Thalassemias

Rodak's Hematology Clinical Principles and Applications

PART IV

Erythrocyte Disorders

CHAPTER 28

Elaine M. Keohane*

OUTLINE

Definitions and History

Epidemiology

Genetics of Globin Synthesis

Categories of Thalassemia

Genetic Defects Causing Thalassemia

Pathophysiology

Mechanisms in b-Thalassemias

Μεψηανισμσ ιν α‴Τηαλασσεμια

b-Globin Gene Cluster Thalassemias

Clinical Syndromes of b-Thalassemia

Other Thalassemias Caused by Defects in the b-Globin Gene Cluster

Screening for b-Thalassemia Minor

a-Thalassemias

Clinical Syndromes of a-Thalassemia

Thalassemia Associated with Structural Hemoglobin Variants

Hemoglobin S-Thalassemia

Hemoglobin C-Thalassemia

Hemoglobin E-Thalassemia

Diagnosis of Thalassemia

History and Methods Examination

Laboratory Methods

Differential Diagnosis of Thalassemia Minor and Iron Deficiency Anemia

Objectives

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

Describe the hemoglobin defect found in thalassemias.
Discuss the geographic distribution of thalassemia and its association with malaria.
Name the chromosomes that contain the α-globin gene and the β-globin gene clusters and the globin chains produced by each.
Describe the type of genetic mutations that result in α- and β-thalassemias.
Explain the pathophysiologic effects caused by the imbalance of globin chain synthesis in α-and β-thalassemias.
Describe the four major clinical syndromes of β-thalassemia and the clinical expression of each heterozygous and homozygous form.
Recognize the pattern of laboratory findings in heterozygous and homozygous β-thalassemias, including hereditary persistence of fetal hemoglobin (HPFH).
Describe the treatment of homozygous β-thalassemias, the risks involved, and the reason it is necessary to monitor iron levels.
Correlate the clinical syndromes of α-thalassemia with the number of α genes present.
Recognize the laboratory findings associated with various α-thalassemia syndromes.
Describe the clinical syndromes of thalassemia associated with common structural hemoglobin variants.
Specify tests that are used for screening for β-thalassemia carriers.
Discuss the role of the complete blood count, peripheral blood film review, supravital stain, hemoglobin fraction quantification (using hemoglobin electrophoresis, high-performance liquid chromatography, and/or capillary zone electrophoresis), and molecular genetic testing in diagnosis of thalassemia syndromes.
Differentiate β-thalassemia minor from iron deficiency anemia.

CASE STUDY

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

A 24-year-old male medical student in the United States was found to have a hemoglobin level of 10.2 g/dL in a hematology laboratory class. During discussion of the family history with this student, a hematologist at the university discovered that his mother had always been anemic, had periodically been given iron therapy, and had a history of several acute episodes of gallbladder disease (attacks). Both of the student’s parents had been born in Sicily. A cousin on his mother’s side had two children who died of thalassemia major at the ages of 4 and 5 years and had a third young daughter with thalassemia major who was being treated with regular blood transfusions. The student’s laboratory test results were as follows:

	Patient Results	Reference Interval
RBC (× 10¹²/L)	5.74	4.60–6.00
HGB (g/dL)	10.2	14.0–18.0
HCT (%)	35	40–54
MCV (fL)	61.0	80–100
MCH (pg)	17.8	26–32
MCHC (g/dL)	29.1	32–36

Peripheral blood RBCs exhibited moderate microcytosis, slight hypochromia, and slight poikilocytosis with occasional target cells, and several RBCs had basophilic stippling. Hb A₂was 4.9% of total hemoglobin by high-performance liquid chromatography (reference interval, 0% to 3.5%). Serum ferritin level was 320 ng/mL (reference interval, 15 to 400 ng/mL).

