Rodak's Hematology Clinical Principles and Applications

PART IV

Erythrocyte Disorders

CHAPTER 27

Hemoglobinopathies (structural defects in hemoglobin)

Tim R. Randolph

OUTLINE

Structure of Globin Genes

Hemoglobin Development

Genetic Mutations

Zygosity

Pathophysiology

Nomenclature

Hemoglobin S

Sickle Cell Anemia

Sickle Cell Trait

Hemoglobin C

Prevalence, Etiology, and Pathophysiology

Hemoglobin C-Harlem (Hemoglobin C-Georgetown)

Hemoglobin E

Prevalence, Etiology, and Pathophysiology

Clinical Features

Hemoglobin O-Arab

Hemoglobin D and Hemoglobin G

Compound Heterozygosity with Hemoglobin S and Another b-Globin Gene Mutation

Hemoglobin SC

Hemoglobin S b-Thalassemia

Hemoglobin SD and Hemoglobin SG-Philadelphia

Hemoglobin S/O-Arab and HbS/D-Punjab

Hemoglobin S-Korle Bu

Concomitant Cis Mutations with Hemoglobin S

Hemoglobin C-Harlem

Hemoglobin S-Antilles and Hemoglobin S-Oman

Hemoglobin M

Unstable Hemoglobin Variants

Clinical Features

Treatment and Prognosis

Hemoglobins with Increased and Decreased Oxygen Affinity

Hemoglobins with Increased Oxygen Affinity

Hemoglobins with Decreased Oxygen Affinity

Global Burden of Hemoglobinopathies

Objectives

After completion of this chapter, the reader will be able to:

  1. Explain the difference between structural hemoglobin disorders and thalassemias, and describe the types of mutations found in the structural disorders.
  2. Describe globin gene structure and the development of normal human hemoglobins throughout prenatal and postnatal life.
  3. Differentiate between homozygous and heterozygous states and the terms “disease” and “trait” as they relate to the hemoglobinopathies.
  4. Given the hemoglobin genotypes of parents involving common β chain variants, determine the possible genotypes of their children using a Punnett square.
  5. Describe the general geographic distribution of common hemoglobin variants and the relationship of that distribution with the prevalence of malaria and glucose-6-phosphate dehydrogenase deficiency.
  6. For disorders involving Hb S and Hb C, describe the genetic mutation, the effect of the mutation on the hemoglobin molecule, the inheritance pattern, pathophysiology, symptoms, clinical findings, peripheral blood findings, laboratory diagnosis, and genetic counseling and treatment considerations.
  7. Describe the genetic mutation, clinical findings, and laboratory diagnosis for disorders involving Hb C-Harlem, Hb E, Hb O-Arab, Hb D, and Hb G.
  8. Describe the clinical and laboratory findings for the compound heterozygous disorders of Hb S with Hb C, β-thalassemia, Hb D, Hb O-Arab, Hb Korle Bu, and Hb C-Harlem.
  9. Describe the electrophoretic mobility of Hb A, Hb F, Hb S, and Hb C at an alkaline pH, and explain how other methods (including the Hb S solubility test, citrate agar electrophoresis at acid pH, and high-performance liquid chromatography) are used to distinguish Hb S and Hb C from other hemoglobins with the same mobility.
  10. Describe the genetic mutations, inheritance patterns, pathophysiology, and clinical and laboratory findings in hemoglobin variants that result in methemoglobinemia.
  11. Describe the inheritance patterns, causes, and clinical and laboratory findings of unstable hemoglobin variants.
  12. Discuss the pathophysiology of hemoglobin variants with increased and decreased oxygen affinities, and explain how they differ from unstable hemoglobins.
  13. Given a case history and clinical and laboratory findings, interpret test results to identify the hemoglobin variants present in the patient.

CASE STUDY

After studying the material in this chapter, the reader should be able to respond to the following case study:

An 18-year-old African-American woman was seen in the emergency department for fever and abdominal pain. The following results were obtained on a complete blood count:

 

Patient Results

Reference Interval

WBCs (×109/L)

11.9

3.6–10.6

RBCs (×1012/L)

3.67

4.00–5.40

HGB (g/dL)

10.9

12.0–15.0

HCT (%)

32.5

35–49

RDW (%)

19.5

11.5–14.5

Platelets (×109/L)

410

150–450

Segmented neutrophils (%)

75

50–70

Lymphocytes (%)

18

18–42

Monocytes (%)

3

2–11

Eosinophils (%)

3

1–3

Basophils (%)

1

0–2

Reticulocytes (%)

3.1

0.5–2.5

A typical field in the patient’s peripheral blood film is shown in Figure 27-1. Electrophoresis on cellulose acetate at alkaline pH showed 50.9% Hb S and 49.1% Hb C.

  1. Select confirmatory tests that should be performed 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 diagnosis is suggested?
  4. If this patient were to marry a person of genotype Hb AS, what would be the expected frequency of genotypes for each of four children?

 
FIGURE 27-1 Peripheral blood film for the patient in the case study (×1000).  Source:  (Courtesy Ann Bell, University of Tennessee, Memphis.)

Hemoglobinopathy refers to a disease state (opathy) involving the hemoglobin (Hb) molecule. Hemoglobinopathies are the most common genetic diseases, affecting approximately 7% of the world’s population.1 Approximately 300,000 children are born each year with some form of inherited hemoglobin disorder.2 All hemoglobinopathies result from a genetic mutation in one or more genes that affect hemoglobin synthesis. The genes that are mutated can code for either the proteins that make up the hemoglobin molecule (globin or polypeptide chains) or the proteins involved in synthesizing or regulating synthesis of the globin chains. Regardless of the mutation encountered, all hemoglobinopathies affect hemoglobin synthesis in one of two ways: qualitatively or quantitatively. In qualitative hemoglobinopathies, hemoglobin synthesis occurs at a normal or near-normal rate, but the hemoglobin molecule has an altered amino acid sequence within the globin chains. This change in amino acid sequence alters the structure of the hemoglobin molecule (structural defect) and its function (qualitative defect). In contrast, thalassemias result in a reduced rate of hemoglobin synthesis (quantitative) but do not affect the amino acid sequence of the globin chains. A reduction in the amount of hemoglobin synthesized produces an anemia and stimulates the production of other hemoglobins not affected by the mutation in an attempt to compensate for the anemia. Based on this distinction, hematologists divide hemoglobinopathies into two categories: structural defects (qualitative) and thalassemias (quantitative). To add confusion to the classification scheme, many hematologists also refer to only the structural defects as hemoglobinopathies. This chapter describes the structural or qualitative defects that are referred to as hemoglobinopathies; the quantitative defects (thalassemias) are described in Chapter 28.

Structure of globin genes

As discussed in Chapter 10, there are six functional human globin genes located on two different chromosomes. Two of the globin genes, α and ζ, are located on chromosome 16 and are referred to as α-like genes. The remaining four globin genes, β, γ, δ, and ε, are located on chromosome 11 and are referred to as β-like genes. In the human genome, there is one copy of each globin gene per chromatid, for a total of two genes per diploid nucleus, with the exception of α and γ. There are two copies of the α and γ genes per chromatid, for a total of four genes per diploid nucleus. Each globin gene codes for the corresponding globin chain: the α-globin genes (HBA1 and HBA2) are used as the template to synthesize the α-globin chains, the β-globin gene (HBB) codes for the β-globin chain, the γ-globin genes (HBG1 and HBG2) code for the γ-globin chains, and the δ-globin gene (HBD) codes for the δ-globin chain.

Hemoglobin development

Each human hemoglobin molecule is composed of four globin chains: a pair of α-like chains and a pair of β-like chains. During the first 3 months of embryonic life, only one α-like gene (ζ) and one β-like gene (ε) are activated, which results in the production of ζ and ε globin chains that pair to form hemoglobin Gower-1 (ζ2ε2). Shortly thereafter, α and γ chain synthesis begins, which leads to the production of Hb Gower-2 (α2ε2) and Hb Portland (ζ2γ2). Later in fetal development, ζ and ε synthesis ceases; this leaves α and γ chains, which pair to produce Hb F (α2γ2), also known as fetal hemoglobin. During the 6 months after birth, γ chain synthesis gradually decreases and is replaced by β chain synthesis so that Hb A (α2β2), also known asadult hemoglobin, is produced. Recent evidence suggests BCL11A and KLF1, zinc-finger transcriptional repressors, are necessary to silence the γ-globin gene and mutations in the gene that codes for either factor results in elevated HbF levels.3 The remaining globin gene, δ, becomes activated around birth, producing δ chains at low levels that pair with α chains to produce the second adult hemoglobin, Hb A2 (α2δ2). Normal adults produce Hb A (95%), Hb A2 (less than 3.5%), and Hb F (less than 1% to 2%).

Genetic mutations

More than 1000 structural hemoglobin variants (hemoglobinopathies) are known to exist throughout the world, and more are being discovered regularly (Table 27-1).4,  5 Each of these hemoglobin variants results from one or more genetic mutations that alter the amino acid sequence. Some of these changes alter the molecular structure of the hemoglobin molecule, ultimately affecting hemoglobin function. The types of genetic mutations that occur in the hemoglobinopathies include point mutations, deletions, insertions, and fusions involving one or more of the adult globin genes—α, β, γ, and δ.5

TABLE 27-1

Molecular Abnormalities of Hemoglobin Variants

 

NUMBER OF VARIANTS BY GLOBIN CHAIN

 

α

β

δ

γ

Total

Amino acid substitution

413

535

65

96

1109

Deletions or insertions

22

48

1

1

72

Total

435

583

66

97

1181

Fusions

9*

* Seven fusions involve the β and δ chains; two fusions involve the β and γ chains.

Data from Patrinos GP, Giardine B, Riemer W, 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.xhtml. Accessed November 29, 2013.

The table is designed to provide a relative distribution of mutation types and just includes structural variants. Fifty-one of the variants are also categorized as thalassemias. Mutations are being added regularly.

Point mutation is the most common type of genetic mutation occurring in the hemoglobinopathies. Point mutation is the replacement of one original nucleotide in the normal gene with a different nucleotide. Because one nucleotide is replaced by one nucleotide, the codon triplet remains intact, and the reading frame is unaltered. This results in the substitution of one amino acid in the globin chain product at the position corresponding to the location of the original point mutation. As can be seen in Table 27-1, 1109 of the 1181 known hemoglobin variants result from a point mutation that causes an amino acid substitution. It also is possible to have two point mutations occurring in the same globin gene, which results in two amino acid substitutions within the same globin chain. Over 35 mutations occur by this mechanism.5

Deletions involve the removal of one or more nucleotides, whereas insertions result in the addition of one or more nucleotides. Usually deletions and insertions are not divisible by three and disrupt the reading frame, which leads to the nullification of synthesis of the corresponding globin chain. This is the case for the quantitative thalassemias (Chapter 28). In hemoglobinopathies, the reading frame usually remains intact, however; the result is the addition or deletion of one or more amino acids in the globin chain product, which sometimes affects the structure and function of the hemoglobin molecule. Of the 1181 variants described in Table 27-1, 72 variants result from deletions or insertions, or both.5

Chain extensions occur when the stop codon is mutated so that translation continues beyond the typical last codon. Amino acids continue to be added until a stop codon is reached by chance. This process produces globin chains that are longer than normal. Significant globin chain extensions usually result in degradation of the globin chain and a quantitative defect. If the extension of the globin chain is insufficient to produce significant degradation, however, the defect is qualitative and is classified as a hemoglobinopathy. Hemoglobin molecules with extended globin chains fold inappropriately, which affects hemoglobin structure and function.

Gene fusions occur when two normal genes break between nucleotides, switch positions, and anneal to the opposite gene. For example, if a β-globin gene and a δ-globin gene break in similar locations, switch positions, and reanneal, the resultant genes would be βδ and δβ fusion genes in which the head of the fusion gene is from one original gene and the tail is from the other. As long as the reading frames are not disrupted and the globin chain lengths are similar, the genes are transcribed and translated into hybrid globin chains. The fusion chains fold differently, however, and affect the corresponding hemoglobin function. Nine fusion globin chains have been identified (see Table 27-1).

Zygosity

Zygosity refers to the association between the number of gene mutations and the level of severity of the resultant genetic defect. Generally, there is a level of severity associated with each gene that is normally used to synthesize the globin chain product. For the normal adult globin genes, there are four copies of the α and γ genes and two copies of the β and δ genes. In theory, this could result in four levels of severity for α and γ gene mutations and two levels of severity for the β and δ gene mutations. Expressed another way, if all things were equal, it would require twice as many mutations within the α and γ genes to produce the same physiologic effect as mutations within the β and δ genes. Because the γ and δ genes are transcribed and translated at such low levels in adults, however, mutations of either gene would have little impact on overall hemoglobin function. In addition, because the dominant hemoglobin in adults, Hb A, is composed of α and β chains, β gene mutations would affect overall hemoglobin function to a greater extent than the same number of α gene mutations. This partially explains the greater number of identified β chain variants compared with α chain variants, because a single β gene mutation would be more likely to create a clinical condition than would a single α gene mutation.

