State Hb Bibba: a2136Prob2
A.  Hemoglobins Associated with Methemoglobinemia Hb Hammersmith: a2b242Ser
and Cyanosis Hb Bristol-Alesha: a2b267Asp or 67Met
1 . Hb M-Boston: a258Tyrb2 Hb Olmsted: a2b2141Arg
2. Hb M-Iwate: a287Tyrb2 b. Severe hemolysis: improvement after splenectomy
3. Hb Auckland: a287Asnb2 Hb Torino: a243Valb2
4. Hb Chile: a2b228Met Hb Ann Arbor: a280Argb2
5. Hb M-Saskatoon: a2b263Tyr Hb Genova: a2b228Pro
6. Hb M-Milwaukee-1: a2b267Glu Hb Shepherds Bush: a2b274Asp
7. Hb M-Milwaukee-2: a2b292Tyr Hb Köln: a2b298Met
8. Hb F-M-Osaka: a2g263Tyr Hb Wien: a2b2130Asp
9. Hb F-M-Fort Ripley: a2g292Tyr c. Mild hemolysis: intermittent exacerbations
Hb Hasharon: a247Hisb2
B.  Hemoglobins Associated with Altered Oxygen Hb Leiden: a2b26 or 7 (Glu deleted)
Affinity Hb Freiburg: a2b223 (Val deleted)
1. Increased affinity and erythrocytosis Hb Seattle: a2b270Asp
a. Hb Chesapeake: a292Leub2 Hb Louisville: a2b242Leu
b. Hb J-Capetown: a292Glnb2 Hb Zurich: a2b263Arg
c. Hb Malmo: a2b297Gln Hb Gun Hill: a2b291-95 (5 amino acids deleted)
d. Hb Yakima: a2b299His d. No disease
e. Hb Kempsey: a2b299Asn Hb Etobicoke: a284Argb2
f. Hb Ypsi (Ypsilanti): a2b299Tyr Hb Sogn: a2b214Arg
g. Hb Hiroshima: a2b2146Asp Hb Tacoma: a2b230Ser
h. Hb Rainier: a2b2145Cys 2. Tetramers of normal chains; appear in thalassemias
i. Hb Bethesda: a2b2145His Hb Bart: g4
2. Decreased affinity—may have mild anemia or cyanosis Hb H: b4
a. Hb Kansas: a2b2102Thr
b. Hb Titusville: a294Asnb2

From Elghetany MT, Banki K: Erythrocyte disorders. In McPherson RA, Pincus MR: Henry’s clinical diagnosis and management by laboratory methods, ed 22, Philadelphia,
2011, Elsevier, Saunders, p. 578. Originally modified from Winslow RM, Anderson WF: The hemoglobinopathies. In Stanbury JB, Wyngaarden JB, Fredrickson DS, et al,
editors: The metabolic basis of inherited disease, ed 5, New York, 1983, McGraw-Hill, pp. 2281-2317. Updated from 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, Nucl Acids Res 32
(database issue):D537-541, 2004. Available at: http://globin.cse.psu.edu/hbvar/menu.html. Accessed November 30, 2013.
Arg, Arginine; Asn, asparagine; Asp, aspartic acid; Cys, cysteine; Gln, glutamine; Glu, glutamic acid; His, histidine; Leu, leucine; Lys, lysine; Met, methionine; Pro,
proline; Ser serine; Thr, threonine; Tyr, tyrosine; Val, valine.

occurring early in childhood between the time that passively The term sickle cell diseases is used to describe a group of symp-
acquired immunity dissipates and active immunity develops.10 tomatic hemoglobinopathies that have in common sickle cell for-
In 1949, Pauling showed that when Hb S is subjected to electro- mation and the associated crises. Patients with SCD are either ho-
phoresis, it migrates differently than does Hb A. This difference mozygous for Hb S (SS) or are compound heterozygotes expressing
was shown to be caused by an amino acid substitution in the Hb S in combination with another hemoglobin b chain mutation
globin chain. Pauling and coworkers defined the genetics of the like Hb C or b-thalassemia. SCDs are the most common form of
disorder and clearly distinguished heterozygous sickle trait (Hb hemoglobinopathy, with Hb SS and the variants Hb SC and Hb
AS) from the homozygous state (Hb SS).4 S–b-thalassemia (Hb S–b-thal) occurring most frequently.
 CHAPTER 27  Hemoglobinopathies (Structural Defects in Hemoglobin) 431

A S S S A S
BOX 27-2  ​Clinically Important Hemoglobin (Hb)
Variants A AA AS A AS AS A AA AS
I. Sickle syndromes
A. Sickle cell trait (AS) A AA AS A AS AS S AS SS
B. Sickle cell disease
1. SS A B C
2. SC
3. SD-Punjab (Los Angeles) S S A S
4. SO-Arab
A AS AS A AA AS
5. S–b-Thalassemia
6. S–hereditary persistence of fetal hemoglobin
S SS SS C AC SC
7. SE
II. Unstable hemoglobinsncongenital Heinz body anemia (.140
variants) D E
III. Hemoglobins with abnormal oxygen affinity Figure 27-2  ​Punnett square illustrating the standard method for predicting
A. High affinitynfamilial erythrocytosis (.90 variants) the inheritance of abnormal hemoglobins. Each parent contributes one gene.
B. Low affinitynfamilial cyanosis (Hbs Kansas, Beth Israel,
Yoshizuka, Agenogi, Titusville, Providence)
Figure 27-2 illustrates the inheritance of abnormal hemoglo-
IV. M hemoglobinsnfamilial cyanosis (9 variants): Hb M-Boston,
bins involving mutations in the b gene.
Hb M-Iwate, Hb Auckland, Hb Chile, Hb M-Saskatoon, Hb M-
Milwaukee-1, Hb M-Milwaukee-2 (Hyde Park), Hb FM-Osaka, Hb
Prevalence
FM-Fort Ripley
The highest frequency of the sickle cell gene is found in sub-
V. Structural variants that result in a thalassemic phenotype
Saharan Africa, where each year approximately 230,000 babies
A. b-Thalassemia phenotype
are born with sickle cell disease (Hb SS), representing 0.74% of
1. Hb Lepore (db fusion)
all live births occurring in this area.11 In contrast, approximately
2. Hb E
2600 babies are born annually with sickle cell disease in North
3. Hb-Indianapolis, Hb-Showa-Yakushiji, Hb-Geneva
America and 1300 in Europe.11 Globally, the sickle cell gene
B. a-Thalassemia phenotype chain termination mutants (e.g., Hb
occurs at the highest frequency in five geographic areas: sub-
Constant Spring)
Saharan Africa, Arab-India, the Americas, Eurasia, and Southeast
Modified from Lukens JN: Abnormal hemoglobins: general principles (chap 39); Asia. In 2010, these five geographic areas accounted for 64.4%,
Wong WC: Sickle cell anemia and other sickling syndromes (chap 40); Lukens JN: 22.7%, 7.4%, 5.4%, and 0.1%, respectively, of all neonates born
Unstable hemoglobin disease (chap 41). In Greer JP, Foerster J, Lukens JN, et al, globally with sickle cell trait, and 75.5%, 16.9%, 4.6%, 3.0%,
editors: Wintrobe’s clinical hematology, ed 11, Philadelphia, 2004, Lippincott and 0%, respectively, of all neonates born globally with sickle
Williams & Wilkins. Updated from Patrinos GP, Giardine B, Riemer C, et al: Im-
cell disease. Three countries accounted for approximately 50%
provements in the HbVar database of human hemoglobin variants and thalasse-
mia mutations for population and sequence variation studies, Nucl Acids Res of neonates with SS and AS genotypes: Nigeria, India, and DR
32(database issue):D537-541, 2004. Available at: http://globin.cse.psu.edu/ Congo.12 Although in the United States, SCD is found mostly in
hbvar/menu.html. Accessed November 30, 2013. individuals of African descent, it also has been found in indi-
viduals from the Middle East, India, and the Mediterranean area
(Figure 27-3). SCD can also be found in individuals from the
Caribbean and Central and South America.13 The sickle cell mu-
Inheritance Pattern tation is becoming more prominent in southern India, particu-
As stated earlier, the genes that code for the globin chains are larly in certain tribes.14 It is estimated that 25,000 babies are
located at specific loci on chromosomes 16 and 11. The a-like born annually with sickle cell anemia in India.2
genes (a and z) are located on the short arm of chromosome
16, whereas the b-like genes (b, g, d, and e) are located on the Etiology and Pathophysiology
short arm of chromosome 11. With the exception of the Hb S is defined by the structural formula a2b26GlunVal, which
g genes, which have four loci, each b-like gene has two loci. indicates that on the b chain at position 6, glutamic acid is
b-hemoglobin variants are inherited as autosomal codominants, replaced by valine. The mutation occurs in nucleotide 17,
with one gene inherited from each parent.4 where thymine is changed to adenine, resulting in a change in
Patients with SCD (Hb SS), Hb SC, or Hb S–b-thal have codon 6 and the substitution of valine for glutamic acid at
inherited a sickle (S) gene from one parent and an S, C, or amino acid position 6.11 Glutamic acid has a net charge of
b-thalassemia gene from the other. Among patients with SCD, (21), whereas valine has a net charge of (0). This amino acid
individuals who are homozygotes (Hb SS) have more severe substitution produces a change in charge of (11), which af-
disease than individuals who are compound heterozygotes for fects the electrophoretic mobility of the hemoglobin molecule.
Hb S (Hb SC or Hb S–b-thal). Heterozygotes (Hb AS) are This amino acid substitution also affects the way the hemoglo-
generally asymptomatic. Using Hb S and Hb C as examples, bin molecules interact with one another within the erythrocyte
432 PART IV  Erythrocyte Disorders

Thalassemia

Sickle cell anemia

Hb C

Hb D

Hb E

Figure 27-3  ​Geographic distribution of common inherited structural hemoglobin variants and the thalassemias. (From Hoffbrand AV, Pettit JE: Essential
haematology, ed 3, Oxford, 1993, Blackwell Scientific.)

cytosol. The nonpolar (hydrophobic) valine amino acid has and sickle cell formation slow blood flow. In addition to a
been placed in the position that the polar glutamic acid once decrease in oxygen tension, there is a reduction in the pH and
held. Because glutamic acid is polar, the b chain folds in such an increase in 2,3-bisphosphoglycerate. Reduced blood flow
a way that glutamic acid extends outward from the surface of prolongs the exposure of Hb S-containing erythrocytes to a
the hemoglobin tetramer to bind water and contribute to he- hypoxic environment, and the lower tissue pH decreases the
moglobin solubility in the cytosol. Therefore, the hydrophobic oxygen affinity, which further promotes sickling. The end result
valine is also extended outward, but instead of binding water, is occlusion of capillaries and arterioles by sickled RBCs and
it seeks a hydrophobic niche with which to bind. When Hb S infarction of surrounding tissue.
is fully oxygenated, the quaternary structure of the molecule Sickle cells occur in two forms: reversible sickle cells
does not produce a hydrophobic pocket for valine to bind to, and irreversible sickle cells.20 Reversible sickle cells are Hb
which allows the hemoglobin molecules to remain soluble in S–containing erythrocytes that change shape in response to oxy-
the erythrocyte cytosol like Hb A and maintains the normal gen tension. Reversible sickle cells circulate as normal biconcave
biconcave disc shape of the RBCs. However, the natural alloste- discs when fully oxygenated but undergo hemoglobin polym-
ric change that occurs upon deoxygenation creates a hydropho- erization, show increased viscosity, and change shape on de-
bic pocket in the area of phenylalanine 85 and leucine 88, oxygenation. The vasoocclusive complications of SCD are
which allows the valine from an adjacent hemoglobin mole- thought to be due to reversible sickle cells that are able to travel
cule to bind. This hemoglobin pairing creates an orientation into the microvasculature in the biconcave disk conformation
that helps other hemoglobin molecules to form electrostatic due to their normal rheologic properties when oxygenated and
bonds between amino acids and becomes the seed for polymer then become distorted and viscous as they become deoxygen-
formation. Other hemoglobin pairs polymerize, forming a ated, converting to the sickle cell configuration in the vessel.
hemoglobin core composed of four hemoglobin molecules In contrast, irreversible sickle cells do not change their
that elongate in a helical formation. An outer layer of 10 hemo- shape regardless of the change in oxygen tension or degree of
globin molecules forms around the 4-hemoglobin-molecule hemoglobin polymerization. These cells are seen on the pe-
core, creating the long, slender Hb S polymer.15-18 Hb S mole- ripheral blood film as elongated sickle cells with a point at
cules within the RBCs become less soluble, forming tactoids or each end. It is thought that irreversible sickle cells are recog-
liquid crystals of Hb S polymers that grow in length beyond the nized as abnormal by the spleen and removed from circula-
diameter of the RBC, causing sickling. In homozygotes, the tion, which prevents them from entering the microcirculation
sickling process begins when oxygen saturation decreases to and causing vasoocclusion.
less than 85%. In heterozygotes, sickling does not occur unless Not only the oxygen tension but also the level of intracellular
the oxygen saturation of hemoglobin is reduced to less than hydration affects the sickling process. When RBCs containing
40%.19 The blood becomes more viscous when polymers are Hb S are exposed to a low oxygen tension, hemoglobin polym-
formed and sickle cells are created.19 Increased blood viscosity erization occurs. Polymerized deoxyhemoglobin S activates a
 CHAPTER 27  Hemoglobinopathies (Structural Defects in Hemoglobin) 433