Why was the family history so important in this case, and what diagnosis did it suggest?
What laboratory values helped confirm the diagnosis?
From what other disorders should this anemia be differentiated? What laboratory tests would be helpful? Why is differentiation important?
If this individual was planning to have children, what genetic counseling should be done?

Definitions and history

The thalassemias are a diverse group of inherited disorders caused by genetic mutations that reduce or prevent the synthesis of one or more of the globin chains of the hemoglobin (Hb) tetramer. In 1925, Cooley and Lee first described four children with anemia, splenomegaly, mild hepatomegaly, and mongoloid facies.¹ These characteristics would later become typical findings in young children with untreated β-thalassemia major, often referred to as Cooley’s anemia. Seven years later, Whipple and Bradford published a paper outlining the detailed autopsy studies of children who died of this disorder.² Because of the high incidence of patients of Mediterranean descent with this disorder, Whipple called the disease Thalassic (Greek for “great sea”) anemia, which was subsequently changed to thalassemia.² Several investigators in the 1940s demonstrated the genetic basis for this anemia and were able to show that in patients who were homozygous for this condition (thalassemia major), the disease had a severe course. The heterozygotes, however, not only were carriers but also had a milder anemia (thalassemia minor). In the 1950s, thalassemias resulting from defects in the α-globin chain were described.²

Thalassemia results from a reduced or absent synthesis of one or more of the globin chains of hemoglobin. A wide variety of mutations in hemoglobin genes lead to clinical outcomes that are extremely wide ranging, with certain mutations causing no anemia and others leading to death in utero, childhood, or early adulthood. Thalassemias are named according to the chain with reduced or absent synthesis. Mutations affecting the α- or β-globin gene are most clinically significant because Hb A (α₂β₂) is the major adult hemoglobin. The decreased or absent synthesis of one of the chains not only leads to a decreased production of hemoglobin but results in an imbalance in the α/β chain ratio.³ The unaffected gene continues to produce globin chains at normal levels, and the accumulation of the unpaired normal chains damages the red blood cells (RBCs) or their precursors resulting in their premature destruction. This exacerbates the anemia and makes some forms of thalassemia particularly severe.

Epidemiology

The morbidity and mortality due to thalassemia significantly contributes to the global health burden. Approximately 5% of the world’s population is a carrier of a clinically significant mutation for a hemoglobinopathy or thalassemia.⁴ Annually an estimated 56,000 infants are born with a form of thalassemia major.⁴^,⁵Although thalassemia occurs in all parts of the world, its distribution is concentrated in the “thalassemia belt” that extends from the Mediterranean east through the Middle East and India to Southeast Asia and south to Northern Africa (Figure 27-3).⁶ The carrier frequency of β-thalassemia depends on the region, with Sardinia, Cyprus, and Greece having the highest frequency in Europe (6% to 19%) and India, Thailand, and Indonesia having the highest frequency in Asia and Southeast Asia (0.3% to 15%).⁶ The carrier frequency of α-thalassemia varies considerably. In Europe, Cyprus has the highest carrier frequency at 14%.⁶ The carrier frequency reaches 50% to 60% in Eastern Saudi Arabia and parts of Asia and Africa, and may be as high as 75% to 80% in certain groups in Nepal, India, Thailand, and Papua New Guinea.⁶

The geographic location of the thalassemia belt coincides with areas in which malaria is prevalent (Figure 27-3). Thalassemia minor (heterozygous thalassemia) appears to impartresistance to malaria. This allowed the selective advantage that established thalassemia in high frequency in areas in which malaria is endemic.⁷^,⁸ Several case-control studies evaluated the incidence of thalassemia in patients with severe malaria compared with a control population and consistently found a lower incidence of thalassemia in the population with malaria than in the population without malaria. One study demonstrated that the risk of death from malaria was 40% lower in patients with αα/– α thalassemia and more than 60% lower in those with – α/– α thalassemia.⁸ The mechanism of this resistance is still not fully elucidated; however, two major theories have been put forward: defective growth of the parasite in the affected cell and increased phagocytosis of the infected cell.⁹^,¹⁰ Although the exact mechanism is not known, the geographic distribution and the case-control studies corroborate the protective nature of the thalassemias in promoting resistance to malaria.