The inheritance pattern of β chain variants is referred to as heterozygous when only one β gene is mutated and homozygous when both β genes are mutated. The terms disease and trait are also commonly used to refer to the homozygous (disease) and heterozygous (trait) states.

Pathophysiology

Pathophysiology refers to the manner in which a disorder translates into clinical symptoms. The impact of point mutations on hemoglobin function depends on the chemical nature of the substituted amino acid, where it is located in the globin chain, and the number of genes mutated (zygosity). The charge and size of the substituted amino acid may alter the manner in which the globin chain folds. A change in charge affects the interaction of the substituted amino acid with adjacent amino acids. In addition, the size of the substituted amino acid makes the globin chain either more or less bulky. Therefore, the charge and the size of the substituted amino acid determine its impact on hemoglobin structure by potentially altering the tertiary structure of the globin chain and the quaternary structure of the hemoglobin molecule. Changes in hemoglobin structure usually affect function. Location of the substitution within the globin chain also has an impact on the degree of structural alteration and hemoglobin function based on its positioning within the molecule and the interactions with the surrounding amino acids. In the case of the sickle cell mutation, one amino acid substitution results in hemoglobin polymerization, leading to the formation of long hemoglobin crystals that stretch the red blood cell (RBC) membrane and produce the characteristic crescent moon or sickle cell shape.

Zygosity also affects the pathophysiology of the disease. In β-hemoglobinopathies, zygosity predicts two severities of disease. In homozygous β-hemoglobinopathies, in which both β genes are mutated, the variant hemoglobin becomes the dominant hemoglobin type, and normal hemoglobin (Hb A) is absent. Examples are sickle cell disease (SCD, Hb SS) and Hb C disease (Hb CC). In heterozygous β-hemoglobinopathies, one β gene is mutated and the other is normal, which suggests a 50/50 distribution. In an attempt to minimize the impact of the abnormal hemoglobin, however, the variant hemoglobin is usually present in lesser amounts than Hb A. Nevertheless, in some cases they may be present in equal amounts. Examples are Hb S trait (Hb AS) and Hb C trait (Hb AC). Patients with homozygous sickle cell disease (Hb SS) inherit a severe form of the disease that occurs less frequently but requires lifelong medical intervention, which must begin early in life, whereas heterozygotes (Hb AS) are much more common but rarely experience symptoms.

Fishleder and Hoffman6 divided the structural hemoglobins into four groups: abnormal hemoglobins that result in hemolytic anemia, such as Hb S and the unstable hemoglobins; abnormal hemoglobins that result in methemoglobinemia, such as Hb M; hemoglobins with either increased or decreased oxygen affinity; and abnormal hemoglobins with no clinical or functional effect. Imbalanced chain production also may be associated in rare instances with a structurally abnormal chain, such as Hb Lepore,4 because of the reduced production of the abnormal chain. The functional classification of selected hemoglobin variants is summarized in Box 27-1.

BOX 27-1

Functional Classification of Selected Hemoglobin (Hb) Variants

  1. homozygous: hemoglobin polymorphisms: The variants that are most common

Hb S: α2β26Val—severe hemolytic anemia; sickling

Hb C: α2β26Lys—mild hemolytic anemia

Hb D-Punjab: α2β2121Gln—no anemia

Hb E: α2β226Lys—mild microcytic anemia

  1. heterozygous: Hemoglobin variants causing functional aberrations or hemolytic anemia in the heterozygous state

A. hemoglobins associated with methemoglobinemia and cyanosis

  1. Hb M-Boston: α258Tyrβ2
  2. Hb M-Iwate: α287Tyrβ2
  3. Hb Auckland: α287Asnβ2
  4. Hb Chile: α2β228Met
  5. Hb M-Saskatoon: α2β263Tyr
  6. Hb M-Milwaukee-1: α2β267Glu
  7. Hb M-Milwaukee-2: α2β292Tyr
  8. Hb F-M-Osaka: α2γ263Tyr
  9. Hb F-M-Fort Ripley: α2γ292Tyr

B. hemoglobins associated with altered oxygen affinity

  1. Increased affinity and erythrocytosis
  2. Hb Chesapeake: α292Leuβ2
  3. Hb J-Capetown: α292Glnβ2
  4. Hb Malmo: α2β297Gln
  5. Hb Yakima: α2β299His
  6. Hb Kempsey: α2β299Asn
  7. Hb Ypsi (Ypsilanti): α2β299Tyr
  8. Hb Hiroshima: α2β2146Asp
  9. Hb Rainier: α2β2145Cys
  10. Hb Bethesda: α2β2145His
  11. Decreased affinity—may have mild anemia or cyanosis
  12. Hb Kansas: α2β2102Thr
  13. Hb Titusville: α294Asnβ2
  14. Hb Providence: α2β282Asn
  15. Hb Agenogi: α2β290Lys
  16. Hb Beth Israel: α2β2102Ser
  17. Hb Yoshizuka: α2β2108Asp

C. unstable hemoglobins

  1. Hemoglobin may precipitate as Heinz bodies after splenectomy (congenital Heinz body anemia)
  2. Severe hemolysis: no improvement after splenectomy

Hb Bibba: α2136Proβ2

Hb Hammersmith: α2β242Ser

Hb Bristol-Alesha: α2β267Asp or 67Met

Hb Olmsted: α2β2141Arg

  1. Severe hemolysis: improvement after splenectomy

Hb Torino: α243Valβ2

Hb Ann Arbor: α280Argβ2

Hb Genova: α2β228Pro

Hb Shepherds Bush: α2β274Asp

Hb Köln: α2β298Met

Hb Wien: α2β2130Asp

  1. Mild hemolysis: intermittent exacerbations

Hb Hasharon: α247Hisβ2

Hb Leiden: α2β26 or 7 (Glu deleted)

Hb Freiburg: α2β223 (Val deleted)

Hb Seattle: α2β270Asp

Hb Louisville: α2β242Leu

Hb Zurich: α2β263Arg

Hb Gun Hill: α2β2 (5 amino acids deleted)

  1. No disease

Hb Etobicoke: α284Argβ2

Hb Sogn: α2β214Arg

Hb Tacoma: α2β230Ser

  1. Tetramers of normal chains; appear in thalassemias

Hb Bart: γ4

Hb H: β4

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.

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.

Many of the variants are clinically insignificant because they do not show any physiologic effect. As discussed previously, most clinical abnormalities are associated with the β chain followed by the α chain. Involvement of the γ and δ chains does occur, but because of the small amount of hemoglobin involved, it is rarely detected and is usually of no consequence.Box 27-2 lists clinically significant abnormal hemoglobins. The most frequently occurring of the abnormal hemoglobins and the most severe is Hb S.

BOX 27-2

Clinically Important Hemoglobin (Hb) Variants

  1. Sickle syndromes
  2. Sickle cell trait (AS)
  3. Sickle cell disease
  4. SS
  5. SC
  6. SD-Punjab (Los Angeles)
  7. SO-Arab
  8. S–β-Thalassemia
  9. S–hereditary persistence of fetal hemoglobin
  10. SE
  11. Unstable hemoglobins→congenital Heinz body anemia (> 140 variants)

III. Hemoglobins with abnormal oxygen affinity

  1. High affinity→familial erythrocytosis (> 90 variants)
  2. Low affinity→familial cyanosis (Hbs Kansas, Beth Israel, Yoshizuka, Agenogi, Titusville, Providence)
  3. M hemoglobins→familial cyanosis (9 variants): Hb M-Boston, Hb M-Iwate, Hb Auckland, Hb Chile, Hb M-Saskatoon, Hb M-Milwaukee-1, Hb M-Milwaukee-2 (Hyde Park), Hb FM-Osaka, Hb FM-Fort Ripley
  4. Structural variants that result in a thalassemic phenotype
  5. β-Thalassemia phenotype
  6. Hb Lepore (δβ fusion)
  7. Hb E
  8. Hb-Indianapolis, Hb-Showa-Yakushiji, Hb-Geneva
  9. α-Thalassemia phenotype chain termination mutants (e.g., Hb Constant Spring)

Modified from Lukens JN: Abnormal hemoglobins: general principles (chap 39); Wong WC: Sickle cell anemia and other sickling syndromes (chap 40); Lukens JN: Unstable hemoglobin disease (chap 41). In Greer JP, Foerster J, Lukens JN, et al, editors: Wintrobe’s clinical hematology, ed 11, Philadelphia, 2004, Lippincott Williams & Wilkins. 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.xhtml. Accessed November 30, 2013.

Nomenclature

As hemoglobins were reported in the literature, they were designated by letters of the alphabet. Normal adult hemoglobin and fetal hemoglobin were called Hb A and Hb F. By the time the middle of the alphabet was reached, however, it became apparent that the alphabet would be exhausted before all mutations were named. Currently, some abnormal hemoglobins are assigned a common designation and a scientific designation. The common name is selected by the discoverer and usually represents the geographic area where the hemoglobin was identified. A single capital letter is used to indicate a special characteristic of the hemoglobin variants, such as hemoglobins demonstrating identical electrophoretic mobility but containing different amino acid substitutions, as in Hb G-Philadelphia, Hb G-Copenhagen, and Hb C-Harlem. The variant description also can involve scientific designations that indicate the variant chain, the sequential and the helical number of the abnormal amino acid, and the nature of the substitution. The designation [β6 (A3) Glu→Val] for the Hb S mutation indicates the substitution of valine for glutamic acid in the A helix in the β chain at position 6.4

Hemoglobin s

Sickle cell anemia

History

Although the origin of sickle cell anemia has not been identified, symptoms of the disease have been traced in one Ghanaian family back to 1670.7 Sickle cell anemia was first reported by a Chicago cardiologist, Herrick, in 1910 in a West Indian student with severe anemia. In 1917, Emmel recorded that sickling occurred in nonanemic patients and in patients who were severely anemic. In 1927, Hahn and Gillespie described the pathologic basis of the disorder and its relationship to the hemoglobin molecule. These investigators showed that sickling occurred when a solution of RBCs was deficient in oxygen and that the shape of the RBCs was reversible when that solution was oxygenated again.4,  8 In 1946, Beet reported that malarial parasites were present less frequently in blood films from patients with SCD than in individuals without SCD.9 It was determined that the sickle cell trait confers a resistance against infection with Plasmodium falciparum occurring early in childhood between the time that passively acquired immunity dissipates and active immunity develops.10 In 1949, Pauling showed that when Hb S is subjected to electrophoresis, it migrates differently than does Hb A. This difference was shown to be caused by an amino acid substitution in the globin chain. Pauling and coworkers defined the genetics of the disorder and clearly distinguished heterozygous sickle trait (Hb AS) from the homozygous state (Hb SS).4

The term sickle cell diseases is used to describe a group of symptomatic hemoglobinopathies that have in common sickle cell formation and the associated crises. Patients with SCD are either homozygous for Hb S (SS) or are compound heterozygotes expressing Hb S in combination with another hemoglobin β chain mutation like Hb C or β-thalassemia. SCDs are the most common form of hemoglobinopathy, with Hb SS and the variants Hb SC and Hb S–β-thalassemia (Hb S–β-thal) occurring most frequently.

Inheritance pattern

As stated earlier, the genes that code for the globin chains are located at specific loci on chromosomes 16 and 11. The α-like genes (α and ζ) are located on the short arm of chromosome 16, whereas the β-like genes (β, γ, δ, and ε) are located on the short arm of chromosome 11. With the exception of the γ genes, which have four loci, each β-like gene has two loci. β-hemoglobin variants are inherited as autosomal codominants, with one gene inherited from each parent.4

Patients with SCD (Hb SS), Hb SC, or Hb S–β-thal have inherited a sickle (S) gene from one parent and an S, C, or β-thalassemia gene from the other. Among patients with SCD, individuals who are homozygotes (Hb SS) have more severe disease than individuals who are compound heterozygotes for Hb S (Hb SC or Hb S–β-thal). Heterozygotes (Hb AS) are generally asymptomatic. Using Hb S and Hb C as examples, Figure 27-2 illustrates the inheritance of abnormal hemoglobins involving mutations in the β gene.

 
FIGURE 27-2 Punnett square illustrating the standard method for predicting the inheritance of abnormal hemoglobins. Each parent contributes one gene.