membrane channel called Psickle that is otherwise inactive in nor- membrane phospholipids is accomplished by adenosine
mal RBCs. These membrane channels open when the blood triphosphate–dependent enzymes called translocases or flippases.
partial pressure of oxygen decreases to less than 50 mm Hg. Inhibition of flippases and activation of an enzyme called
Open Psickle channels allow the influx of Ca21, raising the intra- scramblase cause a more random distribution of membrane
cellular calcium levels and activating a second membrane chan- phospholipids, which increases the number of choline phos-
nel called the Gardos channel. An activated Gardos channel pholipids on the interior half of the membrane and the number
causes the efflux of K1, which stimulates the efflux of Cl– of aminophospholipids on the exterior membrane surface. The
through another membrane channel to maintain charge equilib- sickle cells of homozygotes (Hb SS) express 2.1% PS on eryth-
rium across the RBC membrane. The efflux of these ions leads to rocyte exterior surfaces compared with 0.2% for normal Hb AA
water efflux and intracellular dehydration, effectively increasing controls.21,22 It is hypothesized that Hb S polymerization may
the intracellular concentration of Hb S and intensifying polym- produce microparticles and iron complexes that adhere to the
erization. Another contributor to K1 and Cl– efflux and the resul- RBC membrane and generate reactive oxygen species, which,
tant dehydration is the K1/Cl– cotransporter system. Ironically, along with increased intracellular calcium or protein kinase C
this system is activated by dehydration and positively charged activation, may contribute to flippase inhibition and scram-
hemoglobins such as Hb S and Hb C. The K1/Cl– cotransporter blase activation.23,24 PS on the exterior surface of RBCs binds
pathway is also activated by the low pH encountered in the spleen thrombospondin on vascular endothelial cells,25 enhancing
and kidneys. One potential explanation for the altered function of adherence between RBCs and the vessel wall and contributing
the membrane channels is oxidative damage triggered by Hb S to vasoocclusive crisis, activation of coagulation, and decreased
polymerization. Injury to the RBC membrane induces adherence RBC survival.26,27 In addition, RBCs with PS on the external
to endothelial surfaces, which causes RBC aggregation, produces membrane surface are vulnerable to hydrolysis by secretory
ischemia, and exacerbates Hb S polymerization.10 phospholipase A2 (sPLA2), which generates lysophospholipids
Another important factor in the pathophysiology of SCD and fatty acids like lysophosphatidic acid. This results in vascu-
involves the redistribution of phospholipids in the RBC mem- lar damage that contributes to acute chest syndrome.28,29
brane, which contributes to hemolysis, vasoocclusive crisis,
stroke, and acute chest syndrome. In the bilayer membranes of Clinical Features
normal RBCs, choline phospholipids like sphingomyelin and The clinical manifestations of SCD can vary from no symptoms
phosphatidylcholine are located on the outer plasma layer, to a potentially lethal state. Symptoms also vary between eth-
whereas aminophospholipids like phosphatidylserine (PS) and nic groups with Indian patients expressing a much milder dis-
phosphatidylethanolamine are primarily on the inner cytoplas- ease than their African counterparts.14 People with SCD can
mic layer of the membrane. This asymmetrical distribution of develop a variety of symptoms as listed in Box 27-3. Over a

BOX 27-3  ​Clinical Features of Sickle Cell Disease

I. Vasoocclusion 5. Penis:
A. Causes: Priapism
Acidosis 6. Eyes:
Hypoxia Retinal hemorrhage
Dehydration 7. Central nervous system
Infection 8. Urinary tract:
Fever Renal papillary necrosis
Extreme cold 9. Leg ulcers
B. Clinical manifestations II. Bacterial infections
1. Bones: A. Sepsis
Pain B. Pneumonia
Hand-foot dactylitis C. Osteomyelitis
Infection (osteomyelitis) III. Hematologic defects
2. Lungs: A. Chronic hemolytic anemia
Pneumonia B. Megaloblastic episodes
Acute chest syndrome C. Aplastic episodes
3. Liver: IV. Cardiac defects
Hepatomegaly A. Enlarged heart
Jaundice B. Heart murmurs
4. Spleen: V. Other clinical features
Sequestration splenomegaly A. Stunted growth
Autosplenectomy B. High-risk pregnancy
434 PART IV  Erythrocyte Disorders

thousand hemoglobin variants are known; however, only eight INFECTION/INFLAMMATION C-reactive protein

genotypes cause severe disease: Hb SS, Hb S–b0-thal, severe Hb Tissue factor expression
S–b1-thal, Hb SD-Punjab, Hb SO-Arab, Hb SC-Harlem, Hb CS- Release of biologic modifiers

Antilles, and Hb S-Quebec-CHORI. These eight clinically sig- COAGULATION ACTIVATION

nificant forms are listed in the order of severity and can have WBC ACTIVATION
Thrombin generation
high morbidity and mortality rates. Three additional genotypes ENDOTHELIAL MODULATION
produce moderate disease: Hb SC, moderate Hb S-b1-thal, and PLATELET ACTIVATION
Hb AS-Oman. Three produce mild disease: mild Hb S-bsilent-
thal, Hb SE, and Hb SA-Jamaica Plain. Two produce very mild RBC ADHESION TO ENDOTHELIUM Thrombospondin release

disease: Hb S-HPFH and Hb S with a variety of mild variants.11

Symptom variability in patients with sickle cell disease and VASCULAR OBSTRUCTION
von Willebrand factor release
across the genotypes listed above are largely due to the intracel-
lular ratio of Hb S to Hb F, as well as factors that affect vessel POOR RBC DEFORMABILITY
Altered vascular tone
tone and cellular activation.30 Individuals affected with SCD Acidosis
CLINICAL DEHYDRATION
are characteristically symptom free until the second half of the
first year of life because of the protective effect of Hb F.31 To- RBC dehydration RBC SICKLING HYPOXIA
ward the end of the first 6 months of life, mutated b chains
begin to be produced and gradually replace normal g chains, Delayed blood flow
which causes Hb S levels to increase and Hb F levels to de- Figure 27-4  ​Numerous risk factors for vasoocclusion are highly interre-
crease. Erythrocytes containing Hb S become susceptible to lated physiologically, as shown here. RBC, Red blood cell; WBC, white blood
hemolysis, and a progressive hemolytic anemia and spleno- cell.  (From Embury SH, Hebbel RP, Mohandas N, et al: Sickle cell disease:
megaly may become evident. basic principles and clinical practice, Philadelphia, 1994, Lippincott Williams
Many individuals with SCD undergo episodes of recurring & Wilkins, p. 322.)
pain termed crises. Sickle cell crises were described by Diggs as
“any new syndrome that develops rapidly in patients with
SCD owing to the inherited abnormality.”32 The pathogenesis the patient organ by organ, through the destructive and debili-
of the acute painful episode first described by Diggs is not tative effects of cumulative infarcts. Approximately 8% to 10%
fully understood. Various crises may occur: vasoocclusive or of SCD patients develop cutaneous manifestations in the form
“painful,” aplastic, megaloblastic, sequestration, and chronic of ulcers or sores on the lower leg.11
hemolytic. The abnormal interaction between sickle cells and vascular
The hallmark of SCD is vasoocclusive crisis (VOC), which endothelium seems to have a great impact on the vasoocclusive
accounts for most hospital and emergency department visits. event. Endothelial adherence correlates significantly with the
This acute, painful aspect of SCD occurs with great predictabil- severity of painful episodes. In addition, sickle cell adherence
ity and severity in many individuals and can be triggered by to vascular endothelium results in intimal hyperplasia that can
acidosis, hypoxia, dehydration, infection and fever, and expo- slow blood flow.36 Cells of patients with Hb SC disease pro-
sure to extreme cold. Painful episodes manifest most often in duce less sickling with fewer adherent RBCs.8,37
the bones, lungs, liver, spleen, penis, eyes, central nervous sys- The frequency of painful episodes varies from none to six
tem, and urinary tract. per year.8 On average, each episode persists for 4 to 5 days,
The pathogenesis of vasoocclusion in SCD is not fully un- although protracted episodes may last for weeks. Repeated
derstood, but Hb S polymerization and sickling of RBCs play a splenic infarcts produce scarring resulting in diminished
major role, with other factors also affecting this process. Most splenic tissue and abnormal function. Splenic sequestration
VOC events occur in capillaries and postcapillary venules.6,33 is characterized by a sudden trapping of blood in the spleen,
The list of possible risk factors includes polymerization, de- which leads to a rapid decline in hemoglobin, often to less
creased deformability, sickle cell–endothelial cell adherence, than 6 g/dL.37 This phenomenon occurs most often in in-
endothelial cell activation, white blood cell (WBC) and plate- fants and young children whose spleens are chronically en-
let activation, hemostatic activation, and altered vascular larged. Children experiencing splenic sequestration episodes
tone.33 The interrelationships among these risk factors is shown may have earlier onset of splenomegaly and a lower level of
in Figure 27-4. Vasoocclusion can be triggered by any of these Hb F at 6 months of age.8 Crises are often associated with
factors under various circumstances. During inflammation, respiratory tract infections. Gradual loss of splenic function
increased WBCs interacting with endothelium, platelet activa- is referred to as autosplenectomy and is evidenced by the pres-
tion causing elevation of thrombospondin level, or clinical ence of Howell-Jolly and Pappenheimer bodies in RBCs on
dehydration resulting in an increase in von Willebrand factor the peripheral blood film. In the lungs, pulmonary infarction
can trigger RBC adherence to endothelium, precipitating vascu- from sickling in the microvasculature causes acute chest
lar obstruction. Another mechanism of obstruction can be syndrome.
dense cells, which are less deformable and are at greatest risk Acute chest syndrome is characterized by fever, chest pain, and
for intracellular polymerization because of their higher Hb S presence of pulmonary infiltrates on the chest radiograph and is
concentration.34,35 Vasoocclusive episodes gradually consume the leading cause of death among adults with SCD. Over 10% of
 CHAPTER 27  Hemoglobinopathies (Structural Defects in Hemoglobin) 435