Genetics of globin synthesis

The normal hemoglobin molecule is a tetramer of two α-like chains (α or ζ) with two β-like chains (β, γ, δ, or ε). Combinations of these chains produce six normal hemoglobins. Three are embryonic hemoglobins: Hb Gower-1 (ζ₂ε₂), Hb Gower-2 (α₂ε₂), and Hb Portland (ζ₂γ₂). The others are fetal hemoglobin (Hb F, α₂γ₂) and two adult hemoglobins (Hb A, α₂β₂, and Hb A₂, α₂δ₂). The α-like globin gene cluster is located on chromosome 16, whereas the β-like globin gene cluster is on chromosome 11. The α-like globin gene cluster contains three functional genes: HBZ (ζ-globin), HBA1 (α₁-globin), and HBA2 (α₂-globin).¹¹^,¹² The β-like globin gene cluster contains five functional genes: HBE (ε-globin), HBG2 (^Gγ-globin), HBG1 (^Aγ-globin),HBD (δ-globin), and HBB (β-globin).³^,¹¹^,¹² These genes are positioned in the order that corresponds with their developmental stage of expression.¹¹ During the first 2 months of gestation, the genes for the embryonic ζ and ε chains are expressed, generating Hb Gower-1 (ζ₂ε₂). Expression of the genes for the α and γ chains begins in the second month of gestation, generating two additional embryonic hemoglobins: Hb Gower-2 (α₂ε₂) and Hb Portland (ζ₂γ₂). By 10 weeks gestation, the genes for the embryonic ζ and ε chains are switched off and silenced, while the genes for α and γ chains are upregulated (called the ζ to α switch on chromosome 16, and the ε to γ switch on chromosome 11).¹¹ The γ chains combine with the α chains to make Hb F (α₂γ₂), the predominant hemoglobin of fetal life. The gene for the β chain is initially activated during the second month of gestation, but β chain production occurs at low levels throughout most of fetal life.¹³ Shortly before birth, however, the expression of the γ-globin gene is downregulated, while expression of the β-globin gene is upregulated (called the γ to β switch), so by 6 months of age and through adult life, Hb A (α₂β₂) is the predominant hemoglobin. The gene for the δ chain is activated shortly before birth, but owing to its weak promoter, only produces a relatively small amount of δ chain, resulting in a low level of Hb A₂ (α₂δ₂) (Chapter 10 and Figure 10-6).³^,¹¹ Table 28-1 contains the reference intervals for the normal hemoglobins in adults.

TABLE 28-1

Reference Intervals for Normal Hemoglobins in Adults

Hb A (α₂β₂)	95%–100%
Hb A₂ (α₂δ₂)	0%–3.5%
Hb F (α₂γ₂)	0%–2%

The γ-globin genes code for two globin chains (^Gγ and ^Aγ) that differ at position 136 by a single amino acid (glycine and alanine, respectively).¹³ Both of these globin chains are found in Hb F, with no functional difference identified between them. Similarly, the α-globin gene loci are duplicated on each chromosome 16 and also code for two globin chains (α₁ and α₂). Either of these genes can contribute to the two α-globin chains in the hemoglobin tetramer, and no functional difference has been identified between the two. Interspersed between the functional genes on these chromosomes are four functionless gene-like loci or pseudogenes that are designated by the prefixed symbol ψ. The purpose of these pseudogenes is unknown.¹³The organization of these genes on chromosomes 16 and 11 is shown in Figure 28-1.