Prevalence

The highest frequency of the sickle cell gene is found in sub-Saharan Africa, where each year approximately 230,000 babies are born with sickle cell disease (Hb SS), representing 0.74% of all live births occurring in this area.11 In contrast, approximately 2600 babies are born annually with sickle cell disease in North America and 1300 in Europe.11 Globally, the sickle cell gene occurs at the highest frequency in five geographic areas: sub-Saharan Africa, Arab-India, the Americas, Eurasia, and Southeast Asia. In 2010, these five geographic areas accounted for 64.4%, 22.7%, 7.4%, 5.4%, and 0.1%, respectively, of all neonates born globally with sickle cell trait, and 75.5%, 16.9%, 4.6%, 3.0%, and 0%, respectively, of all neonates born globally with sickle cell disease. Three countries accounted for approximately 50% of neonates with SS and AS genotypes: Nigeria, India, and DR Congo.12 Although in the United States, SCD is found mostly in individuals of African descent, it also has been found in individuals 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 mutation is becoming more prominent in southern India, particularly in certain tribes.14 It is estimated that 25,000 babies are born annually with sickle cell anemia in India.2

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

Etiology and pathophysiology

Hb S is defined by the structural formula α2β26GluVal, which indicates that on the β chain at position 6, glutamic acid is replaced by valine. The mutation occurs in nucleotide 17, where thymine is changed to adenine, resulting in a change in codon 6 and the substitution of valine for glutamic acid at amino acid position 6.11 Glutamic acid has a net charge of (−1), whereas valine has a net charge of (0). This amino acid substitution produces a change in charge of (+1), which affects the electrophoretic mobility of the hemoglobin molecule. This amino acid substitution also affects the way the hemoglobin molecules interact with one another within the erythrocyte cytosol. The nonpolar (hydrophobic) valine amino acid has been placed in the position that the polar glutamic acid once held. Because glutamic acid is polar, the β chain folds in such a way that glutamic acid extends outward from the surface of the hemoglobin tetramer to bind water and contribute to hemoglobin solubility in the cytosol. Therefore, the hydrophobic valine is also extended outward, but instead of binding water, it seeks a hydrophobic niche with which to bind. When Hb S is fully oxygenated, the quaternary structure of the molecule does not produce a hydrophobic pocket for valine to bind to, which allows the hemoglobin molecules to remain soluble in the erythrocyte cytosol like Hb A and maintains the normal biconcave disc shape of the RBCs. However, the natural allosteric change that occurs upon deoxygenation creates a hydrophobic pocket in the area of phenylalanine 85 and leucine 88, which allows the valine from an adjacent hemoglobin molecule to bind. This hemoglobin pairing creates an orientation that helps other hemoglobin molecules to form electrostatic bonds between amino acids and becomes the seed for polymer formation. Other hemoglobin pairs polymerize, forming a hemoglobin core composed of four hemoglobin molecules that elongate in a helical formation. An outer layer of 10 hemoglobin molecules forms around the 4-hemoglobin-molecule core, creating the long, slender Hb S polymer.15-18 Hb S molecules within the RBCs become less soluble, forming tactoids or liquid crystals of Hb S polymers that grow in length beyond the diameter of the RBC, causing sickling. In homozygotes, the sickling process begins when oxygen saturation decreases to less than 85%. In heterozygotes, sickling does not occur unless the oxygen saturation of hemoglobin is reduced to less than 40%.19 The blood becomes more viscous when polymers are formed and sickle cells are created.19 Increased blood viscosity and sickle cell formation slow blood flow. In addition to a decrease in oxygen tension, there is a reduction in the pH and an increase in 2,3-bisphosphoglycerate. Reduced blood flow prolongs the exposure of Hb S-containing erythrocytes to a hypoxic environment, and the lower tissue pH decreases the oxygen affinity, which further promotes sickling. The end result is occlusion of capillaries and arterioles by sickled RBCs and infarction of surrounding tissue.

Sickle cells occur in two forms: reversible sickle cells and irreversible sickle cells.20 Reversible sickle cells are Hb S–containing erythrocytes that change shape in response to oxygen tension. Reversible sickle cells circulate as normal biconcave discs when fully oxygenated but undergo hemoglobin polymerization, show increased viscosity, and change shape on deoxygenation. The vasoocclusive complications of SCD are thought to be due to reversible sickle cells that are able to travel into the microvasculature in the biconcave disk conformation due to their normal rheologic properties when oxygenated and then become distorted and viscous as they become deoxygenated, converting to the sickle cell configuration in the vessel.

In contrast, irreversible sickle cells do not change their shape regardless of the change in oxygen tension or degree of hemoglobin polymerization. These cells are seen on the peripheral blood film as elongated sickle cells with a point at each end. It is thought that irreversible sickle cells are recognized as abnormal by the spleen and removed from circulation, which prevents them from entering the microcirculation and causing vasoocclusion.

Not only the oxygen tension but also the level of intracellular hydration affects the sickling process. When RBCs containing Hb S are exposed to a low oxygen tension, hemoglobin polymerization occurs. Polymerized deoxyhemoglobin S activates a membrane channel called P sickle that is otherwise inactive in normal RBCs. These membrane channels open when the blood partial pressure of oxygen decreases to less than 50 mm Hg. Open Psickle channels allow the influx of Ca2+, raising the intracellular calcium levels and activating a second membrane channel called the Gardos channel. An activated Gardos channel causes the efflux of K+, which stimulates the efflux of Cl through another membrane channel to maintain charge equilibrium across the RBC membrane. The efflux of these ions leads to water efflux and intracellular dehydration, effectively increasing the intracellular concentration of Hb S and intensifying polymerization. Another contributor to K+ and Cl efflux and the resultant dehydration is the K+/Cl cotransporter system. Ironically, this system is activated by dehydration and positively charged hemoglobins such as Hb S and Hb C. The K+/Cl cotransporter pathway is also activated by the low pH encountered in the spleen and kidneys. One potential explanation for the altered function of the membrane channels is oxidative damage triggered by Hb S polymerization. Injury to the RBC membrane induces adherence to endothelial surfaces, which causes RBC aggregation, produces ischemia, and exacerbates Hb S polymerization.10

Another important factor in the pathophysiology of SCD involves the redistribution of phospholipids in the RBC membrane, which contributes to hemolysis, vasoocclusive crisis, stroke, and acute chest syndrome. In the bilayer membranes of normal RBCs, choline phospholipids like sphingomyelin and phosphatidylcholine are located on the outer plasma layer, whereas aminophospholipids like phosphatidylserine (PS) and phosphatidylethanolamine are primarily on the inner cytoplasmic layer of the membrane. This asymmetrical distribution of membrane phospholipids is accomplished by adenosine triphosphate–dependent enzymes called translocases or flippases. Inhibition of flippases and activation of an enzyme calledscramblase cause a more random distribution of membrane phospholipids, which increases the number of choline phospholipids on the interior half of the membrane and the number of aminophospholipids on the exterior membrane surface. The sickle cells of homozygotes (Hb SS) express 2.1% PS on erythrocyte exterior surfaces compared with 0.2% for normal Hb AA controls.21,  22 It is hypothesized that Hb S polymerization may produce microparticles and iron complexes that adhere to the RBC membrane and generate reactive oxygen species, which, along with increased intracellular calcium or protein kinase C activation, may contribute to flippase inhibition and scramblase activation.23,  24 PS on the exterior surface of RBCs binds thrombospondin on vascular endothelial cells,25 enhancing adherence between RBCs and the vessel wall and contributing to vasoocclusive crisis, activation of coagulation, and decreased RBC survival.26,  27 In addition, RBCs with PS on the external membrane surface are vulnerable to hydrolysis by secretory phospholipase A2 (sPLA2), which generates lysophospholipids and fatty acids like lysophosphatidic acid. This results in vascular damage that contributes to acute chest syndrome.28,  29

Clinical features

The clinical manifestations of SCD can vary from no symptoms to a potentially lethal state. Symptoms also vary between ethnic groups with Indian patients expressing a much milder disease than their African counterparts.14 People with SCD can develop a variety of symptoms as listed in Box 27-3. Over a thousand hemoglobin variants are known; however, only eight genotypes cause severe disease: Hb SS, Hb S–β0-thal, severe Hb S–β+-thal, Hb SD-Punjab, Hb SO-Arab, Hb SC-Harlem, Hb CS-Antilles, and Hb S-Quebec-CHORI. These eight clinically significant forms are listed in the order of severity and can have high morbidity and mortality rates. Three additional genotypes produce moderate disease: Hb SC, moderate Hb S-β+-thal, and Hb AS-Oman. Three produce mild disease: mild Hb S-βsilent-thal, Hb SE, and Hb SA-Jamaica Plain. Two produce very mild disease: Hb S-HPFH and Hb S with a variety of mild variants.11 Symptom variability in patients with sickle cell disease and across the genotypes listed above are largely due to the intracellular ratio of Hb S to Hb F, as well as factors that affect vessel tone and cellular activation.30 Individuals affected with SCD are characteristically symptom free until the second half of the first year of life because of the protective effect of Hb F.31 Toward the end of the first 6 months of life, mutated β chains begin to be produced and gradually replace normal γ chains, which causes Hb S levels to increase and Hb F levels to decrease. Erythrocytes containing Hb S become susceptible to hemolysis, and a progressive hemolytic anemia and splenomegaly may become evident.

BOX 27-3

Clinical Features of Sickle Cell Disease

  1. Vasoocclusion
  2. Causes:

Acidosis

Hypoxia

Dehydration

Infection

Fever

Extreme cold

  1. Clinical manifestations
  2. Bones:

Pain

Hand-foot dactylitis

Infection (osteomyelitis)

  1. Lungs:

Pneumonia

Acute chest syndrome

  1. Liver:

Hepatomegaly

Jaundice

  1. Spleen:

Sequestration splenomegaly

Autosplenectomy

  1. Penis:

Priapism

  1. Eyes:

Retinal hemorrhage

  1. Central nervous system
  2. Urinary tract:

Renal papillary necrosis

  1. Leg ulcers
  2. Bacterial infections
  3. Sepsis
  4. Pneumonia
  5. Osteomyelitis

III. Hematologic defects

  1. Chronic hemolytic anemia
  2. Megaloblastic episodes
  3. Aplastic episodes
  4. Cardiac defects
  5. Enlarged heart
  6. Heart murmurs
  7. Other clinical features
  8. Stunted growth
  9. High-risk pregnancy

Many individuals with SCD undergo episodes of recurring 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 of the acute painful episode first described by Diggs is not fully understood. Various crises may occur: vasoocclusive or “painful,” aplastic, megaloblastic, sequestration, and chronic hemolytic.

The hallmark of SCD is vasoocclusive crisis (VOC), which accounts for most hospital and emergency department visits. This acute, painful aspect of SCD occurs with great predictability and severity in many individuals and can be triggered by acidosis, hypoxia, dehydration, infection and fever, and exposure to extreme cold. Painful episodes manifest most often in the bones, lungs, liver, spleen, penis, eyes, central nervous system, and urinary tract.

The pathogenesis of vasoocclusion in SCD is not fully understood, but Hb S polymerization and sickling of RBCs play a major role, with other factors also affecting this process. Most VOC events occur in capillaries and postcapillary venules.6,  33 The list of possible risk factors includes polymerization, decreased deformability, sickle cell–endothelial cell adherence, endothelial cell activation, white blood cell (WBC) and platelet activation, hemostatic activation, and altered vascular tone.33 The interrelationships among these risk factors is shown inFigure 27-4. Vasoocclusion can be triggered by any of these factors under various circumstances. During inflammation, increased WBCs interacting with endothelium, platelet activation causing elevation of thrombospondin level, or clinical dehydration resulting in an increase in von Willebrand factor can trigger RBC adherence to endothelium, precipitating vascular obstruction. Another mechanism of obstruction can be dense cells, which are less deformable and are at greatest risk for intracellular polymerization because of their higher Hb S concentration.34,  35 Vasoocclusive episodes gradually consume the patient organ by organ, through the destructive and debilitative effects of cumulative infarcts. Approximately 8% to 10% of SCD patients develop cutaneous manifestations in the form of ulcers or sores on the lower leg.11

 
FIGURE 27-4 Numerous risk factors for vasoocclusion are highly interrelated physiologically, as shown here. RBC, Red blood cell; WBC, white blood cell.  Source:  (From Embury SH, Hebbel RP, Mohandas N, et al: Sickle cell disease: basic principles and clinical practice, Philadelphia, 1994, Lippincott Williams & Wilkins, p. 322.)

The abnormal interaction between sickle cells and vascular endothelium seems to have a great impact on the vasoocclusive event. Endothelial adherence correlates significantly with the severity of painful episodes. In addition, sickle cell adherence to vascular endothelium results in intimal hyperplasia that can slow blood flow.36 Cells of patients with Hb SC disease produce less sickling with fewer adherent RBCs.8,  37

The frequency of painful episodes varies from none to six per year.8 On average, each episode persists for 4 to 5 days, although protracted episodes may last for weeks. Repeated splenic infarcts produce scarring resulting in diminished splenic tissue and abnormal function. Splenic sequestration is characterized by a sudden trapping of blood in the spleen, which leads to a rapid decline in hemoglobin, often to less than 6 g/dL.37 This phenomenon occurs most often in infants and young children whose spleens are chronically enlarged. Children experiencing splenic sequestration episodes may have earlier onset of splenomegaly and a lower level of Hb F at 6 months of age.8 Crises are often associated with respiratory tract infections. Gradual loss of splenic function is referred to as autosplenectomy and is evidenced by the presence of Howell-Jolly and Pappenheimer bodies in RBCs on the peripheral blood film. In the lungs, pulmonary infarction from sickling in the microvasculature causes acute chest syndrome.