adults with acute chest syndrome die from complications linked patients.21 RBC hemolysis releases free hemoglobin, which
to chronic lung disease and pulmonary hypertension.38 In chil- disrupts the arginine-nitric oxide pathway, resulting in the
dren, acute chest syndrome generally is precipitated by infection sequestration and lowering of nitric oxide.31,45 Decreased
characterized by fever, cough, and tachypnea. Acute chest syn- NO leads to endothelial cell activation, vasoconstriction, ad-
drome is also linked with sPLA2, discussed previously. The level herence of RBCs to the endothelium, and pulmonary hyper-
of sPLA2 has been shown to be a predictor of acute chest syn- tension previously discussed.45 Another major sequelae of
drome in patients with SCD39 in that sPLA2 rises 24 to 48 hours hemolysis is renal dysfunction, which can be detected early
before symptoms of acute chest syndrome begin.40 In addition, a by an increased glomerular filtration rate of 140 mL/min
high sPLA2 level correlates with the degree of lung damage. per 1.73 m3 found in 71% of patients with SCD.46 Progression
Pulmonary hypertension (PHT) is a serious and potentially of renal dysfunction can be identified by detecting microalbu-
fatal sequela of SCD. Among patients with SCD, PHT has a minuria (.4.5 mg/mmol), followed by proteinuria and termi-
prevalence of about 33%, with 10% of patients manifesting a nating in elevated BUN and creatinine levels. Angiotensin-
more severe version.41 The mortality rate for sickle cell patients converting enzyme inhibitors (ACEI) have been shown to
who develop PHT is 40% at 40 months.41 An association has lower proteinuria in SCD patients.33
been documented between the development of PHT and the Megaloblastic episodes result from the sudden arrest of
nitrous oxide (NO) pathway. NO is produced from the action erythropoiesis due to folate depletion. Folic acid deficiency as
of endothelial NO synthase (eNOS) on arginine, which causes a cause of exaggerated anemia in SCD is extremely rare in the
vasodilation. Patients with SCD have a decrease in NO, and United States. It is common practice to prescribe prophylactic
this leads to vasoconstriction and hypertension.4,41 In addition, folic acid for patients with SCD, however.8
low NO levels in the blood fail to inhibit endothelin-1, a Aplastic episodes (bone marrow failure) are the most
potent vasoconstrictor, which results in additional vasocon- common life-threatening hematologic complications and are
striction and hypertension.38 The connection between NO and usually associated with infection, particularly parvovirus in-
SCD involves the hemolytic crisis. Erythrocyte hemolysis re- fection.34 Aplastic episodes present clinical problems similar
leases high levels of arginase, which degrades arginine; the re- to those seen with other hemolytic disorders.47 Sickle cell
sult is less NO production from eNOS.42,43 In addition, the free patients usually can compensate for the decrease in RBC
hemoglobin released from hemolyzed RBCs scavenges NO, survival by increasing bone marrow output. When the bone
which further reduces the levels and exacerbates the vasocon- marrow is suppressed temporarily by bacterial or viral infec-
striction and hypertension.41 Blood arginine and NO levels tions, however, the hematocrit decreases substantially with
drop a few days before the onset of acute chest syndrome,37 a no reticulocyte compensation. The spontaneous recovery
finding suggesting that the NO pathway is a connection be- phase is characterized by the presence of nucleated RBCs and
tween SCD, PHT, and asthma.38,44 Treatment with large doses an increase in the number of reticulocytes in the peripheral
of arginine reduces pulmonary artery pressure, but the effect is blood. Most aplastic episodes are short-lived and require no
not sustainable and does not reduce mortality. An increased therapy. If anemia is severe and the bone marrow remains
tricuspid regurgitation velocity (TRV) and blood NT-ProBNP aplastic, transfusions become necessary. If patients are not
levels above 160 ng/L were found to be good predictors of transfused in a timely fashion, death can occur.47
pulmonary hypertension and are associated with a higher mor- Patients also experience cardiac defects, including enlarged
tality rate.33 Bosentan is the treatment of choice for pulmonary heart and heart murmurs. In patients with severe anemia, car-
hypertension, but liver enzymes should be monitored for liver diomegaly can develop as the heart works harder to maintain
toxicity.33 adequate blood flow and tissue oxygenation. Increased cardiac
Bacterial infections pose a major problem for SCD patients. workload along with increased bone marrow erythropoiesis
These patients have increased susceptibility to life-threatening increases calorie burning, contributing to a reduced growth
infection from Staphylococcus aureus, Streptococcus pneumoniae, rate.48 When patients enter childbearing age, pregnancy be-
and Haemophilus influenzae. Acute infections are common comes risky.4
causes of hospitalization and have been the most frequent Impaired blood supply to the head of the femur and hu-
cause of death, especially in the first 3 years of life.1 Bacterial merus results in a condition called avascular necrosis (AVN).
infections of the blood (septicemia) are exacerbated by the About 50% of patients with SCD develop AVN by 35 years of
autosplenectomy effect as the spleen gradually loses its ability age.49 Physical therapy and surgery to relieve intramedullary
to function as a secondary lymphoid tissue to effectively clear pressure within the head of the long bones are effective, but
organisms from the blood. hip and/or shoulder implants become necessary in most pa-
Chronic hemolytic anemia is characterized by shortened tients experiencing AVN.49 Similarly, leg ulcers are a common
RBC survival of between 16 and 20 days,45 with a correspond- complication of SCD. Ulcers tend to heal slowly, develop un-
ing decrease in hemoglobin and hematocrit, an elevated re- stable scars, and recur at the same site, becoming a chronic
ticulocyte count, and jaundice. Continuous screening and re- problem, with associated chronic pain.47
moval of sickle cells by the spleen perpetuate the chronic Microstrokes can lead to headaches, poor school perfor-
hemolytic anemia and autosplenectomy effect. Because other mance, reduced intelligence quotient (IQ), and overt central
conditions, such as hepatitis and gallstones, may cause jaun- nervous system dysfunction. A neurologic examination followed
dice, chronic hemolysis is difficult to diagnose in sickle cell by magnetic resonance imaging and, if available, transcranial
436 PART IV  Erythrocyte Disorders

Doppler ultrasonography or magnetic resonance angiography is Laboratory Diagnosis

recommended to detect microstrokes.48 The anemia of SCD is a chronic hemolytic anemia, classified mor-
phologically as normocytic, normochromic. The characteristic
Incidence with Malaria and Glucose-6-Phosphate diagnostic cell observed on a Wright-stained peripheral blood
Dehydrogenase Deficiency film is a long, curved cell with a point at each end (Figure 27-5).
The sickle gene occurs with greatest frequency in Central Africa, Because of its appearance, the cell was named a sickle cell.32 The
the Near East, the region around the Mediterranean, and parts peripheral blood film shows marked poikilocytosis and aniso-
of India. The frequency of the gene parallels the incidence of P. cytosis with normal RBCs, sickle cells, target cells, nucleated
falciparum and seems to offer some protection against cerebral RBCs along with a few spherocytes, basophilic stippling, Pap-
falciparum malaria in young patients. Malarial parasites are penheimer bodies, and Howell-Jolly bodies. The presence of
living organisms within the RBCs that use the oxygen within sickle cells and target cells is the hallmark of SCD. There is
the cells. This reduced oxygen tension causes the cells to sickle, moderate to marked polychromasia with a reticulocyte count
which results in injury to the cells. These injured cells tend to between 10% and 25%, corresponding with the hemolytic state
become trapped within the blood vessels of the spleen and and the resultant bone marrow response. The RBC distribution
other organs, where they are easily phagocytized by scavenger width (RDW) is increased owing to moderate anisocytosis. The
WBCs. Selective destruction of RBCs containing parasites de- mean cell volume (MCV) is not as elevated as one would ex-
creases the number of malarial organisms and increases the pect, however, given the elevated reticulocyte count. An aplastic
time for immunity to develop. One explanation for this phe- crisis can be heralded by a decreased reticulocyte count. Moder-
nomenon is that the infected cell is uniquely sickled and de- ate leukocytosis is usually present (sometimes 40 to 50 3 109
stroyed, probably in an area of the spleen or liver, where WBC/L) with neutrophilia and a mild shift toward immature
phagocytic cells are plentiful, and the oxygen tension is signifi- granulocytes. The leukocyte alkaline phosphatase score is not
cantly decreased.50 elevated when neutrophilia is caused by sickle cell crisis alone
Because of the high incidence of glucose-6-phosphate when no underlying infection is present. Thrombocytosis is
dehydrogenase (G6PD) deficiency in patients with SCD, it usually present. The bone marrow shows erythroid hyperplasia,
has been suggested that G6PD deficiency has a protective reflecting an attempt to compensate for the anemia, which re-
effect in these patients,51 although this correlation has not sults in polychromasia and an increase in reticulocytes and
been confirmed through studies. It also has been postulated nucleated RBCs in the peripheral blood. Levels of immuno-
that hemolytic episodes are more common in these patients. globulins, particularly immunoglobulin A, are elevated in all
In the first 42 months of life, patients with SCD and G6PD forms of SCD. Serum ferritin levels are normal in young pa-
deficiency had lower steady-state hemoglobin levels, higher tients but tend to be elevated later in life; however, hemochro-
reticulocyte counts, three times more acute anemia events, matosis is rare. Chronic hemolysis is evidenced by elevated
and more frequent blood transfusions—vasoocclusive levels of indirect and total bilirubin with the accompanying
and infectious events than matched sickle cell patients with- jaundice.
out G6PD deficiency.30 Because of the presence of young The diagnosis of SCD is generally a two-step process by first
cells rich in G6PD, however, the increased hemolysis is demonstrating the insolubility of deoxygenated Hb S in solu-
more likely caused by the enzyme abnormality when the tion followed by confirmation of its presence using hemoglo-
population is shifted to the oldest cells during an aplastic bin electrophoresis, high-performance liquid chromatography
crisis.52 (HPLC), or capillary electrophoresis. For more complicated

A B
Figure 27-5  ​A, Peripheral blood film for a patient with sickle cell disease (SCD) showing anisocytosis, polychromasia, three sickle cells, target cells, and
normal platelets (31000). B, Peripheral blood film for an SCD patient showing anisocytosis, poikilocytosis, sickle cells, target cells, and one nucleated RBC
(31000). Platelets are not present in this field, but their numbers were adequate in this patient. (Courtesy Ann Bell, University of Tennessee, Memphis.)
 CHAPTER 27  Hemoglobinopathies (Structural Defects in Hemoglobin) 437

cases, isoelectric focusing, tandem mass spectrometry, or DNA unrelated substitution. Hb S-Antilles is particularly impor-
analysis may be needed. An older screening test detects Hb S tant because it can cause sickling in the heterozygous state.
insolubility by inducing sickle cell formation on a glass slide. Alkaline hemoglobin electrophoresis is a common first step
A drop of blood is mixed with a drop of 2% sodium metabi- in the confirmation of hemoglobinopathies, including SCD.
sulfite (a reducing agent) on a slide, and the mixture is sealed Electrophoresis is based on the separation of hemoglobin mol-
under a coverslip. The hemoglobin inside the RBCs is reduced ecules in an electric field due primarily to differences in total
to the deoxygenated form; this induces polymerization and molecular charge. In alkaline electrophoresis, hemoglobin
the resultant sickle cell formation, which can be identified molecules assume a negative charge and migrate toward the
microscopically. This method is slow and cumbersome and is anode (positive pole). Historically, alkaline hemoglobin elec-
rarely used. trophoresis was performed on cellulose acetate medium but is
The most common screening test for Hb S, called the being replaced by electrophoresis on agarose medium. None-
hemoglobin solubility test, capitalizes on the decreased theless, because some hemoglobins have the same charge and,
solubility of deoxygenated Hb S in solution, producing tur- therefore, the same electrophoretic mobility patterns, hemo-
bidity. Blood is added to a buffered salt solution containing globins that exhibit an abnormal electrophoretic pattern at an
a reducing agent, such as sodium hydrosulfite (dithionite), alkaline pH may be subjected to electrophoresis at an acid pH
and a detergent-based lysing agent (saponin). The saponin for definitive separation. In an acid pH some hemoglobins as-
dissolves membrane lipids, causing the release of hemoglo- sume a negative charge and migrate toward the anode, while
bin from the RBCs, and the dithionite reduces the iron from others are positively charged and migrate toward the cathode
the ferrous to the ferric oxidation state. Ferric iron is unable (negative pole). For example, Hb S migrates with Hb D and Hb
to bind oxygen, converting the hemoglobin to the deoxy- G on alkaline electrophoresis but separates from Hb D and Hb
genated form. Deoxygenated Hb S polymerizes in solution, G on acid electrophoresis. Similarly, Hb C migrates with Hb E
which renders it turbid, whereas solutions containing non- and Hb O on alkaline electrophoresis but separates on acid
sickling hemoglobins remain clear (Figure 27-6). False- electrophoresis. Figure 27-7 shows electrophoretic patterns for
positive results for Hb S can occur with hyperlipidemia, a
few rare hemoglobinopathies, and when too much blood
is added to the test solution; false-negative results can occur
in infants less than 6 months of age and with low hemato-
crits. Other hemoglobins that give a positive result on
the solubility test include Hb C-Harlem (Georgetown), Hb
C-Ziguinchor, Hb S-Memphis, Hb S-Travis, Hb S-Antilles,
Hb S-Providence, Hb S-Oman, Hb Alexander, and Hb Porte-
Alegre.4,8 All of these hemoglobins have two amino acid
substitutions: the Hb S substitution (b6GlunVal) and another