FIGURE 28-1 Chromosome organization of globin genes and their expression during development. The light blue boxes indicate functional globin genes; the tan boxes indicate pseudogenes. The scale of the depicted chromosomal segments is in kilobases of DNA. The switch from embryonic to fetal hemoglobin (Hb) occurs by 10 weeks of gestation, and the switch from fetal to adult hemoglobin occurs in the third trimester. Source: (From Cunningham MJ, Sankaran VG, Nathan DG, et al: Chapter 20. The Thalassemias. In: Orkin SH, Nathan DG, Ginsberg D, et al (eds). Nathan and Oski ‘s hematology of infancy and childhood, ed 7, 2009, Saunders, an imprint of Elsevier, page 1017.)

An individual inherits one cluster of the five functional genes on chromosome 11 from each parent. The genotype for normal β chain synthesis is designated β/β. Because two α-globin genes (α₁ and α₂) are inherited on each chromosome 16, a normal genotype is designated αα/αα.

Categories of thalassemia

The thalassemias are divided into β-thalassemias, which include all the disorders of reduced globin chain production arising from the β-globin gene cluster on chromosome 11, and α-thalassemias, which involve the genes for the α₁ and α₂ chains on chromosome 16. Various deletional and non-deletional mutations can cause each of these disorders, and individuals with similar clinical manifestations are often heterogeneous at the genetic level.³^,¹¹^,¹³

The β-thalassemias affect mainly the β chain production but also may involve the δ, ^Gγ, ^Aγ, and ε chains. In the β-thalassemias, β⁰ is the designation for the various mutations in the β-globin gene in which no β chains are produced. In the homozygous state (β⁰/β⁰), an individual does not produce Hb A (α₂β₂). β⁺ is the designation for the various mutations in the β-globin gene that result in a partial deficiency of β chains (5% to 30% of normal) and a decrease in production of Hb A.³ Some mutations in the β-globin gene lead to minimal reductions in β chain production and are associated with mild or silent clinical states. The designation β^silent for silent carrier has been used for those mutations. The designation δβ⁰ is used for mutations in the δ- or β-globin genes in which no δ or β chains are produced. In the homozygous state (δβ⁰/δβ⁰), no Hb A (α₂β₂) or Hb A₂ (α₂δ₂) are produced. The designation δβ^Lepore indicates a fusion of the δ- and β-globin genes that produces Hb Lepore.

The most common mutations in α-thalassemia are deletions involving the α₁- and/or α₂-globin genes.¹¹^,¹² The designation α⁺ is used to indicate a deletion of either the α₁- or the α₂-globin gene on chromosome 16 (also called the – α haplotype). This results in a decreased production of α chains from that chromosome. The designation α⁰ is used to indicate a deletion of both the α₁- and α₂-globin genes on chromosome 16 (also called the – – haplotype). This results in no production of α chains from that chromosome.³^,¹¹^,¹³ Non-deletional mutations in the α-globin gene can also result in α-thalassemia, but these are less common.¹² The designation α^T is used for these mutations.¹³ The major gene designations in thalassemia are summarized in Table 28-2.

TABLE 28-2

Genetic Designations in Thalassemia

Designation	Definition
Designations for Normal β-Globin and α-Globin Genes
β	Normal β-globin gene; normal amount of β chains produced; one gene located on each chromosome 11
αα	Normal α₁- and α₂-globin genes on one chromosome (haplotype αα); normal amount of α chains produced; two genes located on each chromosome 16
Designations for the Major Thalassemic Genes
β0	β-globin gene mutation in which no β chains are produced
β+	β-globin gene mutation that results in 5% to 30% decrease in β chain production
β^silent	β-globin gene mutation that results in mildly decreased β chain production
δβ0	δβ-globin gene deletional or non-deletional mutation in which no δ or β chains are produced; accompanied by some increase in γ chain production
δβ^Lepore	δβ-globin gene fusion that produces a small amount of fusion product, hemoglobin Lepore; no δ or β chains are produced; accompanied by some increase in γ chain production
HPFH	Hereditary persistence of fetal hemoglobin; δβ-globin gene deletional or non-deletional mutation in γ-globin gene promoter in which no δ or β chains are produced; accompanied by increase in γ chain production
α⁰	Deletion of both α-globin genes on one chromosome (haplotype, − −) that results in no α chain production
α⁺	Deletion of one α-globin gene on one chromosome (haplotype, − α) that results in decreased α chain production
α^T	Non-deletional mutation in one α-globin gene on one chromosome (haplotype α^Tα) that results in decreased α chain production (T denotes thalassemia)