Acute chest syndrome is characterized by fever, chest pain, and presence of pulmonary infiltrates on the chest radiograph and is the leading cause of death among adults with SCD. Over 10% of adults with acute chest syndrome die from complications linked to chronic lung disease and pulmonary hypertension.38 In children, acute chest syndrome generally is precipitated by infection characterized by fever, cough, and tachypnea. Acute chest syndrome is also linked with sPLA2, discussed previously. The level of sPLA2 has been shown to be a predictor of acute chest syndrome in patients with SCD39 in that sPLA2 rises 24 to 48 hours before symptoms of acute chest syndrome begin.40 In addition, a high sPLA2 level correlates with the degree of lung damage.

Pulmonary hypertension (PHT) is a serious and potentially fatal sequela of SCD. Among patients with SCD, PHT has a prevalence of about 33%, with 10% of patients manifesting a more severe version.41 The mortality rate for sickle cell patients who develop PHT is 40% at 40 months.41 An association has been documented between the development of PHT and the nitrous oxide (NO) pathway. NO is produced from the action of endothelial NO synthase (eNOS) on arginine, which causes vasodilation. Patients with SCD have a decrease in NO, and this leads to vasoconstriction and hypertension.4,  41 In addition, low NO levels in the blood fail to inhibit endothelin-1, a potent vasoconstrictor, which results in additional vasoconstriction and hypertension.38 The connection between NO and SCD involves the hemolytic crisis. Erythrocyte hemolysis releases high levels of arginase, which degrades arginine; the result is less NO production from eNOS.42,  43 In addition, the free hemoglobin released from hemolyzed RBCs scavenges NO, which further reduces the levels and exacerbates the vasoconstriction and hypertension.41 Blood arginine and NO levels drop a few days before the onset of acute chest syndrome,37 a finding suggesting that the NO pathway is a connection between SCD, PHT, and asthma.38,  44 Treatment with large doses of arginine reduces pulmonary artery pressure, but the effect is not sustainable and does not reduce mortality. An increased tricuspid regurgitation velocity (TRV) and blood NT-ProBNP levels above 160 ng/L were found to be good predictors of pulmonary hypertension and are associated with a higher mortality rate.33 Bosentan is the treatment of choice for pulmonary hypertension, but liver enzymes should be monitored for liver toxicity.33

Bacterial infections pose a major problem for SCD patients. These patients have increased susceptibility to life-threatening infection from Staphylococcus aureus, Streptococcus pneumoniae,and Haemophilus influenzae. Acute infections are common causes of hospitalization and have been the most frequent cause of death, especially in the first 3 years of life.1 Bacterial infections of the blood (septicemia) are exacerbated by the autosplenectomy effect as the spleen gradually loses its ability to function as a secondary lymphoid tissue to effectively clear organisms from the blood.

Chronic hemolytic anemia is characterized by shortened RBC survival of between 16 and 20 days,45 with a corresponding decrease in hemoglobin and hematocrit, an elevated reticulocyte count, and jaundice. Continuous screening and removal of sickle cells by the spleen perpetuate the chronic hemolytic anemia and autosplenectomy effect. Because other conditions, such as hepatitis and gallstones, may cause jaundice, chronic hemolysis is difficult to diagnose in sickle cell patients.21 RBC hemolysis releases free hemoglobin, which disrupts the arginine-nitric oxide pathway, resulting in the sequestration and lowering of nitric oxide.31,  45 Decreased NO leads to endothelial cell activation, vasoconstriction, adherence of RBCs to the endothelium, and pulmonary hypertension previously discussed.45 Another major sequelae of hemolysis is renal dysfunction, which can be detected early by an increased glomerular filtration rate of 140 mL/min per 1.73 m3 found in 71% of patients with SCD.46 Progression of renal dysfunction can be identified by detecting microalbuminuria (> 4.5 mg/mmol), followed by proteinuria and terminating in elevated BUN and creatinine levels. Angiotensin-converting enzyme inhibitors (ACEI) have been shown to lower proteinuria in SCD patients.33

Megaloblastic episodes result from the sudden arrest of erythropoiesis due to folate depletion. Folic acid deficiency as a cause of exaggerated anemia in SCD is extremely rare in the United States. It is common practice to prescribe prophylactic folic acid for patients with SCD, however.8

Aplastic episodes (bone marrow failure) are the most common life-threatening hematologic complications and are usually associated with infection, particularly parvovirus infection.34Aplastic episodes present clinical problems similar to those seen with other hemolytic disorders.47 Sickle cell patients usually can compensate for the decrease in RBC survival by increasing bone marrow output. When the bone marrow is suppressed temporarily by bacterial or viral infections, however, the hematocrit decreases substantially with no reticulocyte compensation. The spontaneous recovery phase is characterized by the presence of nucleated RBCs and an increase in the number of reticulocytes in the peripheral blood. Most aplastic episodes are short-lived and require no therapy. If anemia is severe and the bone marrow remains aplastic, transfusions become necessary. If patients are not transfused in a timely fashion, death can occur.47

Patients also experience cardiac defects, including enlarged heart and heart murmurs. In patients with severe anemia, cardiomegaly can develop as the heart works harder to maintain adequate blood flow and tissue oxygenation. Increased cardiac workload along with increased bone marrow erythropoiesis increases calorie burning, contributing to a reduced growth rate.48 When patients enter childbearing age, pregnancy becomes risky.4

Impaired blood supply to the head of the femur and humerus results in a condition called avascular necrosis (AVN). About 50% of patients with SCD develop AVN by 35 years of age.49Physical therapy and surgery to relieve intramedullary pressure within the head of the long bones are effective, but hip and/or shoulder implants become necessary in most patients experiencing AVN.49 Similarly, leg ulcers are a common complication of SCD. Ulcers tend to heal slowly, develop unstable scars, and recur at the same site, becoming a chronic problem, with associated chronic pain.47

Microstrokes can lead to headaches, poor school performance, reduced intelligence quotient (IQ), and overt central nervous system dysfunction. A neurologic examination followed by magnetic resonance imaging and, if available, transcranial Doppler ultrasonography or magnetic resonance angiography is recommended to detect microstrokes.48

Incidence with malaria and glucose-6-phosphate dehydrogenase deficiency

The sickle gene occurs with greatest frequency in Central Africa, the Near East, the region around the Mediterranean, and parts of India. The frequency of the gene parallels the incidence of P. falciparum and seems to offer some protection against cerebral falciparum malaria in young patients. Malarial parasites are living organisms within the RBCs that use the oxygen within the cells. This reduced oxygen tension causes the cells to sickle, which results in injury to the cells. These injured cells tend to become trapped within the blood vessels of the spleen and other organs, where they are easily phagocytized by scavenger WBCs. Selective destruction of RBCs containing parasites decreases the number of malarial organisms and increases the time for immunity to develop. One explanation for this phenomenon is that the infected cell is uniquely sickled and destroyed, probably in an area of the spleen or liver, where phagocytic cells are plentiful, and the oxygen tension is significantly decreased.50

Because of the high incidence of glucose-6-phosphate dehydrogenase (G6PD) deficiency in patients with SCD, it has been suggested that G6PD deficiency has a protective effect in these patients,51 although this correlation has not been confirmed through studies. It also has been postulated that hemolytic episodes are more common in these patients. In the first 42 months of life, patients with SCD and G6PD deficiency had lower steady-state hemoglobin levels, higher reticulocyte counts, three times more acute anemia events, and more frequent blood transfusions—vasoocclusive and infectious events than matched sickle cell patients without G6PD deficiency.30 Because of the presence of young cells rich in G6PD, however, the increased hemolysis is more likely caused by the enzyme abnormality when the population is shifted to the oldest cells during an aplastic crisis.52

Laboratory diagnosis

The anemia of SCD is a chronic hemolytic anemia, classified morphologically as normocytic, normochromic. The characteristic diagnostic cell observed on a Wright-stained peripheral blood film is a long, curved cell with a point at each end (Figure 27-5). Because of its appearance, the cell was named a sickle cell.32 The peripheral blood film shows marked poikilocytosis and anisocytosis with normal RBCs, sickle cells, target cells, nucleated RBCs along with a few spherocytes, basophilic stippling, Pappenheimer bodies, and Howell-Jolly bodies. The presence of sickle cells and target cells is the hallmark of SCD. There is moderate to marked polychromasia with a reticulocyte count between 10% and 25%, corresponding with the hemolytic state and the resultant bone marrow response. The RBC distribution width (RDW) is increased owing to moderate anisocytosis. The mean cell volume (MCV) is not as elevated as one would expect, however, given the elevated reticulocyte count. An aplastic crisis can be heralded by a decreased reticulocyte count. Moderate leukocytosis is usually present (sometimes 40 to 50 × 109 WBC/L) with neutrophilia and a mild shift toward immature granulocytes. The leukocyte alkaline phosphatase score is not elevated when neutrophilia is caused by sickle cell crisis alone when no underlying infection is present. Thrombocytosis is usually present. The bone marrow shows erythroid hyperplasia, reflecting an attempt to compensate for the anemia, which results in polychromasia and an increase in reticulocytes and nucleated RBCs in the peripheral blood. Levels of immunoglobulins, particularly immunoglobulin A, are elevated in all forms of SCD. Serum ferritin levels are normal in young patients but tend to be elevated later in life; however, hemochromatosis is rare. Chronic hemolysis is evidenced by elevated levels of indirect and total bilirubin with the accompanying jaundice.

 
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 (×1000). B, Peripheral blood film for an SCD patient showing anisocytosis, poikilocytosis, sickle cells, target cells, and one nucleated RBC (×1000). Platelets are not present in this field, but their numbers were adequate in this patient.  Source:  (Courtesy Ann Bell, University of Tennessee, Memphis.)

The diagnosis of SCD is generally a two-step process by first demonstrating the insolubility of deoxygenated Hb S in solution followed by confirmation of its presence using hemoglobin electrophoresis, high-performance liquid chromatography (HPLC), or capillary electrophoresis. For more complicated cases, isoelectric focusing, tandem mass spectrometry, or DNA analysis may be needed. An older screening test detects Hb S insolubility by inducing sickle cell formation on a glass slide. A drop of blood is mixed with a drop of 2% sodium metabisulfite (a reducing agent) on a slide, and the mixture is sealed under a coverslip. The hemoglobin inside the RBCs is reduced to the deoxygenated form; this induces polymerization and the resultant sickle cell formation, which can be identified microscopically. This method is slow and cumbersome and is rarely used.

The most common screening test for Hb S, called the hemoglobin solubility test, capitalizes on the decreased solubility of deoxygenated Hb S in solution, producing turbidity. Blood is added to a buffered salt solution containing a reducing agent, such as sodium hydrosulfite (dithionite), and a detergent-based lysing agent (saponin). The saponin dissolves membrane lipids, causing the release of hemoglobin from the RBCs, and the dithionite reduces the iron from the ferrous to the ferric oxidation state. Ferric iron is unable to bind oxygen, converting the hemoglobin to the deoxygenated form. Deoxygenated Hb S polymerizes in solution, which renders it turbid, whereas solutions containing nonsickling hemoglobins remain clear (Figure 27-6). False-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 hematocrits. 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 (β6GluVal) and another unrelated substitution. Hb S-Antilles is particularly important because it can cause sickling in the heterozygous state.

 
FIGURE 27-6 Tube solubility screening test for the presence of hemoglobin S. In a negative test result (left), the solution is clear and the lines behind the tube are visible. In a positive test result (right), the solution is turbid and the lines are not visible.  Source:  (Courtesy Ann Bell, University of Tennessee, Memphis.)

Alkaline hemoglobin electrophoresis is a common first step in the confirmation of hemoglobinopathies, including SCD. Electrophoresis is based on the separation of hemoglobin molecules in an electric field due primarily to differences in total molecular charge. In alkaline electrophoresis, hemoglobin molecules assume a negative charge and migrate toward the anode (positive pole). Historically, alkaline hemoglobin electrophoresis was performed on cellulose acetate medium but is being replaced by electrophoresis on agarose medium. Nonetheless, because some hemoglobins have the same charge and, therefore, the same electrophoretic mobility patterns, hemoglobins that exhibit an abnormal electrophoretic pattern at an alkaline pH may be subjected to electrophoresis at an acid pH for definitive separation. In an acid pH some hemoglobins assume a negative charge and migrate toward the anode, while others are positively charged and migrate toward the cathode (negative pole). For example, Hb S migrates with Hb D and Hb G on alkaline electrophoresis but separates from Hb D and Hb G on acid electrophoresis. Similarly, Hb C migrates with Hb E and Hb O on alkaline electrophoresis but separates on acid electrophoresis. Figure 27-7 shows electrophoretic patterns for normal and abnormal hemoglobins. Figure 27-8 shows the electrophoretic separation of a normal adult and a patient with sickle cell disease (Hb SS) at an alkaline pH. HPLC and capillary 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 observed with electrophoresis (Figure 27-9).