Figure 27-7  ​Relative mobilities of normal and variant hemoglobins in

various conditions measured by electrophoresis on cellulose acetate at an
alkaline pH and citrate agar at an acid pH. The relative amount of hemoglobin
is not proportional to the size of the band; for example, in sickle cell trait (Hb
Figure 27-6  ​Tube solubility screening test for the presence of hemoglobin AS), the bands may appear equal, but the amount of Hb A exceeds that of Hb
S. In a negative test result (left), the solution is clear and the lines behind the S. (From Schmidt RM, Brosious EF: Basic laboratory methods of hemoglobin-
tube are visible. In a positive test result (right), the solution is turbid and the opathy detection, ed 6, HEW Pub No (CDC) 77-8266, Atlanta, 1976, Centers
lines are not visible. (Courtesy Ann Bell, University of Tennessee, Memphis.) for Disease Control and Prevention.)
438 PART IV  Erythrocyte Disorders

normal and abnormal hemoglobins. Figure 27-8 shows the 30% A S

electrophoretic separation of a normal adult and a patient with
sickle cell disease (Hb SS) at an alkaline pH. HPLC and capil-
lary electrophoresis are gaining in popularity because these
methods are more automated, the instruments are more user
friendly, and they can be used to confirm hemoglobin variants
20%
observed with electrophoresis (Figure 27-9).
HPLC separates hemoglobin types in a cation exchange
column and usually requires only one sample injection. Un-
like electrophoresis, HPLC can identify and quantitate low
levels of Hb A2 and Hb F, but comigration of Hb A2 and Hb E
occurs. Therefore, HPLC is best used in the diagnosis of thal- 10%
assemias rather than hemoglobinopathies because quantita- A2
tion of low levels of normal and abnormal hemoglobin levels
is necessary to distinguish thalassemias. HPLC is also com- F
monly used to quantitate Hb A1c levels to monitor diabetic
patients. 0
Capillary electrophoresis, like agarose electrophoresis, sepa- 0 1 2 3 4 5 6
rates hemoglobin types based on charge in an alkaline buffer
Time (min)
but does so using smaller volumes and produces better separa-
tion than traditional agarose electrophoresis. Semiautomated Figure 27-9  ​Ion-exchange high-performance liquid chromatography
systems like the Capillarys® system (Sebia, Evry, France) allow (HPLC) separation of hemoglobins (Hbs) in a patient with sickle cell trait
demonstrating Hbs F, A, A2, and an abnormal Hb in the S window (Bio-Rad
for the testing of up to eight samples in parallel with computer-
Variant Classic Hb Testing System, BioRad Laboratories, Philadelphia). 
ized analysis of results. Capillary electrophoresis is also eco-
(Modified from Elghetany MT, Banki K: Erythrocytic disorders. In McPherson
nomical, since each capillary can accommodate at least 3000 RA, Pincus MR: Henry’s Clinical Diagnosis and Management by Laboratory
runs.1 In 2009 hemoglobin electrophoresis in agarose medium Methods, ed 22, Philadelphia, 2011, Elsevier, p. 578.)
was still the most commonly used technique to identify hemo-
globin variants, but capillary electrophoresis and HPLC are
gaining in popularity.53
Isoelectric focusing (IEF) is a confirmatory technique
that is expensive and complex, requiring well-trained and
experienced laboratory personnel. The method uses an elec-
tric current to push the hemoglobin molecules across a pH
 gradient. The charge of the molecules change as they migrate
through the pH gradient until the hemoglobin species
reaches its isoelectric point (net charge of zero). With a net
A charge of zero, migration stops and the hemoglobin mole-
F cules accumulate at their isoelectric position. Molecules with
S isoelectric point differences of as little as 0.02 pH units can
be effectively separated.1
Neonatal screening requires a more sophisticated approach,
C often using three techniques: adapted IEF, HPLC, and reversed-
phase HPLC. This multisystem approach is needed to distin-
guish not only the multitude of hemoglobin variants but also
the numerous thalassemias. The more progressive laboratories
use a combination of two or more techniques to improve iden-
tification of hemoglobin variants. Some reference laboratories
may use mass spectroscopy, matrix-assisted laser desorption-
1 2 3 4 5 6 7  ionization time-of-flight (MALDI-TOF) mass spectrometry, or
isoelectric focusing to separate hemoglobin types, or nucleic
Figure 27-8  ​Electrophoretic separation of hemoglobins (Hb) at alkaline
acid identification of the genetic mutation.1,54
pH. 1, Normal adult; 2 and 3, 17-year-old patient with sickle cell anemia (Hb
Patients with Hb SS or Hb SC disease lack normal b-globin
SS); 5 and 6, patient with sickle cell anemia, recently transfused (note the
presence of Hb A from the transfused red blood cells); 4 and 7, Hbs A/F/S/C chains, so they have no Hb A. In Hb SS, the Hb S level is
standard (Hydragel 7 Hemoglobin/Hydrasys System, Sebia Electrophoresis, usually greater than 80%. The Hb F level is usually increased
Norcross, GA).  (Modified from Elghetany MT, Banki K: Erythrocytic disorders. (1% to 20%), and when Hb F constitutes more than 20%
In McPherson RA, Pincus MR: Henry’s Clinical Diagnosis and Management of hemoglobin, it has a tendency to modulate the severity
by Laboratory Methods, ed 22, Philadelphia, 2011, Elsevier, p. 578.) of the disease. This is especially true in newborns and in
 CHAPTER 27  Hemoglobinopathies (Structural Defects in Hemoglobin) 439

patients with hereditary persistence of fetal hemoglobin.31 Acute VOC attacks are often treated with morphine in the
The Hb A2 level is normal or slightly increased (2% to 5%), emergency department or when in transit. Blood exchange
and Hb A2 quantitation is useful in differentiating Hb SS transfusion (BET) is the treatment of choice for severe VOC
from Hb S–b0-thalassemia, in which Hb A2 is increased attacks and acute chest syndrome (ACS).33 Painful crises tend
(Chapter 28). Hb G, Hb D, and Hb S all migrate in the same to increase with age, and physicians must be aware of opiate
position on alkaline cellulose acetate and alkaline agarose tolerance and rebound pain following opiate therapy, called
electrophoresis, but Hb G and Hb D do not give a positive central sensitization. Repeated painful crises can result in hy-
result on the tube solubility test. persensitivity to repeated pain by increasing peripheral inflam-
The typical sequelae of SCD may be predicted, and the ef- mation, increased neurotransmitter release, increased calcium
fectiveness of treatment monitored, if reliable biomarkers of influx into postsynaptic junctions, and other pathways that
inflammation can be identified. Among the common indicators increase pain signals to the brain.45 This phenomenon can be
of inflammation, WBC is a good predictor of sickle cell events misinterpreted as drug intolerance, causing inappropriate dose
and mortality, whereas the erythrocyte sedimentation rate and escalation, or as drug-seeking behavior, causing inappropriate
C-reactive protein (CRP) level exhibit variability too great to termination of treatment. The most appropriate response to
reliably predict events. However, CRP and sPLA2 are both ele- opioid tolerance and central sensitization is a gradual dose re-
vated during vasoocclusive crisis and acute chest syndrome. duction to reset the pain receptors followed by switching to
Other markers such as interleukin-6 (IL-6), IL-10, and protein S opioids such as methadone and buprenorphine that are less
are showing promise as useful indicators in clinical practice.55 sensitive to this phenomenon.45 The patient should be exam-
Annexin A5, a protein bound to lipids in the plasma membrane ined on a regular basis, and routine testing should be done to
of endothelial cells and platelets, has been shown to elevate establish baseline values for the patient during nonsickling
before and during VOC.56 Lipid damage from oxidative stress periods.
can be predicted by plasma elevations of malondialdehyde Children younger than 3 years often experience hand-­­
(MDA) and depleted a-tocopherol. In addition, a-tocopherol foot syndrome, characterized by pain and swelling in the
rises with CRP during bouts of inflammation. Of all the bio- hands and feet.33 Treatment usually consists of increasing
markers evaluated, IL-6, IL-10, vascular cell adhesion molecule intake of fluids and giving analgesics for pain.
1 (VCAM-1), and sPLA2 are the most promising at predicting Pneumococcal disease has been a leading cause of mor-
impending crisis.57 bidity and mortality in children, especially children younger
than 6 years. With immunization and prophylactic antibi-
Treatment otics, however, this is now a preventable complication.7
Supportive care has been the mainstay of therapy for SCD. Immunization with heptavalent conjugated pneumococcal
New therapies have evolved, however, that are actually modi- vaccine is recommended at 2, 4, and 6 months of age. The
fying the genetic pathogenesis of the disease. Neonatal screen- 23-valent pneumococcal vaccine is recommended at 2 years,
ing, childhood prophylactic penicillin therapy, bone marrow with a booster at the age of 5 years. Standard childhood
transplantation, and treatment with hydroxyurea (hydroxy- vaccinations should be given as scheduled. In addition,
carbamide) in adults may extend the life of the SCD patient annual administration of influenza vaccine is recommended
further. beginning at 6 months of age.42 The risk of bacterial infec-
The main components of supportive therapy include ade- tion probably increases in mature patients with Hb SC
quate hydration, prophylactic vitamin therapy, avoidance of disease and homozygous SCD.20
low-oxygen environments, analgesia for pain, and aggressive Transfusions can be used to prevent the complications of
antibiotic therapy with the first signs of infection. Hydration SCD. More specifically, periodic transfusions, given at a fre-
maintains good blood flow and reduces vasoocclusive crises. quency of eight or more per year, are effective at preventing
Prophylactic oral penicillin V at a dose of 125 mg twice per day stroke, symptomatic anemia, brain injury, priapism, leg ulcers,
by the age of 3 months to 3 years of age is recommended to PHT, delayed pubescence, splenomegaly, and chronic pain and
avoid infection and the associated morbidity and mortality. improving school attendance, IQ, energy, exercise tolerance,
The penicillin dosage is increased to 250 mg twice per day mood, and sense of well-being. In other circumstances, such as
from 3 to 5 years of age.58 When infections occur, prompt an- central nervous system infarction, hypoxia with infection,
tibiotic treatment reduces the associated morbidity and mor- stroke, episodes of acute chest syndrome, and preparation for
tality.9 Avoidance of strenuous exercise, high altitudes, and surgery, transfusions are used to decrease blood viscosity and
unpressurized air travel maintains high oxygen tensions and the percentage of circulating sickle cells. Before all but simple
reduces the sickling phenomenon. Treatment for painful epi- surgeries, Hb SS patients are transfused with normal Hb AA
sodes includes ensuring optimal hydration, rapidly treating blood to bring the volume of Hb S to less than 50% or to
associated infection, oxygen therapy, and effectively relieving achieve a hemoglobin of 10 g/dL in an effort to prevent com-
pain. Analgesics are the foundation of pain management, with plications in surgery.58,60 Maintenance transfusions should be
nonsteroidal antiinflammatory drugs like paracetamol (ace­ given in pregnancy if the mother experiences vasoocclusive or
taminophen) and nefopam administered to manage mild is­ anemia-related problems or if there are signs of fetal distress
chemic attacks. Opioids like meperidine (pethidine or demerol) or poor growth.20 Nonetheless, transfusion therapy has the
or tramadol are recommended when pain becomes chronic.59 potential to cause transfusion reactions, transfusion-related
440 PART IV  Erythrocyte Disorders