Genetic defects causing thalassemia

Types of genetic defects that cause a decrease or absent production of a particular globin chain include single nucleotide (or point) mutations, small insertions or deletions, or large deletions.¹²^,¹³ The mechanisms³^,¹¹^,¹³ by which these mutations interfere with globin chain production include:

Reduced or absent transcription of messenger ribonucleic acid (mRNA)due to mutations in the promoter region or initiation codon of a globin gene, as well as mutations in polyadenylation sites that decrease mRNA stability
mRNA processing errorsdue to mutations that add or remove splice sites resulting in no globin chain or altered globin chain production
Translation errorsdue to mutations that change the codon reading frame (frameshift mutations), substitute an incorrect amino acid codon (missense mutations), add a stop codon causing premature chain termination (nonsense mutations), or remove a stop codon, which results in an elongated and unstable mRNA that produces a dysfunctional globin chain
Deletion of one or more globin genesresulting in the lack of production of the corresponding globin chains

All of these heterogeneous genetic mutations cause a reduction or lack of synthesis of one or more globin chains, resulting in the thalassemia syndromes (Figure 28-2).

FIGURE 28-2 The transcription unit of the β-globin gene. The nucleotide sequence of the DNA template is transcribed into a complementary pre-mRNA. The pre-mRNA is processed by removing introns and splicing together the protein coding exons (orange). The DNA sequences required for expression of a functional β-globin chain are indicated in different colors. Mutations in any of these sequences can lead to decreased or absent β-globin chain production. Source: (From Corden JL, Chapter 15. Gene Expression. In Pollard TD, Earnshaw WC. Cell Biology, ed 2. Philadelphia, 2008, Saunders, An imprint of Elsevier, Figure 15-2.)

Pathophysiology

The clinical manifestations of thalassemia stem from:

A reduced or absent production of a particular globin chain, which diminishes hemoglobin synthesis and produces microcytic, hypochromic RBCs; and
An unequal production of the α- or β-globin chains causing an imbalance in the α/β chain ratio; this leads to a markedly decreased survival of RBCs and their precursors.³^,¹¹^,¹³

The α/β chain imbalance is more significant and determines the clinical severity of the thalassemia.¹¹ The mechanism and the degree of shortened RBC survival are different for the β-thalassemias and α-thalassemias.

Mechanisms in β-thalassemias

In the β-thalassemias, the unpaired, excess α chains precipitate in the developing RBCs, forming inclusion bodies; this causes oxidative stress and damage to cellular membranes.¹⁴Apoptosis is triggered, and the damaged and apoptotic RBC precursors are subsequently phagocytized and destroyed in the bone marrow by activated macrophages.¹⁴ In addition, iron accumulation in the RBC precursors (discussed below) and inflammatory cytokines may also contribute to the apoptosis.¹⁴ The premature death of RBC precursors in the bone marrow is called ineffective erythropoiesis.¹¹^,¹⁴ In this situation, the bone marrow attempts to produce RBCs but is not able to release sufficient viable cells into the circulation. The cells that are released into the periphery are laden with inclusion bodies and are rapidly sequestered and destroyed by macrophages in the spleen (extravascular hemolysis).¹¹ Therefore, in β-thalassemia the anemia is multifactorial and results from ineffective production and increased destruction. Typically, individuals with severe β-thalassemia are asymptomatic during fetal life and through approximately 6 months of age because Hb F (α₂γ₂) is the predominant circulating hemoglobin. Symptoms usually begin to appear between 6 and 24 months of age, after completion of the γ to β switch.³^,¹³^,¹⁵ To compensate for the decreased expression of the β-globin gene, the γ- and/or δ-globin genes are usually upregulated, but in β-thalassemia major, this increase is insufficient to correct the α/β chain imbalance.³