 
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 AS), the bands may appear equal, but the amount of Hb A exceeds that of Hb S.  Source:  (From Schmidt RM, Brosious EF: Basic laboratory methods of hemoglobinopathy detection, ed 6, HEW Pub No (CDC) 77-8266, Atlanta, 1976, Centers for Disease Control and Prevention.)

 
FIGURE 27-8 Electrophoretic separation of hemoglobins (Hb) at alkaline pH. 1, Normal adult; 2 and 3, 17-year-old patient with sickle cell anemia (Hb 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 standard (Hydragel 7 Hemoglobin/Hydrasys System, Sebia Electrophoresis, Norcross, GA).  Source:  (Modified from Elghetany MT, Banki K: Erythrocytic disorders. In McPherson RA, Pincus MR: Henry’s Clinical Diagnosis and Management by Laboratory Methods, ed 22, Philadelphia, 2011, Elsevier, p. 578.)

 
FIGURE 27-9 Ion-exchange high-performance liquid chromatography (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 Variant Classic Hb Testing System, BioRad Laboratories, Philadelphia).  Source:  (Modified from Elghetany MT, Banki K: Erythrocytic disorders. In McPherson RA, Pincus MR: Henry’s Clinical Diagnosis and Management by Laboratory Methods, ed 22, Philadelphia, 2011, Elsevier, p. 578.)

HPLC separates hemoglobin types in a cation exchange column and usually requires only one sample injection. Unlike 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 thalassemias rather than hemoglobinopathies because quantitation of low levels of normal and abnormal hemoglobin levels is necessary to distinguish thalassemias. HPLC is also commonly used to quantitate Hb A1c levels to monitor diabetic patients.

Capillary electrophoresis, like agarose electrophoresis, separates hemoglobin types based on charge in an alkaline buffer but does so using smaller volumes and produces better separation than traditional agarose electrophoresis. Semiautomated systems like the Capillarys® system (Sebia, Evry, France) allow for the testing of up to eight samples in parallel with computerized analysis of results. Capillary electrophoresis is also economical, since each capillary can accommodate at least 3000 runs.1 In 2009 hemoglobin electrophoresis in agarose medium was still the most commonly used technique to identify hemoglobin 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 electric 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 charge of zero, migration stops and the hemoglobin molecules accumulate at their isoelectric position. Molecules with isoelectric point differences of as little as 0.02 pH units can be effectively separated.1

Neonatal screening requires a more sophisticated approach, often using three techniques: adapted IEF, HPLC, and reversed-phase HPLC. This multisystem approach is needed to distinguish 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 identification of hemoglobin variants. Some reference laboratories may use mass spectroscopy, matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry, or isoelectric focusing to separate hemoglobin types, or nucleic acid identification of the genetic mutation.1,  54

Patients with Hb SS or Hb SC disease lack normal β-globin chains, so they have no Hb A. In Hb SS, the Hb S level is usually greater than 80%. The Hb F level is usually increased (1% to 20%), and when Hb F constitutes more than 20% of hemoglobin, it has a tendency to modulate the severity of the disease. This is especially true in newborns and in patients with hereditary persistence of fetal hemoglobin.31 The Hb A2 level is normal or slightly increased (2% to 5%), and Hb A2 quantitation is useful in differentiating Hb SS from Hb S–β0-thalassemia, in which Hb A2 is increased (Chapter 28). Hb G, Hb D, and Hb S all migrate in the same position on alkaline cellulose acetate and alkaline agarose electrophoresis, but Hb G and Hb D do not give a positive result on the tube solubility test.

The typical sequelae of SCD may be predicted, and the effectiveness of treatment monitored, if reliable biomarkers of inflammation can be identified. Among the common indicators of inflammation, WBC is a good predictor of sickle cell events and mortality, whereas the erythrocyte sedimentation rate and C-reactive protein (CRP) level exhibit variability too great to reliably predict events. However, CRP and sPLA2 are both elevated during vasoocclusive crisis and acute chest syndrome. Other markers such as interleukin-6 (IL-6), IL-10, and protein S are showing promise as useful indicators in clinical practice.55 Annexin A5, a protein bound to lipids in the plasma membrane of endothelial cells and platelets, has been shown to elevate before and during VOC.56 Lipid damage from oxidative stress can be predicted by plasma elevations of malondialdehyde (MDA) and depleted α-tocopherol. In addition, α-tocopherol rises with CRP during bouts of inflammation. Of all the biomarkers evaluated, IL-6, IL-10, vascular cell adhesion molecule 1 (VCAM-1), and sPLA2 are the most promising at predicting impending crisis.57

Treatment

Supportive care has been the mainstay of therapy for SCD. New therapies have evolved, however, that are actually modifying the genetic pathogenesis of the disease. Neonatal screening, childhood prophylactic penicillin therapy, bone marrow transplantation, and treatment with hydroxyurea (hydroxycarbamide) in adults may extend the life of the SCD patient further.

The main components of supportive therapy include adequate hydration, prophylactic vitamin therapy, avoidance of low-oxygen environments, analgesia for pain, and aggressive antibiotic therapy with the first signs of infection. Hydration maintains good blood flow and reduces vasoocclusive crises. Prophylactic oral penicillin V at a dose of 125 mg twice per day by the age of 3 months to 3 years of age is recommended to avoid infection and the associated morbidity and mortality. The penicillin dosage is increased to 250 mg twice per day from 3 to 5 years of age.58 When infections occur, prompt antibiotic treatment reduces the associated morbidity and mortality.9 Avoidance of strenuous exercise, high altitudes, and unpressurized air travel maintains high oxygen tensions and reduces the sickling phenomenon. Treatment for painful episodes includes ensuring optimal hydration, rapidly treating associated infection, oxygen therapy, and effectively relieving pain. Analgesics are the foundation of pain management, with nonsteroidal antiinflammatory drugs like paracetamol (acetaminophen) and nefopam administered to manage mild ischemic attacks. Opioids like meperidine (pethidine or demerol) or tramadol are recommended when pain becomes chronic.59 Acute VOC attacks are often treated with morphine in the emergency department or when in transit. Blood exchange transfusion (BET) is the treatment of choice for severe VOC attacks and acute chest syndrome (ACS).33 Painful crises tend to increase with age, and physicians must be aware of opiate tolerance and rebound pain following opiate therapy, called central sensitization. Repeated painful crises can result in hypersensitivity to repeated pain by increasing peripheral inflammation, increased neurotransmitter release, increased calcium influx into postsynaptic junctions, and other pathways that increase pain signals to the brain.45 This phenomenon can be misinterpreted as drug intolerance, causing inappropriate dose escalation, or as drug-seeking behavior, causing inappropriate termination of treatment. The most appropriate response to opioid tolerance and central sensitization is a gradual dose reduction to reset the pain receptors followed by switching to opioids such as methadone and buprenorphine that are less sensitive to this phenomenon.45 The patient should be examined on a regular basis, and routine testing should be done to establish baseline values for the patient during nonsickling periods.

Children younger than 3 years often experience hand-foot syndrome, characterized by pain and swelling in the hands and feet.33 Treatment usually consists of increasing intake of fluids and giving analgesics for pain.

Pneumococcal disease has been a leading cause of morbidity and mortality in children, especially children younger than 6 years. With immunization and prophylactic antibiotics, however, this is now a preventable complication.7 Immunization with heptavalent conjugated pneumococcal vaccine is recommended at 2, 4, and 6 months of age. The 23-valent pneumococcal vaccine is recommended at 2 years, with a booster at the age of 5 years. Standard childhood vaccinations should be given as scheduled. In addition, annual administration of influenza vaccine is recommended beginning at 6 months of age.42 The risk of bacterial infection probably increases in mature patients with Hb SC disease and homozygous SCD.20

Transfusions can be used to prevent the complications of SCD. More specifically, periodic transfusions, given at a frequency of eight or more per year, are effective at preventing stroke, symptomatic anemia, brain injury, priapism, leg ulcers, PHT, delayed pubescence, splenomegaly, and chronic pain and improving school attendance, IQ, energy, exercise tolerance, mood, and sense of well-being. In other circumstances, such as central nervous system infarction, hypoxia with infection, stroke, episodes of acute chest syndrome, and preparation for surgery, transfusions are used to decrease blood viscosity and the percentage of circulating sickle cells. Before all but simple surgeries, Hb SS patients are transfused with normal Hb AA blood to bring the volume of Hb S to less than 50% or to achieve a hemoglobin of 10 g/dL in an effort to prevent complications in surgery.58,  60 Maintenance transfusions should be given in pregnancy if the mother experiences vasoocclusive or anemia-related problems or if there are signs of fetal distress or poor growth.20 Nonetheless, transfusion therapy has the potential to cause transfusion reactions, transfusion-related infections, and iron overload. Of the three, iron overload is the most frequent.

Iron overload has been associated with endocrine dysfunction61 and cardiac disease.62 Deferoxamine has been effective in treating iron overload by chelating and removing much of the excess iron from the body. Deferoxamine must be administered intravenously, however, and treatment requires at least 8 hours each day for a week. An oral iron chelator, deferasirox, was approved by the Food and Drug Administration in 2005.63 Deferasirox is consumed in the morning as a slurry by dissolving several pills, but its effectiveness is yet to be determined.

Bone marrow or hematopoietic stem cell transplantation has proved successful for some individuals, but few patients qualify due to the lack of HLA-matched, related donors.64 The event-free survival rates for patients receiving transplants from HLA-identical related donors are between 80% and 90% for SCD. Patients chosen for transplantation are generally children younger than age 17 with severe complications of SCD (i.e., stroke, acute chest syndrome, and refractory pain). In addition, morbidity and mortality following transplantation increase with age, which places another restriction on transplantation therapy.69 There is evidence that transplantation restores some splenic function, but its effect on established organ damage is unknown.70 Transplantation of cord blood stem cells from HLA-identical related and unrelated donors is associated with a disease-free survival rate of 90%.71 The primary benefit of using cord blood as a source of stem cells is that banking of cord blood increases the number of units available to achieve an HLA match.71 Some researchers are now focusing on the use of in utero stem cell transplantation to produce engraftment while the immune system of the fetus is prone to HLA tolerance. Others are attempting to genetically alter fetal hematopoietic stems cells to overcome HLA mismatches.72

Hydroxycarbamide (hydroxyurea) therapy has offered some promise in relieving the sickling disorder by increasing the proportion of Hb F in the erythrocytes of individuals with SCD.73 Hydroxyurea, given at 25 to 30 mg/kg, has been shown to reduce symptoms and prolong life, in part by increasing Hb F levels. Daily dosing produces a better HbF response compared to sequential weekly dosing.74 Because Hb F does not copolymerize with Hb S, if the production of Hb F can be sufficiently augmented, the complications of SCD might be avoided. The severity of the disease expression and the number of irreversible sickle cells are inversely proportional to the extent to which Hb F synthesis persists. Individuals in whom Hb F levels stabilize at 12% to 20% of total hemoglobin may have little or no anemia and few, if any, vasoocclusive attacks. Levels of 4% to 5% Hb F may modulate the disease, and levels of 5% to 12% may suppress the severity of hemolysis and lessen the frequency of severe episodes.34 Drug compliance is best monitored by an increasing MCV, while a decreasing LD might be an indicator of treatment response.33 Response to hydroxycarbamide is variable among SCD patients, but high baseline Hb F level, neutrophil levels, and reticulocyte count are the best predictors of Hb F response.75

Prevention of intracellular RBC dehydration reduces intracellular HbS polymerization thus reducing VOC. The uses of senicapoc to inhibit Gardos channels and Mg++ to modulate K+-Cl transport systems show increased hemoglobin levels and decreased numbers of dense RBCs, resulting in reduced hemolysis but no clear reduction in VOC.76-80

Course and prognosis

Proper management of SCD has increased the life expectancy of patients from 14 years in 1973 to the current average life span of 50 years.81 For men and women who are compound heterozygotes for Hb SC, the average life span is 60 and 68 years, respectively, with a few patients living into their seventies.30,  82 Individuals with Hb SS can pursue a wide range of vocations and professions. They are discouraged, however, from jobs that require strenuous physical exertion or exposure to high altitudes or extreme environmental temperature variations.