infections, and iron overload. Of the three, iron overload is the Prevention of intracellular RBC dehydration reduces intra-
most frequent. cellular HbS polymerization thus reducing VOC. The uses of
Iron overload has been associated with endocrine dysfunc- senicapoc to inhibit Gardos channels and Mg11 to modulate
tion61 and cardiac disease.62 Deferoxamine has been effective K1-Cl– transport systems show increased hemoglobin levels
in treating iron overload by chelating and removing much of and decreased numbers of dense RBCs, resulting in reduced
the excess iron from the body. Deferoxamine must be admin- hemolysis but no clear reduction in VOC.76-80
istered intravenously, however, and treatment requires at least
8 hours each day for a week. An oral iron chelator, defera- Course and Prognosis
sirox, was approved by the Food and Drug Administration in Proper management of SCD has increased the life expectancy
2005.63 Deferasirox is consumed in the morning as a slurry of patients from 14 years in 1973 to the current average life
by dissolving several pills, but its effectiveness is yet to be span of 50 years.81 For men and women who are compound
determined. heterozygotes for Hb SC, the average life span is 60 and
Bone marrow or hematopoietic stem cell transplantation 68 years, respectively, with a few patients living into their
has proved successful for some individuals, but few patients seventies.30,82 Individuals with Hb SS can pursue a wide range
qualify due to the lack of HLA-matched, related donors.64 The of vocations and professions. They are discouraged, however,
event-free survival rates for patients receiving transplants from from jobs that require strenuous physical exertion or exposure
HLA-identical related donors are between 80% and 90% for to high altitudes or extreme environmental temperature
SCD.65-68 Patients chosen for transplantation are generally variations.
children younger than age 17 with severe complications of Newborn screening for hemoglobinopathies has signifi-
SCD (i.e., stroke, acute chest syndrome, and refractory pain). cantly reduced mortality in children with SCD by enabling
In addition, morbidity and mortality following transplanta- prompt and comprehensive medical care. The most common
tion increase with age, which places another restriction on form of screening is HPLC followed by confirmation using
transplantation therapy.69 There is evidence that transplanta- hemoglobin electrophoresis and genotyping methods.83
tion restores some splenic function, but its effect on estab-
lished organ damage is unknown.70 Transplantation of cord Sickle Cell Trait
blood stem cells from HLA-identical related and unrelated The term sickle cell trait refers to the heterozygous state (Hb AS)
donors is associated with a disease-free survival rate of 90%.71 and describes a benign condition that generally does not affect
The primary benefit of using cord blood as a source of stem mortality or morbidity except under conditions of extreme ex-
cells is that banking of cord blood increases the number of ertion. The trait occurs in approximately 8% of African Ameri-
units available to achieve an HLA match.71 Some researchers cans. It also can be found in Central Americans, Asians, and
are now focusing on the use of in utero stem cell transplanta- people from the region around the Mediterranean.1
tion to produce engraftment while the immune system of the Individuals with sickle cell trait are generally asymptomatic
fetus is prone to HLA tolerance. Others are attempting to and present with no significant clinical or hematologic mani-
genetically alter fetal hematopoietic stems cells to overcome festations. Under extremely hypoxic conditions, however, sys-
HLA mismatches.72 temic sickling and vascular occlusion with pooling of sickled
Hydroxycarbamide (hydroxyurea) therapy has offered some cells in the spleen, focal necrosis in the brain, rhabdomyolysis,
promise in relieving the sickling disorder by increasing the and even death can occur. In circumstances such as severe re-
proportion of Hb F in the erythrocytes of individuals with spiratory infection, unpressurized flight at high altitudes, and
SCD.73 Hydroxyurea, given at 25 to 30 mg/kg, has been shown anesthesia in which pH and oxygen levels are sufficiently low-
to reduce symptoms and prolong life, in part by increasing Hb ered to cause sickling, patients may develop splenic infarcts.8
F levels. Daily dosing produces a better HbF response com- Failure to concentrate urine is the only consistent abnormality
pared to sequential weekly dosing.74 Because Hb F does not found in patients with sickle cell trait.84 This abnormality is
copolymerize with Hb S, if the production of Hb F can be suf- caused by diminished perfusion of the vasa recta of the kidney,
ficiently augmented, the complications of SCD might be which impairs concentration of urine by the renal tubules.
avoided. The severity of the disease expression and the number Renal papillary necrosis with hematuria has been described in
of irreversible sickle cells are inversely proportional to the ex- some patients.8
tent to which Hb F synthesis persists. Individuals in whom Hb Although much controversy exists as to the potential con-
F levels stabilize at 12% to 20% of total hemoglobin may have nection between strenuous exercise and severe to fatal ad-
little or no anemia and few, if any, vasoocclusive attacks. Levels verse events in patients with sickle cell trait, at least 46 cases
of 4% to 5% Hb F may modulate the disease, and levels of 5% have been documented in the literature (39 military recruits
to 12% may suppress the severity of hemolysis and lessen the and 7 athletes).85 The causes of these deaths were largely due
frequency of severe episodes.34 Drug compliance is best moni- to cardiac failures, renal failures, rhabdomyolysis, and heart
tored by an increasing MCV, while a decreasing LD might be an illness. Opponents of the connection of sickle cell trait
indicator of treatment response.33 Response to hydroxy- and fatal events argue that these events occur in sickle cell–
carbamide is variable among SCD patients, but high baseline negative people, many people with sickle cell trait do not
Hb F level, neutrophil levels, and reticulocyte count are the develop adverse events, fatal sickle crisis cannot be ade-
best predictors of Hb F response.75 quately established in the patients encountering events, and
 CHAPTER 27  Hemoglobinopathies (Structural Defects in Hemoglobin) 441

similar events have not been clearly documented in patients

with sickle cell disease. However, it has been shown that mili-
tary recruits with sickle cell trait have a 21 times greater risk of
exercise-related death than recruits with normal hemoglobin.85
Similar data have not been established in athletes with sickle
cell trait.85
The peripheral blood film of a patient with sickle cell trait
shows normal RBC morphology, with the exception of a few
target cells. No abnormalities in the leukocytes and thrombo-
cytes are seen. The hemoglobin solubility screening test yields
positive results, and sickle cell trait is diagnosed by detecting
the presence of Hb S and Hb A on hemoglobin electrophoresis
or HPLC. In individuals with sickle cell trait, electrophoresis
reveals approximately 40% or less Hb S and approximately
60% or more Hb A, Hb A2 level is normal or slightly increased, Figure 27-10  ​Peripheral blood film for a patient with hemoglobin C
and Hb F level is within the reference interval. Levels of Hb S disease showing one Hb C crystal and target and folded cells (31000).
(Courtesy Ann Bell, University of Tennessee, Memphis.)
less than 40% can be seen in patients who also have a-thalas-
semia or iron or folate deficiency.20 No treatment is required
for this benign condition, and the patient’s life span is not af- crystals appear extracellularly with no evidence of a cell mem-
fected by sickle cell trait. brane.86,87 In some cells, the hemoglobin is concentrated within
the boundary of the crystal. The crystals are densely stained and
vary in size and appear oblong with pyramid-shaped or pointed
HEMOGLOBIN C
ends. These crystals may be seen on wet preparations by washing
Hb C was the next hemoglobinopathy after Hb S to be de- RBCs and resuspending them in a solution of sodium citrate.11
scribed and in the United States is found almost exclusively in Hb C yields a negative result on the hemoglobin solubility
the African-American population. Spaet and Ranney reported test, and definitive diagnosis is made using electrophoresis or
this disease in the homozygous state (Hb CC) in 1953.8 HPLC. No Hb A is present in Hb CC disease. In addition, Hb C
is present at levels of greater than 90%, with Hb F at less than
Prevalence, Etiology, and Pathophysiology 7% and Hb A2 at approximately 2%. In Hb AC trait, about 60%
Hb C is found in 17% to 28% of people of West African extrac- Hb A and 30% Hb C are present. On cellulose acetate electro-
tion and in 2% to 3% of African Americans.4 It is the most phoresis at an alkaline pH, Hb C migrates in the same position
common nonsickling variant encountered in the United States as Hb A2, Hb E, and Hb O-Arab (Figure 27-7). Hb C is separated
and the third most common in the world.4 Hb C is defined by from these other hemoglobins on citrate agar electrophoresis at
the structural formula a2b26GlunLys, in which lysine is substi- an acid pH (Figure 27-7). No specific treatment is required. This
tuted for glutamic acid in position 6 of the b chain. Lysine has disorder becomes problematic only if infection occurs or if
a 11 charge and glutamic acid has a –1 charge, so the result of mild chronic hemolysis leads to gallbladder disease.
this substitution is a net change in charge of 12, which has a
different structural effect on the hemoglobin molecule than
HEMOGLOBIN C-HARLEM (HEMOGLOBIN
the Hb S substitution.
C-GEORGETOWN)
Hb C is inherited in the same manner as Hb S but manifests
as a milder disease. Similar to Hb S, Hb C polymerizes under Hb C-Harlem (Hb C-Georgetown) has a double substitution on
low oxygen tension, but the structure of the polymers differs. the b chain.5,20 The substitution of valine for glutamic acid at
Hb S polymers are long and thin, whereas the polymers in Hb position 6 of the b chain is identical to the Hb S substitution,
C form a short, thick crystal within the RBCs. The shorter Hb C and the substitution at position 73 of aspartic acid for aspara-
crystal does not alter RBC shape to the extent that Hb S does, gine is the same as that in the Hb Korle Bu mutation. The
so there is less splenic sequestration and hemolysis. In addi- double mutation is termed Hb C-Harlem (Hb C-Georgetown)
tion, vasoocclusive crisis does not occur. because the abnormal hemoglobin migrates with Hb C on cel-
lulose acetate electrophoresis at an alkaline pH. Patients hetero-
Laboratory Diagnosis zygous for this anomaly are asymptomatic, but patients with
A mild to moderate, normochromic, normocytic anemia oc- compound heterozygosity for Hb S and Hb C-Harlem have
curs in homozygous Hb C disease. Occasionally, some micro- crises similar to those in Hb SS disease.88
cytosis and mild hypochromia may be present. There is a A positive solubility test result may occur with Hb C-Harlem,
marked increase in the number of target cells, a slight to mod- and hemoglobin electrophoresis or HPLC is necessary to con-
erate increase in the number of reticulocytes, and nucleated firm the diagnosis. On cellulose acetate at pH 8.4, Hb C-Harlem
RBCs may be present in the peripheral blood. migrates in the C position (Figure 27-7). Citrate agar electro-
Hexagonal crystals of Hb C form within the erythrocyte and phoresis at pH 6.2, however, shows migration of Hb C-Harlem
may be seen on the peripheral blood film (Figure 27-10). Many in the S position (Figure 27-7). Because so few cases have been
442 PART IV  Erythrocyte Disorders

identified, the clinical outcome for homozygous individuals

affected with this abnormality is uncertain,88 but heterozygotes
appear normal.