In β-thalassemia major, the profound anemia stimulates an increase in erythropoietin production by the kidney and results in massive (but ineffective) erythroid hyperplasia.¹¹ In untreated or inadequately treated patients, marked bone changes and deformities occur due to the massive bone marrow expansion. A reduction in bone mineral density and a thinning of the cortex of the bone increases the risk of pathologic fractures.³^,¹¹ In children, radiographs of the long bones may exhibit a lacy or lucent appearance.³ Skull radiographs may demonstrate a typical “hair on end” appearance due to vertical striations of bony trabeculae (Figure 28-3).³^,¹¹ A typical facies occurs, with prominence of the forehead (also known as frontal bossing), cheekbones, and upper jaw. Extramedullary erythropoiesis causes hepatosplenomegaly, and foci of hematopoietic tissue can appear in other body areas. Sequestration of blood cells in the enlarged spleen can worsen the anemia and can also cause neutropenia and thrombocytopenia.¹¹ The release of hemoglobin from the excessive destruction of RBCs and their precursors leads to an increase in the level of plasma indirect bilirubin. The bilirubin can diffuse into the tissues, causing jaundice (Chapter 23). Patients also have an increased risk of developing thrombosis.³^,¹¹

FIGURE 28-3 Radiologic abnormalities in a patient with homozygous β-thalassemia who receives blood transfusions infrequently (thalassemia intermedia). A, Skull radiograph illustrating the typical “hair on end” appearance. B, Severe osteoporosis, pseudofractures, thinning of the cortex, and bowing of the femur. Source: (From Cunningham MJ, Sankaran VG, Nathan DG, et al. Chapter 20. The Thalassemias. In: Orkin SH, Nathan DG, Ginsberg D, et al. Nathan and Oski’s hematology of infancy and childhood, ed 7, 2009, Saunders, an imprint of Elsevier.)

Iron accumulation in various organs is a serious complication in β-thalassemia major and is a significant cause of morbidity and mortality in adults.³ In children, excess iron causes growth retardation and absence of sexual maturity; in adults, it causes cardiomyopathy, fibrosis and cirrhosis of the liver, and dysfunction of exocrine glands.³^,¹⁵ The risk of organ damage due to iron accumulation begins to increase after 10 to 11 years of age.¹⁵ The iron overload is predominantly due to the regular RBC transfusions required in β-thalassemia major (discussed later). However, the extreme degree of erythropoiesis also suppresses hepcidin production by the liver, resulting in more iron absorption by the intestinal enterocytes (Chapter 11).³^,¹⁶ This increase in intestinal iron absorption further adds to the iron overload burden.³^,¹⁶ The pathophysiology of β-thalassemia major is summarized in Figure 28-4.

FIGURE 28-4 Pathophysiology of severe forms of β-thalassemia. The diagram outlines the pathogenesis of clinical abnormalities resulting from the primary defect in β-globin chain synthesis. RBC, Red blood cell. Source: (From Giardina PJ, Rivella S: Thalassemia Syndromes. In: Hoffman R, Benz EJ, Jr, Silberstein LE, et al, editors: Hematology: basic principles and practice, ed 6, Philadelphia, 2013, Saunders, an imprint of Elsevier.)