Newborn screening for hemoglobinopathies has significantly reduced mortality in children with SCD by enabling prompt and comprehensive medical care. The most common form of screening is HPLC followed by confirmation using hemoglobin electrophoresis and genotyping methods.83

Sickle cell trait

The term sickle cell trait refers to the heterozygous state (Hb AS) and describes a benign condition that generally does not affect mortality or morbidity except under conditions of extreme exertion. The trait occurs in approximately 8% of African Americans. It also can be found in Central Americans, Asians, and people from the region around the Mediterranean.1

Individuals with sickle cell trait are generally asymptomatic and present with no significant clinical or hematologic manifestations. Under extremely hypoxic conditions, however, systemic sickling and vascular occlusion with pooling of sickled cells in the spleen, focal necrosis in the brain, rhabdomyolysis, and even death can occur. In circumstances such as severe respiratory infection, unpressurized flight at high altitudes, and anesthesia in which pH and oxygen levels are sufficiently lowered to cause sickling, patients may develop splenic infarcts.8 Failure to concentrate urine is the only consistent abnormality found in patients with sickle cell trait.84 This abnormality is caused by diminished perfusion of the vasa recta of the kidney, which impairs concentration of urine by the renal tubules. Renal papillary necrosis with hematuria has been described in some patients.8

Although much controversy exists as to the potential connection between strenuous exercise and severe to fatal adverse events in patients with sickle cell trait, at least 46 cases have been documented in the literature (39 military recruits and 7 athletes).85 The causes of these deaths were largely due to cardiac failures, renal failures, rhabdomyolysis, and heart illness. Opponents of the connection of sickle cell trait and fatal events argue that these events occur in sickle cell–negative people, many people with sickle cell trait do not develop adverse events, fatal sickle crisis cannot be adequately established in the patients encountering events, and similar events have not been clearly documented in patients with sickle cell disease. However, it has been shown that military 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 thrombocytes 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, and Hb F level is within the reference interval. Levels of Hb S less than 40% can be seen in patients who also have α-thalassemia or iron or folate deficiency.20 No treatment is required for this benign condition, and the patient’s life span is not affected by sickle cell trait.

Hemoglobin c

Hb C was the next hemoglobinopathy after Hb S to be described and in the United States is found almost exclusively in the African-American population. Spaet and Ranney reported this disease in the homozygous state (Hb CC) in 1953.8

Prevalence, etiology, and pathophysiology

Hb C is found in 17% to 28% of people of West African extraction and in 2% to 3% of African Americans.4 It is the most common nonsickling variant encountered in the United States and the third most common in the world.4 Hb C is defined by the structural formula α2β26GluLys, in which lysine is substituted for glutamic acid in position 6 of the β chain. Lysine has a +1 charge and glutamic acid has a –1 charge, so the result of this substitution is a net change in charge of +2, which has a different structural effect on the hemoglobin molecule than the Hb S substitution.

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 low oxygen tension, but the structure of the polymers differs. Hb S polymers are long and thin, whereas the polymers in Hb C form a short, thick crystal within the RBCs. The shorter Hb C crystal does not alter RBC shape to the extent that Hb S does, so there is less splenic sequestration and hemolysis. In addition, vasoocclusive crisis does not occur.

Laboratory diagnosis

A mild to moderate, normochromic, normocytic anemia occurs in homozygous Hb C disease. Occasionally, some microcytosis and mild hypochromia may be present. There is a marked increase in the number of target cells, a slight to moderate increase in the number of reticulocytes, and nucleated RBCs may be present in the peripheral blood.

Hexagonal crystals of Hb C form within the erythrocyte and may be seen on the peripheral blood film (Figure 27-10). Many crystals appear extracellularly with no evidence of a cell membrane.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 ends. These crystals may be seen on wet preparations by washing RBCs and resuspending them in a solution of sodium citrate.11

 
FIGURE 27-10 Peripheral blood film for a patient with hemoglobin C disease showing one Hb C crystal and target and folded cells (×1000).  Source:  (Courtesy Ann Bell, University of Tennessee, Memphis.)

Hb C yields a negative result on the hemoglobin solubility test, and definitive diagnosis is made using electrophoresis or 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 7% and Hb A2 at approximately 2%. In Hb AC trait, about 60% Hb A and 30% Hb C are present. On cellulose acetate electrophoresis at an alkaline pH, Hb C migrates in the same position as Hb A2, Hb E, and Hb O-Arab (Figure 27-7). Hb C is separated from these other hemoglobins on citrate agar electrophoresis at an acid pH (Figure 27-7). No specific treatment is required. This disorder becomes problematic only if infection occurs or if mild chronic hemolysis leads to gallbladder disease.

Hemoglobin c-harlem (hemoglobin c-georgetown)

Hb C-Harlem (Hb C-Georgetown) has a double substitution on the β chain.5,  20 The substitution of valine for glutamic acid at position 6 of the β chain is identical to the Hb S substitution, and the substitution at position 73 of aspartic acid for asparagine is the same as that in the Hb Korle Bu mutation. The double mutation is termed Hb C-Harlem (Hb C-Georgetown) because the abnormal hemoglobin migrates with Hb C on cellulose acetate electrophoresis at an alkaline pH. Patients heterozygous for this anomaly are asymptomatic, but patients with compound heterozygosity for Hb S and Hb C-Harlem have crises similar to those in Hb SS disease.88

A positive solubility test result may occur with Hb C-Harlem, and hemoglobin electrophoresis or HPLC is necessary to confirm the diagnosis. On cellulose acetate at pH 8.4, Hb C-Harlem migrates in the C position (Figure 27-7). Citrate agar electrophoresis at pH 6.2, however, shows migration of Hb C-Harlem in the S position (Figure 27-7). Because so few cases have been 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 immigrants 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 β chain variant in which lysine is substituted for glutamic acid in position 26 (α2β226GluLys). As with Hb C, this substitution results in a net change in charge of +2, but because of the position of the substitution, hemoglobin polymerization does not occur. However, the amino acid substitution 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 mutation is both a qualitative defect (due to the amino acid substitution in the globin chain) and a quantitative defect with a β-thalassemia phenotype (due to the decreased production of the globin chain).91

Clinical features

The homozygous state (Hb EE) manifests as a mild anemia with microcytes and target cells. The RBC survival time is shortened. The condition is not associated with clinically observable icterus, hemolysis, or splenomegaly. The main concern in identifying homozygous Hb E is differentiating it from iron deficiency, β-thalassemia trait, and Hb E–β-thal (Chapter 28).91 The disease, Hb EE, resembles thalassemia trait. Because the highest incidence of the Hb E gene is in the areas of Thailand where malaria is most prevalent, it is thought that P. falciparummultiplies more slowly in Hb EE RBCs than in Hb AE or Hb AA RBCs and that the mutation may give some protection against malaria.1 Hb E trait is asymptomatic. When Hb E is combined with β-thalassemia, however, the disease becomes more severe than Hb EE and more closely resembles β-thalassemia major, requiring regular blood transfusions.1

Laboratory diagnosis

Hb E does not produce a positive hemoglobin solubility test result and must be confirmed using electrophoresis or HPLC. In the homozygous state there is greater than 90% Hb E, a very low MCV (55 to 65 fL), few to many target cells, and a normal reticulocyte count. The heterozygous state has a mean MCV of 65 fL, slight erythrocytosis, target cells1 (Figure 27-11), and approximately 30% to 40% Hb E. On cellulose acetate electrophoresis at an alkaline pH, Hb E migrates with Hb C, Hb O, and Hb A2 (Figure 27-7). On citrate agar electrophoresis at an acid pH, Hb E can be separated from Hb C, but it comigrates with Hb A and Hb O (Figure 27-7).

 
FIGURE 27-11 Microcytes and target cells in a patient with hemoglobin E trait.  Source:  (From Hematology tech sample H-1, Chicago, 1991, American Society of Clinical Pathologists.)

Treatment and prognosis

No therapy is required with Hb E disease and trait. Some patients may experience splenomegaly and fatigue, however. Genetic counseling is recommended, and the Hb E gene mutation should be discussed in the same manner as a mild β-thalassemia allele.91

Hemoglobin o-arab

Hb O-Arab is a β chain variant caused by the substitution of lysine for glutamic acid at amino acid position 121 (α2β2121GluLys).5,  20,  32 It is a rare disorder found in Kenya, Israel, Egypt, and Bulgaria and in 0.4% of African Americans. No clinical symptoms are exhibited by individuals who carry this variant, except for a mild splenomegaly in homozygotes.5 When Hb O-Arab is inherited with Hb S, however, severe clinical conditions similar to those in Hb SS result.5

Homozygous individuals have a mild hemolytic anemia, with many target cells on the peripheral blood film and a negative result on the hemoglobin solubility test. The presence of this hemoglobin variant must be confirmed using electrophoresis or HPLC. Because Hb O-Arab migrates with Hb A2, Hb C, and Hb E on cellulose acetate at an alkaline pH, citrate agar electrophoresis at an acid pH is required to differentiate it from Hb C (Figure 27-7). Hb O-Arab is the only hemoglobin to move just slightly away from the point of application toward the cathode on citrate agar at an acid pH. No treatment is generally necessary for individuals with Hb O-Arab.

Hemoglobin d and hemoglobin g

Hb D and Hb G are a group of at least 16 β chain variants (Hb D) and 6 α chain variants (Hb G) that migrate in an alkaline pH at the same electrophoretic position as Hb S.4,  8,  20,  92 This is because their α and β subunits have one fewer negative charge at an alkaline pH than Hb A, as does Hb S. They do not sickle, however, when exposed to reduced oxygen tension.

Most variants are named for the place where they were discovered. Hb D-Punjab and Hb D-Los Angeles are identical hemoglobins in which glutamine is substituted for glutamic acid at position 121 in the β chain (α2β2121GluGln). Hb D-Punjab occurs in about 3% of the population in northwestern India, and Hb D-Los Angeles is seen in fewer than 2% of African Americans.

Hb G-Philadelphia is an α chain variant of the G hemoglobins, with a substitution of asparagine by lysine at position 68 (α268AsnLysβ2).5 The Hb G-Philadelphia variant is the most common G variant encountered in African Americans and is seen with greater frequency than the Hb D variants. The Hb G variant is also found in Ghana.4,  8,  20,  92

Hb D and Hb G do not sickle and yield a negative hemoglobin solubility test result. On alkaline electrophoresis, Hb D and Hb G have the same mobility as Hb S (Figure 27-7). Hb D 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 hemoglobin is encountered that migrates in the S position on alkaline electrophoresis and has a negative result on the hemoglobin solubility test. In the homozygous state (Hb DD), there is greater than 95% Hb D, with normal amounts of Hb A2 and Hb F.30 Hb DD can be confused with the compound heterozygous state for Hb D and β0-thalassemia. The two disorders can be differentiated on the basis of the MCV, levels of Hb A2, and family studies.4,  8,  20,  92

Hb D and Hb G are asymptomatic in the heterozygous state. Hb D disease (Hb DD) is marked by mild hemolytic anemia and chronic nonprogressive splenomegaly. No treatment is required.4,  8,  20,  92

Compound heterozygosity with hemoglobin s and another β-globin gene mutation

Compound heterozygosity is the inheritance of two different mutant genes that share a common genetic locus—in this case the β-globin gene locus. Because there are two β-globin genes, these compound heterozygotes have inherited Hb S from one parent and another β chain hemoglobinopathy or thalassemia from the other parent. Compound heterozygosity of Hb S with Hb C, Hb D, Hb O, or β-thalassemia may produce hemolytic anemia of variable severity. Inheritance of Hb S with other hemoglobins, such as Hb E, Hb G-Philadelphia, and Hb Korle Bu, causes disorders of no clinical consequence.93

Hemoglobin sc

Hb SC is the most common compound heterozygous syndrome that results in a structural defect in the hemoglobin molecule in which different amino acid substitutions are found on each of two β-globin chains. At position 6, glutamic acid is replaced by valine (Hb S) on one β-globin chain and by lysine (Hb C) on the other β-globin chain. The frequency of Hb SC is 25% in West Africa. The incidence in the United States is approximately 1 in 833 births per year.93,  97

Clinical features

Hb SC disease resembles a mild SCD. Growth and development are delayed compared with normal children. Unlike Hb SS, Hb SC usually does not produce significant symptoms until the teenage years. Hb SC disease may cause all the vasoocclusive complications of sickle cell anemia, but the episodes are less frequent, and damage is less disabling. Hemolytic anemia is moderate, and many patients exhibit moderate splenomegaly. Proliferative retinopathy is more common and more severe than in sickle cell anemia.98 Respiratory tract infections with S. pneumoniae are common.8

Patients with Hb SC disease live longer than patients with Hb SS and have fewer painful episodes, but this disorder is associated with considerable morbidity and mortality, especially after age 30.99 In the United States, the median life span for men is 60 years and for women 68 years.30

Laboratory diagnosis

The complete blood count shows a mild normocytic, normochromic anemia with many of the features associated with sickle cell anemia. The hemoglobin level is usually 11 to 13 g/dL, and the reticulocyte count is 3% to 5%. On the peripheral blood film, there are a few sickle cells, target cells, and intraerythrocytic crystalline structures. Crystalline aggregates of hemoglobin (SC crystals) form in some cells, where they protrude from the membrane (Figure 27-12).93,  96 Hb SC crystals often appear as a hybrid of Hb S and Hb C crystals. They are longer than Hb C crystals but shorter and thicker than Hb S polymers and are often branched.