HEMOGLOBIN E
Prevalence, Etiology, and Pathophysiology
Hb E was first described in 1954.89 The variant has a prevalence
of 30% in Southeast Asia. As a result of the influx of immi-
grants from this area, Hb E prevalence has increased in the
United States.90 It occurs infrequently in African Americans
and whites. Hb E is a b chain variant in which lysine is substi-
tuted for glutamic acid in position 26 (a2b226GlunLys). As with
Hb C, this substitution results in a net change in charge of 12,
but because of the position of the substitution, hemoglobin Figure 27-11  ​Microcytes and target cells in a patient with hemoglobin E
polymerization does not occur. However, the amino acid sub- trait. (From Hematology tech sample H-1, Chicago, 1991, American Society
of Clinical Pathologists.)
stitution at codon 26 inserts a cryptic splice site that causes
abnormal alterative splicing and decreased transcription of
functional mRNA for the Hb E globin chain.91 Thus the Hb E Genetic counseling is recommended, and the Hb E gene muta-
mutation is both a qualitative defect (due to the amino acid tion should be discussed in the same manner as a mild b-
substitution in the globin chain) and a quantitative defect with thalassemia allele.91
a b-thalassemia phenotype (due to the decreased production
of the globin chain).91
HEMOGLOBIN O-ARAB
Clinical Features Hb O-Arab is a b chain variant caused by the substitution of lysine
The homozygous state (Hb EE) manifests as a mild anemia for glutamic acid at amino acid position 121 (a2b2121GlunLys).5,20,32
with microcytes and target cells. The RBC survival time is short- It is a rare disorder found in Kenya, Israel, Egypt, and Bulgaria
ened. The condition is not associated with clinically observable and in 0.4% of African Americans. No clinical symptoms are
icterus, hemolysis, or splenomegaly. The main concern in iden- exhibited by individuals who carry this variant, except for a mild
tifying homozygous Hb E is differentiating it from iron defi- splenomegaly in homozygotes.5 When Hb O-Arab is inherited
ciency, b-thalassemia trait, and Hb E–b-thal (Chapter 28).91 with Hb S, however, severe clinical conditions similar to those
The disease, Hb EE, resembles thalassemia trait. Because the in Hb SS result.5
highest incidence of the Hb E gene is in the areas of Thailand Homozygous individuals have a mild hemolytic anemia,
where malaria is most prevalent, it is thought that P. falciparum with many target cells on the peripheral blood film and a
multiplies more slowly in Hb EE RBCs than in Hb AE or Hb AA negative result on the hemoglobin solubility test. The presence
RBCs and that the mutation may give some protection against of this hemoglobin variant must be confirmed using electro-
malaria.1 Hb E trait is asymptomatic. When Hb E is combined phoresis or HPLC. Because Hb O-Arab migrates with Hb A2,
with b-thalassemia, however, the disease becomes more severe Hb C, and Hb E on cellulose acetate at an alkaline pH, citrate
than Hb EE and more closely resembles b-thalassemia major, agar electrophoresis at an acid pH is required to differentiate it
requiring regular blood transfusions.1 from Hb C (Figure 27-7). Hb O-Arab is the only hemoglobin
to move just slightly away from the point of application toward
Laboratory Diagnosis the cathode on citrate agar at an acid pH. No treatment is gen-
Hb E does not produce a positive hemoglobin solubility test erally necessary for individuals with Hb O-Arab.
result and must be confirmed using electrophoresis or HPLC.
In the homozygous state there is greater than 90% Hb E, a very
HEMOGLOBIN D AND HEMOGLOBIN G
low MCV (55 to 65 fL), few to many target cells, and a normal
reticulocyte count. The heterozygous state has a mean MCV of Hb D and Hb G are a group of at least 16 b chain variants
65 fL, slight erythrocytosis, target cells1 (Figure 27-11), and ap- (Hb D) and 6 a chain variants (Hb G) that migrate in an
proximately 30% to 40% Hb E. On cellulose acetate electro- alkaline pH at the same electrophoretic position as Hb
phoresis at an alkaline pH, Hb E migrates with Hb C, Hb O, S.4,8,20,92 This is because their a and b subunits have one
and Hb A2 (Figure 27-7). On citrate agar electrophoresis at an fewer negative charge at an alkaline pH than Hb A, as does
acid pH, Hb E can be separated from Hb C, but it comigrates Hb S. They do not sickle, however, when exposed to reduced
with Hb A and Hb O (Figure 27-7). oxygen tension.
Most variants are named for the place where they were dis-
Treatment and Prognosis covered. Hb D-Punjab and Hb D-Los Angeles are identical he-
No therapy is required with Hb E disease and trait. Some moglobins in which glutamine is substituted for glutamic acid
patients may experience splenomegaly and fatigue, however. at position 121 in the b chain (a2b2121GlunGln). Hb D-Punjab
 CHAPTER 27  Hemoglobinopathies (Structural Defects in Hemoglobin) 443

occurs in about 3% of the population in northwestern India, other hemoglobins, such as Hb E, Hb G-Philadelphia, and Hb
and Hb D-Los Angeles is seen in fewer than 2% of African Korle Bu, causes disorders of no clinical consequence.93
Americans.
Hb G-Philadelphia is an a chain variant of the G hemoglo- Hemoglobin SC
bins, with a substitution of asparagine by lysine at position 68 Hb SC is the most common compound heterozygous syn-
(a268AsnnLysb2).5 The Hb G-Philadelphia variant is the most drome that results in a structural defect in the hemoglobin
common G variant encountered in African Americans and is molecule in which different amino acid substitutions are
seen with greater frequency than the Hb D variants. The Hb G found on each of two b-globin chains. At position 6, glutamic
variant is also found in Ghana.4,8,20,92 acid is replaced by valine (Hb S) on one b-globin chain and by
Hb D and Hb G do not sickle and yield a negative hemoglo- lysine (Hb C) on the other b-globin chain. The frequency of
bin solubility test result. On alkaline electrophoresis, Hb D Hb SC is 25% in West Africa. The incidence in the United
and Hb G have the same mobility as Hb S (Figure 27-7). Hb D States is approximately 1 in 833 births per year.93,97
and Hb G can be separated from Hb S on citrate agar at pH 6.0
(Figure 27-7). These variants should be suspected whenever a Clinical Features
hemoglobin is encountered that migrates in the S position on Hb SC disease resembles a mild SCD. Growth and develop-
alkaline electrophoresis and has a negative result on the hemo- ment are delayed compared with normal children. Unlike Hb
globin solubility test. In the homozygous state (Hb DD), there SS, Hb SC usually does not produce significant symptoms until
is greater than 95% Hb D, with normal amounts of Hb A2 and the teenage years. Hb SC disease may cause all the vasoocclu-
Hb F.30 Hb DD can be confused with the compound heterozy- sive complications of sickle cell anemia, but the episodes are
gous state for Hb D and b0-thalassemia. The two disorders can less frequent, and damage is less disabling. Hemolytic anemia
be differentiated on the basis of the MCV, levels of Hb A2, and is moderate, and many patients exhibit moderate splenomeg-
family studies.4,8,20,92 aly. Proliferative retinopathy is more common and more severe
Hb D and Hb G are asymptomatic in the heterozygous state. than in sickle cell anemia.98 Respiratory tract infections with S.
Hb D disease (Hb DD) is marked by mild hemolytic anemia pneumoniae are common.8
and chronic nonprogressive splenomegaly. No treatment is Patients with Hb SC disease live longer than patients with
required.4,8,20,92 Hb SS and have fewer painful episodes, but this disorder is as-
sociated with considerable morbidity and mortality, especially
after age 30.99 In the United States, the median life span for
COMPOUND HETEROZYGOSITY WITH
men is 60 years and for women 68 years.30
HEMOGLOBIN S AND ANOTHER b-GLOBIN
GENE MUTATION
Laboratory Diagnosis
Compound heterozygosity is the inheritance of two different The complete blood count shows a mild normocytic, normo-
mutant genes that share a common genetic locus—in this case chromic anemia with many of the features associated with sickle
the b-globin gene locus.93-96 Because there are two b-globin cell anemia. The hemoglobin level is usually 11 to 13 g/dL, and
genes, these compound heterozygotes have inherited Hb S the reticulocyte count is 3% to 5%. On the peripheral blood
from one parent and another b chain hemoglobinopathy or film, there are a few sickle cells, target cells, and intraerythrocytic
thalassemia from the other parent. Compound heterozygosity crystalline structures. Crystalline aggregates of hemoglobin
of Hb S with Hb C, Hb D, Hb O, or b-thalassemia may produce (SC crystals) form in some cells, where they protrude from the
hemolytic anemia of variable severity. Inheritance of Hb S with membrane (Figure 27-12).93,96 Hb SC crystals often appear as a

A B
Figure 27-12  ​A and B, Peripheral blood film for a patient with hemoglobin SC. Note intraerythrocytic, blunt-ended SC crystals and target cells (31000). (Courtesy
Ann Bell, University of Tennessee, Memphis.)
444 PART IV  Erythrocyte Disorders

hybrid of Hb S and Hb C crystals. They are longer than Hb C for patients with SCD and is administered according to the
crystals but shorter and thicker than Hb S polymers and are of- severity of the clinical condition.
ten branched.
The result of the hemoglobin solubility screening test is Hemoglobin S/O-Arab and HbS/D-Punjab
positive because of the presence of Hb S. Electrophoreti- Hb S/O-Arab and Hb S/D-Punjab are rare compound heterozy-
cally, Hb C and Hb S migrate in almost equal amounts gous hemoglobinopathies that cause severe chronic hemolytic
(45%) on cellulose acetate, and Hb F is normal. Hb C is anemia with vasoocclusive episodes.8,20,92 Both mutations replace
confirmed on citrate agar at an acid pH, where it is separated glutamic acid at position 121; O-Arab substitutes lysine and D-
from Hb E and Hb O. Hb A2 migrates with Hb C, and its Punjab substitutes glutamine. Glutamic acid at position 121 is
quantitation is of no consequence in Hb SC disease. Deter- located on the outer surface of the hemoglobin tetramer, which
mination of Hb A2 becomes vital, however, if a patient is enhances the polymerization process involving Hb S. Hb S/O-
suspected of having Hb C concurrent with b-thalassemia Arab can be mistaken for Hb SC on cellulose acetate electropho-
(Chapter 28). resis at an alkaline pH because Hb C and Hb O-Arab migrate at
the same position; however, differentiation is easily made on ci-
Treatment and Prognosis trate agar at an acid pH. Therapy for these patients is similar to
Therapy similar to that for SCD is given to individuals with Hb that for patients with SCD. Similarly, Hb D-Punjab comigrates
SC disease.88 with Hb S on alkaline electrophoresis, making this mutation look
like SCD. Hb O-Arab and Hb D-Punjab are not clinically signifi-
Hemoglobin S–b-Thalassemia cant in either the heterozygous or the homozygous form.1
Compound heterozygosity for Hb S and b-thalassemia is the
most common cause of sickle cell syndrome in patients of Hemoglobin S-Korle Bu
Mediterranean descent and is second to Hb SC disease among Hb Korle Bu is a rare hemoglobin variant with substitution of
all compound heterozygous sickle disorders. Hb S–b-thal usu- aspartic acid for asparagine at position 73 of the b chain.20
ally causes a clinical syndrome resembling that of mild or When inherited with Hb S, it interferes with lateral contact
moderate sickle cell anemia. The severity of this compound between Hb S fibers by disrupting the hydrophobic pocket for
heterozygous condition depends on the b chain production of b6 valine, which inhibits Hb S polymerization. The compound
the affected b-thalassemia gene. If there is no b-globin chain heterozygous condition Hb S-Korle Bu is asymptomatic.
production from the b-thalassemia gene (Hb S–b0-thal), the
clinical course is similar to that of homozygous sickle cell ane-
CONCOMITANT CIS MUTATIONS WITH
mia. If there is production of a normal b-globin chain (Hb S–
HEMOGLOBIN S
b1-thal), patients tend to have a milder condition than pa-
tients with Hb SC. These patients can be distinguished from A concomitant cis mutation with Hb S involves a second muta-
individuals with sickle cell trait because of the presence of tion on the same gene along with Hb S. Three cis mutations will
greater amounts of Hb S than of Hb A, increased levels of Hb be described: Hb C-Harlem, Hb S-Antilles, and Hb S-Oman.
A2 and Hb F, microcytosis from the thalassemia, hemolytic
anemia, abnormal peripheral blood morphology, and spleno- Hemoglobin C-Harlem
megaly (Chapter 28).20,93 Hb C-Harlem has two substitutions on the b chain: the sickle
mutation and the Korle Bu mutation. Patients heterozygous for
Hemoglobin SD and Hemoglobin only Hb C-Harlem are asymptomatic. The compound heterozy-
SG-Philadelphia gous Hb S–Hb C-Harlem state resembles Hb SS clinically. Hb
Hb SD is a compound heterozygous and Hb SG-Philadelphia C-Harlem yields a positive result on the hemoglobin solubility
a double heterozygous sickle cell syndrome.20,92 Hb SG- test and migrates to the Hb C position on cellulose acetate elec-
Philadelphia is asymptomatic because Hb G is associated trophoresis at an alkaline pH and to the Hb S position on ci-
with an a gene mutation that still allows for sufficient Hb A trate agar electrophoresis at an acid pH.
to be produced. Hb SD syndrome may cause a mild to severe
hemolytic anemia because both b chains are affected. Some Hemoglobin S-Antilles and Hemoglobin
patients with Hb SD may have severe vasoocclusive complica- S-Oman
tions. The Hb D syndrome in African Americans is usually Hb S-Antilles bears the Hb S mutation (b6GlunVal) along with
due to the interaction of Hb S with Hb D-Los Angeles a substitution of isoleucine for valine at position 23.100 Hb
(Hb D-Punjab). S-Oman also has the Hb S mutation with a second substitu-
The peripheral blood film findings for Hb SD disease are tion of lysine for glutamic acid at position 121.101 In both of
comparable to those seen in less severe forms of Hb SS disease. these hemoglobin variants, the second mutation enhances
Because Hb D and Hb G comigrate with Hb S on cellulose ac- Hb S such that significant sickling can occur even in hetero-
etate electrophoresis at an alkaline pH, citrate agar electropho- zygotes.1
resis at an acid pH is necessary to separate Hb S from Hb D and Table 27-2 summarizes common clinically significant he-
Hb G. The clinical picture is valuable in differentiating Hb SD moglobinopathies, including general characteristics and treat-
and Hb SG. The treatment for Hb SD disease is similar to that ment options.
TABLE 27-2  ​Common Clinically Significant Hemoglobinopathies
Hemoglobin
Hemoglobin Abnormal Structural Groups Primarily Solubility Test Hemoglobins Red Blood Cell Symptoms/Organ
Disorder Hemoglobin Defect Affected Results Present Morphology Defects Treatment
Sickle cell anemia Hb S a2b26GlunVal African, African Positive 0% Hb A, .80% Hb S, Sickle cells, target Vasoocclusion, bacte- Transfusions,
(homozygous) American, Middle 1%—20% Hb F, cells, nucleated rial infections, antibiotics,
Eastern, Indian, 2%—5% Hb A2 RBCs, polychro- hemolytic anemia, analgesics,
Mediterranean masia, Howell- aplastic episodes; bone marrow
Jolly bodies, ba- bones, lungs, liver, transplant,
sophilic stippling spleen, penis, eyes, hydroxyurea
central nervous
system, urinary
tract
Hb C disease Hb C a2b26GlunLys African, African Negative 0% Hb A, .90% Hb C, Hb C crystals, Mild splenomegaly, Usually none,