Mechanisms in α-thalassemia

In α-thalassemia, the decreased production of α chains can manifest in utero because the α chain is a component of both fetal and adult hemoglobins. However, the accumulation of non–α chains has different consequences compared to β-thalassemia. In the fetus and newborn, a decrease in production of α chains results in an excess of γ chains. These γ chains accumulate in proportion to the number of deleted or defective α genes.¹¹^,¹³ The γ chains are more stable and do not precipitate but instead form hemoglobin tetramers (γ₄) called Hb Bart.¹³ After 6 months of age and through adulthood when the γ to β switch is completed, the decrease in α chain production results in excess β chains. The excess β chains are also relatively stable and form tetramers (β₄), called Hb H.

Because Hb H and Hb Bart do not precipitate to any significant degree in the developing RBCs in the bone marrow, patients with α-thalassemia do not have severe ineffective erythropoiesis.¹¹ As the mature RBCs age in the circulation, however, the β₄ tetramers in Hb H eventually precipitate and form inclusion bodies.¹¹ The macrophages in the spleen recognize and remove these abnormal RBCs from the circulation, and the patient manifests a moderate hemolytic anemia.

In addition to the decreased production and shortened RBC survival mechanisms, a third mechanism is involved in the anemia of α-thalassemia. Hb Bart and Hb H cannot deliver oxygen to tissues due to their very high affinity for oxygen.¹³ A fetus cannot survive with only Hb Bart (found with a deletion of all four α-globin genes). The marked tissue hypoxia causes heart failure and massive edema (hydrops fetalis) and hepatomegaly, and the fetus usually dies in utero or shortly after birth.³ This is discussed in the α-thalassemia section later in the chapter.

β-globin gene cluster thalassemias

There is great heterogeneity in the mutations in the β-globin gene cluster that leads to the clinical syndrome of β-thalassemia.¹² More than 300 genetic abnormalities have been discovered, including mutations affecting the β-, δ-, and γ-globin genes individually or in combination.¹²^,¹³ A small subset of mutations, however, accounts for the majority of the mutant alleles within a single ethnic group or geographic area in which β-thalassemia is found.³^,¹⁵ Because multiple mutations are present in each population, most individuals with severe β-thalassemia are compound heterozygotes for two different β-thalassemia mutations.¹¹ A comprehensive list of hemoglobin gene mutations is maintained in the HbVar mutation database, which is available online.¹²

Clinical syndromes of β-thalassemia

β-thalassemia is divided into four categories based on clinical manifestations (Table 28-3):³^,¹³

β-thalassemia silent carrier (heterozygous state) with nohematologic abnormalities or clinical symptoms
β-thalassemia minor (heterozygous state) with mild hemolytic anemia, microcytic/ hypochromic RBCs, and no clinical symptoms
β-thalassemia major (homozygous or compound heterozygous state) with severe hemolytic anemia, microcytic/hypochromic RBCs, severe clinical symptoms, and transfusion-dependence
β-thalassemia intermedia with mild to moderate hemolytic anemia, microcytic/hypochromic RBCs, moderate clinical symptoms, and transfusion independence

TABLE 28-3

Clinical Syndromes of β-Thalassemia with Examples of Genotypes

Genotype	Hb A	Hb A2	Hb F	Hb Lepore
Normal (Normal Hematologic Parameters)
β/β	N	N	N	0
Silent Carrier State (Asymptomatic; Normal Hematologic Parameters)
β^silent/β	N	N	N	0
Thalassemia Minor (Asymptomatic; Mild Hemolytic Anemia; Microcytic, Hypochromic)
β+/β			N to Sl ↑	0
β0/β			N to Sl ↑	0
δβ0/β		N to ↓	5%–20%	0
δβ^Lepore/β				5%–15%
Thalassemia Major (Severe Hemolytic Anemia; Transfusion-Dependent; Microcytic, Hypochromic)
β⁺/β+	↓	V	↑	0
β+/β0	↓↓	V	↑	0
β0/β0	0	V	↑	0
δβ^Lepore/δβ^Lepore	0	0	80%	20%
Thalassemia Intermedia (Mild to Moderate Hemolytic Anemia; Transfusion-Independent; Microcytic, Hypochromic)*
β^silent/β^silent				0
β+/β^silent or β0/β^silent				0
δβ0/δβ0	0	0	100%	0
β0/δβ0	0	N	↑	0

* Patients who are transfusion-independent do not require regular transfusions for survival, but may need transfusions occasionally, such as during pregnancy or infections.