 
FIGURE 27-12 A and B, Peripheral blood film for a patient with hemoglobin SC. Note intraerythrocytic, blunt-ended SC crystals and target cells (×1000).  Source:  (Courtesy Ann Bell, University of Tennessee, Memphis.)

The result of the hemoglobin solubility screening test is positive because of the presence of Hb S. Electrophoretically, Hb C and Hb S migrate in almost equal amounts (45%) on cellulose acetate, and Hb F is normal. Hb C is confirmed on citrate agar at an acid pH, where it is separated from Hb E and Hb O. Hb A2 migrates with Hb C, and its quantitation is of no consequence in Hb SC disease. Determination of Hb A2 becomes vital, however, if a patient is suspected of having Hb C concurrent with β-thalassemia (Chapter 28).

Treatment and prognosis

Therapy similar to that for SCD is given to individuals with Hb SC disease.88

Hemoglobin s–β-thalassemia

Compound heterozygosity for Hb S and β-thalassemia is the most common cause of sickle cell syndrome in patients of Mediterranean descent and is second to Hb SC disease among all compound heterozygous sickle disorders. Hb S–β-thal usually causes a clinical syndrome resembling that of mild or moderate sickle cell anemia. The severity of this compound heterozygous condition depends on the β chain production of the affected β-thalassemia gene. If there is no β-globin chain production from the β-thalassemia gene (Hb S–β0-thal), the clinical course is similar to that of homozygous sickle cell anemia. If there is production of a normal β-globin chain (Hb S–β+-thal), patients tend to have a milder condition than patients with Hb SC. These patients can be distinguished from individuals with sickle cell trait because of the presence of greater amounts of Hb S than of Hb A, increased levels of Hb A2 and Hb F, microcytosis from the thalassemia, hemolytic anemia, abnormal peripheral blood morphology, and splenomegaly (Chapter 28).20,  93

Hemoglobin sd and hemoglobin sg-philadelphia

Hb SD is a compound heterozygous and Hb SG-Philadelphia a double heterozygous sickle cell syndrome.20,  92 Hb SG-Philadelphia is asymptomatic because Hb G is associated with an α gene mutation that still allows for sufficient Hb A to be produced. Hb SD syndrome may cause a mild to severe hemolytic anemia because both β chains are affected. Some patients with Hb SD may have severe vasoocclusive complications. The Hb D syndrome in African Americans is usually due to the interaction of Hb S with Hb D-Los Angeles (Hb D-Punjab).

The peripheral blood film findings for Hb SD disease are comparable to those seen in less severe forms of Hb SS disease. Because Hb D and Hb G comigrate with Hb S on cellulose acetate electrophoresis at an alkaline pH, citrate agar electrophoresis at an acid pH is necessary to separate Hb S from Hb D and Hb G. The clinical picture is valuable in differentiating Hb SD and Hb SG. The treatment for Hb SD disease is similar to that for patients with SCD and is administered according to the severity of the clinical condition.

Hemoglobin s/o-arab and hbs/d-punjab

Hb S/O-Arab and Hb S/D-Punjab are rare compound heterozygous hemoglobinopathies that cause severe chronic hemolytic anemia with vasoocclusive episodes.8,  20,  92 Both mutations replace glutamic acid at position 121; O-Arab substitutes lysine and D-Punjab substitutes glutamine. Glutamic acid at position 121 is located on the outer surface of the hemoglobin tetramer, which enhances the polymerization process involving Hb S. Hb S/O-Arab can be mistaken for Hb SC on cellulose acetate electrophoresis at an alkaline pH because Hb C and Hb O-Arab migrate at the same position; however, differentiation is easily made on citrate agar at an acid pH. Therapy for these patients is similar to that for patients with SCD. Similarly, Hb D-Punjab comigrates with Hb S on alkaline electrophoresis, making this mutation look like SCD. Hb O-Arab and Hb D-Punjab are not clinically significant in either the heterozygous or the homozygous form.1

Hemoglobin s-korle bu

Hb Korle Bu is a rare hemoglobin variant with substitution of aspartic acid for asparagine at position 73 of the β chain.20 When inherited with Hb S, it interferes with lateral contact between Hb S fibers by disrupting the hydrophobic pocket for β6 valine, which inhibits Hb S polymerization. The compound heterozygous condition Hb S-Korle Bu is asymptomatic.

Concomitant CIS mutations with hemoglobin s

A concomitant cis mutation with Hb S involves a second mutation on the same gene along with Hb S. Three cis mutations will be described: Hb C-Harlem, Hb S-Antilles, and Hb S-Oman.

Hemoglobin c-harlem

Hb C-Harlem has two substitutions on the β chain: the sickle mutation and the Korle Bu mutation. Patients heterozygous for only Hb C-Harlem are asymptomatic. The compound heterozygous Hb S–Hb C-Harlem state resembles Hb SS clinically. Hb C-Harlem yields a positive result on the hemoglobin solubility test and migrates to the Hb C position on cellulose acetate electrophoresis at an alkaline pH and to the Hb S position on citrate agar electrophoresis at an acid pH.

Hemoglobin s-antilles and hemoglobin s-oman

Hb S-Antilles bears the Hb S mutation (β6GluVal) along with a substitution of isoleucine for valine at position 23.100 Hb S-Oman also has the Hb S mutation with a second substitution of lysine for glutamic acid at position 121.101 In both of these hemoglobin variants, the second mutation enhances Hb S such that significant sickling can occur even in heterozygotes.1

Table 27-2 summarizes common clinically significant hemoglobinopathies, including general characteristics and treatment options.

TABLE 27-2

Common Clinically Significant Hemoglobinopathies

Hemoglobin Disorder

Abnormal Hemoglobin

Structural Defect

Groups Primarily Affected

Hemoglobin Solubility Test Results

Hemoglobins Present

Red Blood Cell Morphology

Symptoms/Organ Defects

Treatment

Sickle cell anemia (homozygous)

Hb S

α2β26GluVal

African, African American, Middle Eastern, Indian, Mediterranean

Positive

0% Hb A, > 80% Hb S, 1%—20% Hb F, 2%—5% Hb A2

Sickle cells, target cells, nucleated RBCs, polychromasia, Howell-Jolly bodies, basophilic stippling

Vasoocclusion, bacterial infections, hemolytic anemia, aplastic episodes; bones, lungs, liver, spleen, penis, eyes, central nervous system, urinary tract

Transfusions, antibiotics, analgesics, bone marrow transplant, hydroxyurea

Hb C disease (homozygous)

Hb C

α2β26GluLys

African, African American

Negative

0% Hb A, > 90% Hb C, < 7% Hb F, 2% Hb A2

Hb C crystals, target cells, nucleated RBCs, occasionally some microcytes

Mild splenomegaly, mild hemolysis

Usually none, antibiotics

Hb SC-Harlem*(Hb C-Georgetown)

Hb C-Harlem, Hb S

α2β26GluVal and α2β273AspAsn on same gene and α2β26GluVal

Rare, so uncertain; African, African American

Positive

Hb C-Harlem migrates with Hb C at alkaline pH; migrates with Hb S at acid pH

Target cells

Compound heterozygotes with Hb SC-Harlem have symptoms similar to Hb SS

Similar to Hb SS

Hb E disease (homozygous)

Hb E

α2β226GluLys

Southeast Asian, African, African American

Negative

0% Hb A, 95% Hb E, 2%-4% Hb A2; migrates with Hb A2, Hb C, and Hb O at alkaline pH

Target cells, microcytes

Mild anemia, mild splenomegaly, no symptoms

Usually none

Hb O-Arab (homozygous)

Hb O-Arab

α2β2121GluLys

Kenyan, Israeli, Egyptian, Bulgarian, African American

Negative

0% Hb A, 95% Hb O, 2%-4% Hb A2; migrates with Hb A2, Hb C, and Hb E at alkaline pH

Target cells

Mild splenomegaly

Usually none

Hb D disease (rare homozygous)

Hb D-Punjab (Hb-D Los Angeles)

α2β2121GluGln

Middle Eastern, Indian

Negative

95% Hb D, normal Hb A2 and Hb F; migrates with Hb S at alkaline pH

Target cells

Mild hemolytic anemia, mild splenomegaly

Usually none

Hb G disease (rare homozygous)

Hb G, Hb G-Philadelphia

α268AsnLysβ2

African American, Ghanaian

Negative

95% Hb G, normal Hb A2 and Hb F; migrates with S at alkaline pH

Target cells

Mild hemolytic anemia, mild splenomegaly

Usually none

Hb SC* disease

Hb S, Hb C

α2β26GluVal and α2β26GluLys

Same as Hb S

Positive

45% Hb S, 45% Hb C, 2%–4% Hb A2, 1% Hb F

Sickle cells, Hb SC crystals, target cells

Same as those for Hb SS except milder

Similar to that for Hb SS but less intensive

Hb S–β-thalassemia*

Hb S + β-thalassemia mutation

α2β26GluVal and β0 or β+

Same as Hb S

Positive

Hb S variable, some Hb A in β+, increased Hb A2 and Hb F

Sickle cells, target cells, microcytes

Hemolytic anemia, splenomegaly

Similar to that for Hb SS; varies depending on amount of Hb A present

Hb SD* disease

Hb S, Hb D

α2β26GluVal and α2β2121GluGln

Same as Hb S

Positive

45% Hb S, 45% Hb D, 2%–4% Hb A2, 1% Hb F; Hb S and D comigrate at alkaline pH

Sickle cells, target cells

Similar to those for Hb SS but milder

Similar to that for Hb SS but less intensive

Hb SG

Hb S, Hb G

α2β26GluVal and α268AsnLysβ2

Same as Hb S

Positive

45% Hb S, 45% Hb G, 2%–4% Hb A2, 1% Hb F; Hb S and G comigrate at alkaline pH

Target cells

No symptoms

Usually none

Hb SO-Arab*

Hb S, Hb O-Arab

α2β26GluVal and α2β2121GluLys

Same as Hb S

Positive

45% Hb S, 45% Hb O, 2%–4% Hb A2, 1% Hb F

Sickle cells, target cells

Similar to those for Hb SS

Similar to that for Hb SS

* Compound heterozygous.

† Double heterozygous.

Asn, Asparagine; Asp, aspartic acid; Gln, glutamine; Glu, glutamic acid; Hb, hemoglobin; Lys, lysine; Val, valine.

Hemoglobin m

Hb M is caused by a variety of mutations in the α-, β-, and γ-globin genes, all of which result in the production of methemoglobin—hence the Hb M designation.92,  102 These genetic mutations result in a structural abnormality in the globin portion of the molecule. Most M hemoglobins involve a substitution of a tyrosine amino acid for either the proximal (F8) or the distal (E7) histidine amino acid in the α, β, or γ chains. These substitutions cause heme iron to auto-oxidize, which results in methemoglobinemia. Hb M has iron in the ferric state (Fe3+) and is unable to carry oxygen, which produces cyanosis. Seven hemoglobin variants affecting the α or β chains have been classified as M hemoglobins: Hb M-Boston, Hb M-Iwate, and Hb Auckland (α chain variants); and Hb Chile, Hb M-Saskatoon, Hb M-Milwaukee-1, and Hb M-Milwaukee-2 (β chain variants), all named for the locations in which they were discovered.5Two variants affect the γ chain—Hb F-M-Osaka and Hb F-M-Fort Ripley5—but symptoms disappear when Hb A replaces Hb F at 3 to 6 months of age.

Hb M variants have altered oxygen affinity and are inherited as autosomal dominant disorders. Affected individuals have 30% to 50% methemoglobin (healthy individuals have less than 1%) and may appear cyanotic. Ingestion of oxidant drugs, such as sulfonamides, can increase methemoglobin to life-threatening levels. Methemoglobin causes the blood specimen to appear brown. Heinz bodies may be seen sometimes on wet preparations because methemoglobin causes globin chains to precipitate (see Figure 14-11). Diagnosis is made by spectral absorption of the hemolysate or by hemoglobin electrophoresis. The absorption spectrum peaks are determined at various wavelengths. The unique absorption range of each Hb M variant is identified when these are compared with the spectrum of normal blood.

Before electrophoresis, all hemoglobin types are converted to methemoglobin by adding potassium cyanide to the sample so that any migration differences observed are only due to an amino acid substitution, not differences in iron states. On cellulose acetate, Hb M migrates slightly more slowly than Hb A. The electrophoresis should be performed on agar gel at pH 7.1 for clear separation. Further confirmation may be obtained using HPLC or deoxyribonucleic acid (DNA)–based globin gene analysis. No treatment is necessary. Diagnosis is essential to prevent inappropriate treatment for other conditions, such as cyanotic heart disease.