CHAPTER 27  Hemoglobinopathies (Structural Defects in Hemoglobin)

(homozygous) American ,7% Hb F, 2% Hb A2 target cells, mild hemolysis antibiotics
nucleated RBCs,
occasionally
some microcytes
Hb SC-Harlem* Hb C-Harlem, a2b26GlunVal and Rare, so uncertain; Positive Hb C-Harlem migrates Target cells Compound heterozy- Similar to Hb SS
(Hb C-George- Hb S a2b273AspnAsn African, African with Hb C at alkaline gotes with Hb
town) on same gene American pH; migrates with Hb SC-Harlem have
and a2b26GlunVal S at acid pH symptoms similar
to Hb SS
Hb E disease Hb E a2b226GlunLys Southeast Asian, Negative 0% Hb A, 95% Hb E, Target cells, micro- Mild anemia, mild Usually none
(homozygous) African, African 2%-4% Hb A2; cytes splenomegaly, no
American migrates with Hb A2, symptoms
Hb C, and Hb O
at alkaline pH
Hb O-Arab Hb O-Arab a2b2121GlunLys Kenyan, Israeli, Negative 0% Hb A, 95% Hb O, Target cells Mild splenomegaly Usually none
(homozygous) Egyptian, 2%-4% Hb A2;
Bulgarian, African migrates with Hb A2,
American Hb C, and Hb E
at alkaline pH
Hb D disease Hb D-Punjab a2b2121GlunGln Middle Eastern, Negative 95% Hb D, normal Target cells Mild hemolytic ane- Usually none
(rare (Hb-D Los Indian Hb A2 and Hb F; mia, mild spleno-
homozygous) Angeles) migrates with Hb S megaly
at alkaline pH
Continued

445
TABLE 27-2  ​Common Clinically Significant Hemoglobinopathies—cont’d

446
Hemoglobin
Hemoglobin Abnormal Structural Groups Primarily Solubility Test Hemoglobins Red Blood Cell Symptoms/Organ
Disorder Hemoglobin Defect Affected Results Present Morphology Defects Treatment

PART IV  Erythrocyte Disorders

Hb G disease Hb G, Hb a268AsnnLysb2 African American, Negative 95% Hb G, normal Hb Target cells Mild hemolytic Usually none
(rare G-Philadelphia Ghanaian A2 and Hb F; anemia, mild
homozygous) migrates with S at splenomegaly
alkaline pH
Hb SC* disease Hb S, Hb C a2b26GlunVal and Same as Hb S Positive 45% Hb S, 45% Hb C, Sickle cells, Hb SC Same as those for Hb Similar to that for
a2b26GlunLys 2%–4% Hb A2, 1% crystals, target SS except milder Hb SS but less
Hb F cells intensive
Hb S–b- Hb S 1 b-thal- a2b26GlunVal and Same as Hb S Positive Hb S variable, some Hb Sickle cells, target Hemolytic anemia, Similar to that for
thalassemia* assemia mu- b0 or b1 A in b1, increased cells, microcytes splenomegaly Hb SS; varies
tation Hb A2 and Hb F depending on
amount of Hb
A present
Hb SD* disease Hb S, Hb D a2b26GlunVal and Same as Hb S Positive 45% Hb S, 45% Hb D, Sickle cells, target Similar to those for Similar to that for
a2b2121GlunGln 2%–4% Hb A2, 1% cells Hb SS but milder Hb SS but less
Hb F; Hb S and D intensive
comigrate at alkaline
pH
Hb SG† Hb S, Hb G a2b26GlunVal and Same as Hb S Positive 45% Hb S, 45% Hb G, Target cells No symptoms Usually none
a268AsnnLysb2 2%–4% Hb A2, 1%
Hb F; Hb S and G
comigrate at alkaline
pH
Hb SO-Arab* Hb S, Hb O-Arab a2b26GlunVal and Same as Hb S Positive 45% Hb S, 45% Hb O, Sickle cells, target Similar to those for Similar to that for
a2b2121GlunLys 2%–4% Hb A2, 1% cells Hb SS Hb SS
Hb F

*Compound heterozygous.
†Double heterozygous.
Asn, Asparagine; Asp, aspartic acid; Gln, glutamine; Glu, glutamic acid; Hb, hemoglobin; Lys, lysine; Val, valine.
 CHAPTER 27  Hemoglobinopathies (Structural Defects in Hemoglobin) 447

anemia, which varies from compensated mild anemia to severe

HEMOGLOBIN M
hemolytic episodes.
Hb M is caused by a variety of mutations in the a-, b-, and At one time, the anemia was referred to as congenital non­
g-globin genes, all of which result in the production of spherocytic hemolytic anemia or congenital Heinz body anemia. This
methemoglobin—hence the Hb M designation.92,102 These disorder is more properly called unstable hemoglobin disease. The
genetic mutations result in a structural abnormality in the glo- syndrome appears at or just after birth, depending on the glo-
bin portion of the molecule. Most M hemoglobins involve a bin chains involved. It is inherited in an autosomal dominant
substitution of a tyrosine amino acid for either the proximal pattern. All patients are heterozygous; apparently the homozy-
(F8) or the distal (E7) histidine amino acid in the a, b, or gous condition is incompatible with life. The instability of the
g chains. These substitutions cause heme iron to auto-oxidize, hemoglobin molecule may be due to (1) substitution of a
which results in methemoglobinemia. Hb M has iron in the charged for an uncharged amino acid in the interior of the mol-
ferric state (Fe31) and is unable to carry oxygen, which pro- ecule, (2) substitution of a polar for a nonpolar amino acid in
duces cyanosis. Seven hemoglobin variants affecting the a or the hydrophobic heme pocket, (3) substitution of an amino
b chains have been classified as M hemoglobins: Hb M-Boston, acid in the a and b chains at the intersubunit contact points,
Hb M-Iwate, and Hb Auckland (a chain variants); and Hb Chile, (4) replacement of an amino acid with proline in the a helix
Hb M-Saskatoon, Hb M-Milwaukee-1, and Hb M-Milwaukee-2 section of a chain, and (5) deletion or elongation of the primary
(b chain variants), all named for the locations in which structure.
they were discovered.5 Two variants affect the g chain—Hb
F-M-Osaka and Hb F-M-Fort Ripley5—but symptoms disappear Clinical Features
when Hb A replaces Hb F at 3 to 6 months of age. The unstable hemoglobin disorder is usually detected in early
Hb M variants have altered oxygen affinity and are inherited childhood in patients with hemolytic anemia accompanied
as autosomal dominant disorders. Affected individuals have by jaundice and splenomegaly. Fever or ingestion of an oxi-
30% to 50% methemoglobin (healthy individuals have less dant exacerbates the hemolysis. The severity of the anemia
than 1%) and may appear cyanotic. Ingestion of oxidant drugs, depends on the degree of instability of the hemoglobin mol-
such as sulfonamides, can increase methemoglobin to life- ecule. The unstable hemoglobin precipitates in vivo and in
threatening levels. Methemoglobin causes the blood specimen vitro in response to factors that do not affect normal hemo-
to appear brown. Heinz bodies may be seen sometimes on wet globins, such as drug ingestion and exposure to heat or cold.
preparations because methemoglobin causes globin chains to The hemoglobin precipitates in the RBC as Heinz bodies. The
precipitate (see Figure 14-11). Diagnosis is made by spectral precipitated hemoglobin attaches to the cell membrane, caus-
absorption of the hemolysate or by hemoglobin electrophore- ing clustering of band 3, attachment of autologous immuno-
sis. The absorption spectrum peaks are determined at various globulin, and macrophage activation. In addition, Heinz
wavelengths. The unique absorption range of each Hb M vari- bodies can be trapped mechanically in the splenic sieve,
ant is identified when these are compared with the spectrum of which shortens RBC survival. The oxygen affinity of these
normal blood. cells is also abnormal.
Before electrophoresis, all hemoglobin types are converted The most prevalent unstable hemoglobin is Hb Köln. Other
to methemoglobin by adding potassium cyanide to the sample unstable hemoglobins include Hb Hammersmith, Hb Zurich,
so that any migration differences observed are only due to an Hb Gun Hill, and Hb Hammersmith.5 Because of the large
amino acid substitution, not differences in iron states. On cel- variability in the degree of instability in these hemoglobins,
lulose acetate, Hb M migrates slightly more slowly than Hb A. the extent of hemolysis varies greatly. For some of the variants,
The electrophoresis should be performed on agar gel at pH 7.1 such as Hb Zurich, the presence of an oxidant is required for
for clear separation. Further confirmation may be obtained us- any significant hemolysis to occur.
ing HPLC or deoxyribonucleic acid (DNA)–based globin gene
analysis. No treatment is necessary. Diagnosis is essential to Laboratory Diagnosis
prevent inappropriate treatment for other conditions, such as The RBC morphology varies. It may be normal or show slight
cyanotic heart disease. hypochromia and prominent basophilic stippling, which pos-
sibly is caused by excessive clumping of ribosomes. Before
splenectomy, the hemoglobin level ranges from 7 to 12 g/dL,
UNSTABLE HEMOGLOBIN VARIANTS
with a 4% to 20% reticulocyte count. After splenectomy, ane-
Unstable hemoglobin variants result from genetic mutations to mia is corrected, but reticulocytosis persists. Heinz bodies can
globin genes creating hemoglobin products that precipitate in be shown using a supravital stain (see Figure 14-11). After sple-
vivo, producing Heinz bodies and causing a hemolytic ane- nectomy, Heinz bodies are larger and more numerous. Many
mia.92,102 More than 140 variants of unstable hemoglobin ex- patients excrete dark urine that contains dipyrrole.
ist.5 The majority of these are b chain variants, and most others Many unstable hemoglobins migrate in the normal AA pat-
are a chain variants. Only a few are g and d chain variants. Most tern and thus are not detected on electrophoresis. Other tests
unstable hemoglobin variants have no clinical significance, al- used to detect unstable hemoglobins include the isopropanol
though the majority has an increased oxygen affinity. About precipitation test, which is based on the principle that an iso-
25% of unstable hemoglobins are responsible for hemolytic propanol solution at 37° C weakens the bonding forces of the
448 PART IV  Erythrocyte Disorders

hemoglobin molecule. If unstable hemoglobins are present, Hemoglobins with Increased Oxygen Affinity
rapid precipitation occurs in 5 minutes, and heavy flocculation The high-affinity variants, like other structurally abnormal he-
occurs after 20 minutes. Normal hemoglobin does not begin to moglobins, show an autosomal dominant pattern of inheri-
precipitate until after approximately 40 minutes. The heat de- tance. Affected individuals have equal volumes of Hb A and the
naturation test also can be used. When incubated at 50° C for abnormal variant. Exceptions to this are compound heterozy-
1 hour, heat-sensitive unstable hemoglobins show a flocculent gotes for Hb Abruzzo and b-thalassemia and for Hb Crete and
precipitation, whereas normal hemoglobin shows little or no b-thalassemia, in which the proportion of abnormal hemoglo-
precipitation. Significant numbers of Heinz bodies appear after bin is greater than 85%.
splenectomy, but even in individuals with intact spleens, with More than 90 variant hemoglobins with high oxygen affin-
longer incubation and the addition of an oxidative substance ity have been discovered. Such hemoglobins fail to release oxy-
such as acetylphenylhydrazine, unstable hemoglobins form gen on demand, and hypoxia results. The kidneys sense the
more Heinz bodies than does the blood from individuals with hypoxia and respond by increasing the release of erythropoie-
normal hemoglobins. Other techniques, such as isoelectric fo- tin, which leads to a compensatory erythrocytosis. These vari-
cusing, can resolve many hemoglobin variants with only a ants differ from unstable hemoglobin, which also may have
slight alteration in their isoelectric point, and globin chain abnormal oxygen affinity, in that they do not precipitate
analysis can be performed by HPLC or DNA-based globin gene in vivo to produce hemolysis and there is no abnormal RBC
analysis. morphology.
Most individuals are asymptomatic and show no physical
Treatment and Prognosis symptoms except a ruddy complexion. Erythrocytosis is usu-
Patients are treated to prevent hemolytic crises. In severe ally detected during routine examination because the patient
cases, the spleen must be removed to reduce sequestration generally has a high RBC count, hemoglobin, and hematocrit.
and rate of removal of RBCs. Because unstable hemoglobin The WBC count, platelet count, and peripheral blood film find-
disease is rare, prognosis in the affected individuals is un- ings are generally normal. In some cases, hemoglobin electro-
clear. Patients are cautioned against the use of sulfonamides phoresis may establish a diagnosis. An abnormal band that
and other oxidant drugs. They also should be informed separates from the A band is present on cellulose acetate in
of the potential for febrile illnesses to trigger a hemolytic some variants; however, if a band is not found, the diagnosis
episode. of increased oxygen affinity cannot be ruled out. In some cases
the abnormal hemoglobin can be separated by using citrate
agar (pH 6.0) or by gel electrophoresis. Measurement of oxy-
HEMOGLOBINS WITH INCREASED AND
gen affinity is required for definitive diagnosis.
DECREASED OXYGEN AFFINITY
Patients with high-oxygen-affinity hemoglobins live normal
More than 150 hemoglobin variants have been discovered to lives and require no treatment. Diagnosis should be made to
have abnormal oxygen affinity.4,102-104 Most are high-affinity avoid unnecessary treatment of the erythrocytosis as a myelo-
variants and have been associated with familial erythrocytosis. proliferative neoplasm or a secondary erythrocytosis.
The remaining hemoglobin variants are characterized by low
oxygen affinity. Many of these are associated with mild to mod- Hemoglobins with Decreased Oxygen Affinity
erate anemia.2 Hemoglobins with decreased oxygen affinity quickly release
As described in Chapter 10, normal Hb A undergoes a series oxygen to the tissues, which results in normal to decreased
of allosteric conformational changes as it converts from a fully hemoglobin concentration and slight anemia. The best known
deoxygenated to a fully oxygenated form. These conforma- of these hemoglobins is Hb Kansas, which has an amino acid
tional changes affect hemoglobin function and its affinity for substitution of asparagine by threonine at position 102 of the
oxygen. When normal hemoglobin is fully deoxygenated b chain. These hemoglobins may be present when cyanosis
(tense state), it has low affinity for oxygen and other heme li- and a normal arterial oxygen tension coexist, and most may be
gands and high affinity for allosteric effectors, such as Bohr detected by starch gel electrophoresis, HPLC, or DNA-based
protons and 2,3-bisphosphoglycerate. In the oxygenated (re- globin gene analysis.
laxed) state, hemoglobin has a high affinity for heme ligands,
such as oxygen, and a low affinity for Bohr protons and
GLOBAL BURDEN OF
2,3-bisphosphoglycerate. The transition from the tense to the
HEMOGLOBINOPATHIES
relaxed state involves a series of structural changes that have a
marked effect on hemoglobin function. If an amino acid sub- The prevalence of hemoglobinopathies has already been
stitution lowers the stability of the tense structure, the transi- presented in this chapter, and the bulk of these conditions
tion to the relaxed state occurs at an earlier stage in ligand occurs in underdeveloped countries. However, as developing
binding, and the hemoglobin has increased oxygen affinity and countries work to decrease deaths from malnutrition, infec-
decreased heme-heme interaction or cooperativity (Chapter 10). tious diseases, and other conditions, more patients with hemo-
One example of a b chain variant is Hb Kempsey. This unstable globinopathies will survive and remain consumers of the
hemoglobin variant has amino acid substitutions at sites cru- health care system. For example, in 1944 thalassemia was first
cial to hemoglobin function. identified in Cypress. However, during the post–World War II
 CHAPTER 27  Hemoglobinopathies (Structural Defects in Hemoglobin) 449

recovery period, as the death rate decreased, the prevalence of velop prenatal screening and genetic counseling programs have
thalassemias increased.2 In 1970 it was estimated that in the reduced the birth rate of SCD.2 It is clear that hemoglobinopa-
absence of systems to control the disease, within 40 years thies are a worldwide problem requiring planning, investment,
78,000 units of blood would be needed each year, requiring and interventions from around the globe to optimize the im-
that 40% of the population serve as donors.2 If left unchecked, pact on patients with the disease without debilitating the
the cost to maintain thalassemia therapy would exceed the health care systems of developing countries where the disease
country’s total health care budget. In contrast, efforts to de- is prevalent.

SUMMARY
• Hemoglobinopathies are genetic disorders of globin genes that the heterozygous states, these hemoglobinopathies are asymp-
produce structurally abnormal hemoglobins with altered amino tomatic.
acid sequences, which affect hemoglobin function and stability. • Hb C is found primarily in people of African descent.
• Hb S is the most common hemoglobinopathy, resulting from a • On peripheral blood films from patients with Hb CC, hexagonal
substitution of valine for glutamic acid at position 6 of the b globin crystals may be seen with and without apparent RBC membrane
chain, and primarily affects people of African descent. surrounding them.
• Hb S polymerizes in the RBCs because of abnormal interaction • Hb EE results in a microcytic anemia and is found primarily in
with adjacent tetramers when it is in the deoxygenated form, people of Southeast Asian descent.
producing sickle-shaped RBCs. • Other variants, such as unstable hemoglobins and hemoglobins
• In homozygous Hb SS, the polymerization of hemoglobin may re- with altered oxygen affinity, can be identified, and many cause no
sult in severe episodic conditions; however, factors other than clinical abnormality.
hemoglobin polymerization may account for vasoocclusive epi- • Laboratory procedures employed for diagnosis of hemoglobinopa-
sodes in sickle cell patients. thies are the CBC, peripheral blood film evaluation, reticulocyte
• The most clinically significant hemoglobinopathies are Hb SS, Hb SC, count, hemoglobin solubility test, and methods to quantitate normal
and Hb S–b-thalassemia; Hb SS causes the most severe disease. hemoglobins and variants including hemoglobin electrophoresis
• Individuals with sickle cell trait (Hb AS) are clinically asymp- (acid and alkaline pH), high-performance liquid chromatography,
tomatic. and capillary electrophoresis.
• Sickle cell anemia (Hb SS) is a normocytic, normochromic anemia, • Advanced techniques available for hemoglobin identification in-
characterized by a single band in the S position on hemoglobin clude isoelectric focusing and DNA-based analysis of the globin
electrophoresis, a single Hb S peak on HPLC, and a positive hemo- genes.
globin solubility test.
• The median life expectancy of patients with SCD has been ex- Now that you have completed this chapter, go back and
tended to approximately 50 years. read again the case study at the beginning and respond
• Hb C and Hb E are the next most common hemoglobinopathies to the questions presented.
after Hb S and cause mild hemolysis in the homozygous state. In

R E V I E W Q UESTIONS
Answers can be found in the Appendix. 3. Patients with SCD usually do not exhibit symptoms until
6 months of age because:
1. A qualitative abnormality in hemoglobin may involve all of a. The mother’s blood has a protective effect
the following except: b. Hemoglobin levels are higher in infants at birth
a. Replacement of one or more amino acids in a globin c. Higher levels of Hb F are present
chain d. The immune system is not fully developed
b. Addition of one or more amino acids in a globin chain
c. Deletion of one or more amino acids in a globin chain 4. Megaloblastic episodes in SCD can be prevented by prophy-
d. Decreased production of a globin chain lactic administration of:
a. Iron
2. The substitution of valine for glutamic acid at position 6 of b. Folic acid
the b chain of hemoglobin results in hemoglobin that: c. Steroids
a. Is unstable and precipitates as Heinz bodies d. Erythropoietin
b. Polymerizes to form tactoid crystals
c. Crystallizes in a hexagonal shape
d. Contains iron in the ferric (Fe31) state
450 PART IV  Erythrocyte Disorders

5. Which of the following is the most definitive test for Hb S? 11. DNA analysis documents a patient has inherited the sickle
a. Hemoglobin solubility test mutation in both b-globin genes. The two terms that best
b. Hemoglobin electrophoresis at alkaline pH describe this genotype are:
c. Osmotic fragility test a. Homozygous/trait
d. Hemoglobin electrophoresis at acid pH b. Homozygous/disease
c. Heterozygous/trait
6. A patient presents with mild normochromic, normocytic d. Heterozygous/disease
anemia. On the peripheral blood film, there are a few tar-
get cells, rare nucleated RBCs, and hexagonal crystals 12. In which of the following geographic areas is Hb S most
within and lying outside of the RBCs. Which abnormality prevalent?
in the hemoglobin molecule is most likely? a. India
a. Decreased production of b chains b. South Africa
b. Substitution of lysine for glutamic acid at position 6 of c. United States
the b chain d. Sub-Saharan Africa
c. Substitution of tyrosine for the proximal histidine in
the b chain 13. Which hemoglobinopathy is more common in Southeast
d. Double amino acid substitution in the b chain Asian patients?
a. Hb S
7. A well-mixed specimen obtained for a CBC has a brown b. Hb C
color. The patient is being treated with a sulfonamide for a c. Hb O
bladder infection. Which of the following could explain d. Hb E
the brown color?
a. The patient has Hb M. 14. Which of the following Hb S compound heterozygote ex-
b. The patient is a compound heterozygote for Hb S and hibits the mildest symptoms?
thalassemia. a. Hb S-b-Thal
c. The incorrect anticoagulant was used. b. Hb SG
d. Levels of Hb F are high. c. Hb S-C-Harlem
d. Hb SC
8. Through routine screening, prospective parents discover
that they are both heterozygous for Hb S. What percentage 15. A 1-year-old Indian patient presents with anemia, and
of their children potentially could have sickle cell anemia both parents claim to have an “inherited anemia” but can’t
(Hb SS)? remember the type. The peripheral blood shows target
a. 0% cells, and the hemoglobin solubility is negative. Alkaline
b. 25% hemoglobin electrophoresis shows a single band at the
c. 50% “Hb C” position and a small band at the “Hb F” position.
d. 100% Acid hemoglobin electrophoresis shows two bands. The
most likely diagnosis is:
9. Painful crises in patients with SCD occur as a result of: a. Hb CC
a. Splenic sequestration b. Hb AC
b. Aplasia c. Hb CO
c. Vasoocclusion d. Hb SC
d. Anemia
16. Unstable hemoglobins show all of the following findings
10. The screening test for Hb S that uses a reducing agent, such EXCEPT:
as sodium dithionite, is based on the fact that hemoglo- a. Globin chains precipitate intracellularly
bins that sickle: b. Heinz body formation
a. Are insoluble in reduced, deoxygenated form c. Elevated reticulocyte count
b. Form methemoglobin more readily and cause a color d. Only homozygotes are symptomatic
change
c. Are unstable and precipitate as Heinz bodies
d. Oxidize quickly and cause turbidity
 CHAPTER 27  Hemoglobinopathies (Structural Defects in Hemoglobin) 451

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