Unstable hemoglobin variants

Unstable hemoglobin variants result from genetic mutations to globin genes creating hemoglobin products that precipitate in vivo, producing Heinz bodies and causing a hemolytic anemia.92,  102 More than 140 variants of unstable hemoglobin exist.5 The majority of these are β chain variants, and most others are α chain variants. Only a few are γ and δ chain variants. Most unstable hemoglobin variants have no clinical significance, although the majority has an increased oxygen affinity. About 25% of unstable hemoglobins are responsible for hemolytic anemia, which varies from compensated mild anemia to severe hemolytic episodes.

At one time, the anemia was referred to as congenital nonspherocytic hemolytic anemia or congenital Heinz body anemia. This disorder is more properly called unstable hemoglobin disease. The syndrome appears at or just after birth, depending on the globin chains involved. It is inherited in an autosomal dominant pattern. All patients are heterozygous; apparently the homozygous condition is incompatible with life. The instability of the hemoglobin molecule may be due to (1) substitution of a charged for an uncharged amino acid in the interior of the molecule, (2) substitution of a polar for a nonpolar amino acid in the hydrophobic heme pocket, (3) substitution of an amino acid in the α and β chains at the intersubunit contact points, (4) replacement of an amino acid with proline in the α helix section of a chain, and (5) deletion or elongation of the primary structure.

Clinical features

The unstable hemoglobin disorder is usually detected in early childhood in patients with hemolytic anemia accompanied by jaundice and splenomegaly. Fever or ingestion of an oxidant exacerbates the hemolysis. The severity of the anemia depends on the degree of instability of the hemoglobin molecule. The unstable hemoglobin precipitates in vivo and in vitro in response to factors that do not affect normal hemoglobins, such as drug ingestion and exposure to heat or cold. The hemoglobin precipitates in the RBC as Heinz bodies. The precipitated hemoglobin attaches to the cell membrane, causing clustering of band 3, attachment of autologous immunoglobulin, and macrophage activation. In addition, Heinz bodies can be trapped mechanically in the splenic sieve, which shortens RBC survival. The oxygen affinity of these cells is also abnormal.

The most prevalent unstable hemoglobin is Hb Köln. Other unstable hemoglobins include Hb Hammersmith, Hb Zurich, Hb Gun Hill, and Hb Hammersmith.5 Because of the large variability in the degree of instability in these hemoglobins, the extent of hemolysis varies greatly. For some of the variants, such as Hb Zurich, the presence of an oxidant is required for any significant hemolysis to occur.

Laboratory diagnosis

The RBC morphology varies. It may be normal or show slight hypochromia and prominent basophilic stippling, which possibly is caused by excessive clumping of ribosomes. Before splenectomy, the hemoglobin level ranges from 7 to 12 g/dL, with a 4% to 20% reticulocyte count. After splenectomy, anemia is corrected, but reticulocytosis persists. Heinz bodies can be shown using a supravital stain (see Figure 14-11). After splenectomy, Heinz bodies are larger and more numerous. Many patients excrete dark urine that contains dipyrrole.

Many unstable hemoglobins migrate in the normal AA pattern and thus are not detected on electrophoresis. Other tests used to detect unstable hemoglobins include the isopropanol precipitation test, which is based on the principle that an isopropanol solution at 37° C weakens the bonding forces of the hemoglobin molecule. If unstable hemoglobins are present, rapid precipitation occurs in 5 minutes, and heavy flocculation occurs after 20 minutes. Normal hemoglobin does not begin to precipitate until after approximately 40 minutes. The heat denaturation test also can be used. When incubated at 50° C for 1 hour, heat-sensitive unstable hemoglobins show a flocculent precipitation, whereas normal hemoglobin shows little or no precipitation. Significant numbers of Heinz bodies appear after splenectomy, but even in individuals with intact spleens, with longer incubation and the addition of an oxidative substance such as acetylphenylhydrazine, unstable hemoglobins form more Heinz bodies than does the blood from individuals with normal hemoglobins. Other techniques, such as isoelectric focusing, can resolve many hemoglobin variants with only a slight alteration in their isoelectric point, and globin chain analysis can be performed by HPLC or DNA-based globin gene analysis.

Treatment and prognosis

Patients are treated to prevent hemolytic crises. In severe cases, the spleen must be removed to reduce sequestration and rate of removal of RBCs. Because unstable hemoglobin disease is rare, prognosis in the affected individuals is unclear. Patients are cautioned against the use of sulfonamides and other oxidant drugs. They also should be informed of the potential for febrile illnesses to trigger a hemolytic episode.

Hemoglobins with increased and decreased oxygen affinity

More than 150 hemoglobin variants have been discovered to have abnormal oxygen affinity.4,  102-104 Most are high-affinity variants and have been associated with familial erythrocytosis. The remaining hemoglobin variants are characterized by low oxygen affinity. Many of these are associated with mild to moderate anemia.2

As described in Chapter 10, normal Hb A undergoes a series of allosteric conformational changes as it converts from a fully deoxygenated to a fully oxygenated form. These conformational changes affect hemoglobin function and its affinity for oxygen. When normal hemoglobin is fully deoxygenated (tense state), it has low affinity for oxygen and other heme ligands and high affinity for allosteric effectors, such as Bohr protons and 2,3-bisphosphoglycerate. In the oxygenated (relaxed) state, hemoglobin has a high affinity for heme ligands, such as oxygen, and a low affinity for Bohr protons and 2,3-bisphosphoglycerate. The transition from the tense to the relaxed state involves a series of structural changes that have a marked effect on hemoglobin function. If an amino acid substitution lowers the stability of the tense structure, the transition to the relaxed state occurs at an earlier stage in ligand binding, and the hemoglobin has increased oxygen affinity and decreased heme-heme interaction or cooperativity (Chapter 10). One example of a β chain variant is Hb Kempsey. This unstable hemoglobin variant has amino acid substitutions at sites crucial to hemoglobin function.

Hemoglobins with increased oxygen affinity

The high-affinity variants, like other structurally abnormal hemoglobins, show an autosomal dominant pattern of inheritance. Affected individuals have equal volumes of Hb A and the abnormal variant. Exceptions to this are compound heterozygotes for Hb Abruzzo and β-thalassemia and for Hb Crete and β-thalassemia, in which the proportion of abnormal hemoglobin is greater than 85%.

More than 90 variant hemoglobins with high oxygen affinity have been discovered. Such hemoglobins fail to release oxygen on demand, and hypoxia results. The kidneys sense the hypoxia and respond by increasing the release of erythropoietin, which leads to a compensatory erythrocytosis. These variants differ from unstable hemoglobin, which also may have abnormal oxygen affinity, in that they do not precipitate in vivo to produce hemolysis and there is no abnormal RBC morphology.

Most individuals are asymptomatic and show no physical symptoms except a ruddy complexion. Erythrocytosis is usually detected during routine examination because the patient generally has a high RBC count, hemoglobin, and hematocrit. The WBC count, platelet count, and peripheral blood film findings are generally normal. In some cases, hemoglobin electrophoresis may establish a diagnosis. An abnormal band that separates from the A band is present on cellulose acetate in some variants; however, if a band is not found, the diagnosis 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 oxygen affinity is required for definitive diagnosis.

Patients with high-oxygen-affinity hemoglobins live normal lives and require no treatment. Diagnosis should be made to avoid unnecessary treatment of the erythrocytosis as a myeloproliferative neoplasm or a secondary erythrocytosis.

Hemoglobins with decreased oxygen affinity

Hemoglobins with decreased oxygen affinity quickly release oxygen to the tissues, which results in normal to decreased hemoglobin concentration and slight anemia. The best known of these hemoglobins is Hb Kansas, which has an amino acid substitution of asparagine by threonine at position 102 of the β chain. These hemoglobins may be present when cyanosis and a normal arterial oxygen tension coexist, and most may be detected by starch gel electrophoresis, HPLC, or DNA-based globin gene analysis.

Global burden of hemoglobinopathies

The prevalence of hemoglobinopathies has already been presented in this chapter, and the bulk of these conditions occurs in underdeveloped countries. However, as developing countries work to decrease deaths from malnutrition, infectious diseases, and other conditions, more patients with hemoglobinopathies will survive and remain consumers of the health care system. For example, in 1944 thalassemia was first identified in Cypress. However, during the post–World War II recovery period, as the death rate decreased, the prevalence of thalassemias increased.2 In 1970 it was estimated that in the absence of systems to control the disease, within 40 years 78,000 units of blood would be needed each year, requiring that 40% of the population serve as donors.2 If left unchecked, the cost to maintain thalassemia therapy would exceed the country’s total health care budget. In contrast, efforts to develop prenatal screening and genetic counseling programs have reduced the birth rate of SCD.2 It is clear that hemoglobinopathies are a worldwide problem requiring planning, investment, and interventions from around the globe to optimize the impact on patients with the disease without debilitating the health care systems of developing countries where the disease is prevalent.

Summary

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

Now that you have completed this chapter, go back and read again the case study at the beginning and respond to the questions presented.

Review questions

Answers can be found in the Appendix.

  1. A qualitative abnormality in hemoglobin may involve all of the following except:
  2. Replacement of one or more amino acids in a globin chain
  3. Addition of one or more amino acids in a globin chain
  4. Deletion of one or more amino acids in a globin chain
  5. Decreased production of a globin chain
  6. The substitution of valine for glutamic acid at position 6 of the β chain of hemoglobin results in hemoglobin that:
  7. Is unstable and precipitates as Heinz bodies
  8. Polymerizes to form tactoid crystals
  9. Crystallizes in a hexagonal shape
  10. Contains iron in the ferric (Fe3+) state
  11. Patients with SCD usually do not exhibit symptoms until 6 months of age because:
  12. The mother’s blood has a protective effect
  13. Hemoglobin levels are higher in infants at birth
  14. Higher levels of Hb F are present
  15. The immune system is not fully developed
  16. Megaloblastic episodes in SCD can be prevented by prophylactic administration of:
  17. Iron
  18. Folic acid
  19. Steroids
  20. Erythropoietin
  21. Which of the following is the most definitive test for Hb S?
  22. Hemoglobin solubility test
  23. Hemoglobin electrophoresis at alkaline pH
  24. Osmotic fragility test
  25. Hemoglobin electrophoresis at acid pH
  26. A patient presents with mild normochromic, normocytic anemia. On the peripheral blood film, there are a few target cells, rare nucleated RBCs, and hexagonal crystals within and lying outside of the RBCs. Which abnormality in the hemoglobin molecule is most likely?
  27. Decreased production of β chains
  28. Substitution of lysine for glutamic acid at position 6 of the β chain
  29. Substitution of tyrosine for the proximal histidine in the β chain
  30. Double amino acid substitution in the β chain
  31. A well-mixed specimen obtained for a CBC has a brown color. The patient is being treated with a sulfonamide for a bladder infection. Which of the following could explain the brown color?
  32. The patient has Hb M.
  33. The patient is a compound heterozygote for Hb S and thalassemia.
  34. The incorrect anticoagulant was used.
  35. Levels of Hb F are high.
  36. Through routine screening, prospective parents discover that they are both heterozygous for Hb S. What percentage of their children potentially could have sickle cell anemia (Hb SS)?
  37. 0%
  38. 25%
  39. 50%
  40. 100%
  41. Painful crises in patients with SCD occur as a result of:
  42. Splenic sequestration
  43. Aplasia
  44. Vasoocclusion
  45. Anemia
  46. The screening test for Hb S that uses a reducing agent, such as sodium dithionite, is based on the fact that hemoglobins that sickle:
  47. Are insoluble in reduced, deoxygenated form
  48. Form methemoglobin more readily and cause a color change
  49. Are unstable and precipitate as Heinz bodies
  50. Oxidize quickly and cause turbidity
  51. DNA analysis documents a patient has inherited the sickle mutation in both β-globin genes. The two terms that best describe this genotype are:
  52. Homozygous/trait
  53. Homozygous/disease
  54. Heterozygous/trait
  55. Heterozygous/disease
  56. In which of the following geographic areas is Hb S most prevalent?
  57. India
  58. South Africa
  59. United States
  60. Sub-Saharan Africa
  61. Which hemoglobinopathy is more common in Southeast Asian patients?
  62. Hb S
  63. Hb C
  64. Hb O
  65. Hb E
  66. Which of the following Hb S compound heterozygote exhibits the mildest symptoms?
  67. Hb S-β-Thal
  68. Hb SG
  69. Hb S-C-Harlem
  70. Hb SC
  71. A 1-year-old Indian patient presents with anemia, and both parents claim to have an “inherited anemia” but can’t remember the type. The peripheral blood shows target cells, and the hemoglobin solubility is negative. Alkaline hemoglobin electrophoresis shows a single band at the “Hb C” position and a small band at the “Hb F” position. Acid hemoglobin electrophoresis shows two bands. The most likely diagnosis is:
  72. Hb CC
  73. Hb AC
  74. Hb CO
  75. Hb SC
  76. Unstable hemoglobins show all of the following findings EXCEPT:
  77. Globin chains precipitate intracellularly
  78. Heinz body formation
  79. Elevated reticulocyte count
  80. Only homozygotes are symptomatic

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