** Other genotypes are included in this category such as dominantly inherited β-thalassemia (heterozygous for a very severe β-globin gene mutation) and coinheritance of a triplicated α-globin gene (ααα/αα) with thalassemia minor.

↑, Increased; ↓, decreased; 0, absent; Hb, hemoglobin; N, normal; Sl, slight; V, variable.

The clinical manifestations of the various mutations depend on whether one or both of the β-globin genes are affected and the extent to which the affected gene or genes are expressed. Some mutations result in the complete absence of β chain production, and genes with these mutations are designated as β⁰. Other mutations lead to production of the β chains but at a significantly reduced rate, and these are designated as β⁺ mutations. The range of β chain production in these β⁺ mutations varies from 5% to 30% of normal β chain synthesis.³ Still other mutations only minimally reduce β chain production, and genes with those mutations are designated as β^silent (Table 28-2).

β-thalassemia is inherited in an autosomal recessive pattern. If both parents are carriers of a β-thalassemia gene mutation, they have a 25% chance of having a child with two mutated β-globin genes (homozygote or compound heterozygote) and clinical manifestations of β-thalassemia major or intermedia.

Silent carrier state of β-thalassemia

The designation β^silent includes the various heterogeneous β-globin gene mutations that produce only a small decrease in production of the β chains. The silent carrier state (β^silent/β) results in nearly normal α-β chain ratios and no hematologic abnormalities.³^,¹¹^,¹³ It was first recognized through a study of families in which the affected children had a more severe β-thalassemia syndrome than a parent with typical β-thalassemia minor.¹³ The parents had normal levels of Hb A₂ and a slight microcytosis. Some individuals who are homozygous for asilent thalassemia gene mutation (β^silent/β^silent) have been described.¹³^,¹⁷ They present with a mild β-thalassemia intermedia phenotype with an increased level of Hb F and Hb A₂.¹³^,¹⁷

β-thalassemia minor

β-thalassemia minor (also called β-thalassemia trait) results when one β-globin gene is affected by a mutation that decreases or abolishes its expression, whereas the other β-globin gene is normal (heterozygous state). It usually presents as a mild, asymptomatic anemia with hemoglobin ranging from 12.4 to 14.2 g/dL in affected men and 10.8 to 12.8 g/dL in affected women.¹¹ The RBC count is within the reference interval or slightly elevated.³^,¹³ The RBCs are microcytic and hypochromic, with a mean cell volume (MCV) less than 75 fL and a mean cell hemoglobin (MCH) less than 26 pg.³ The reticulocyte count is within the reference interval or slightly increased.³ Some degree of poikilocytosis (including target cells and elliptocytes) and basophilic stippling in the RBCs may be seen on a Wright-stained peripheral blood film (Figure 28-5). The bone marrow shows mild to moderate erythroid hyperplasia, with minimal ineffective erythropoiesis. Hepatomegaly and splenomegaly are seen in a few patients. In the most common β-thalassemia minor syndromes (β⁰/β and β⁺/β), the Hb A level is 92% to 95% and the Hb A₂ level is characteristically elevated and can vary from 3.5% to 7.0%.³^,¹³^,¹⁵^.The Hb F level usually ranges from 1% to 5%.³^,¹³ Less common types of β-thalassemia minor exist, such as δβ⁰/β and δβ^Lepore/β. Other rare types have atypical features, such as Dutch β⁰-thalassemia minor that shows the expected elevation in Hb A₂ level but an Hb F level in the 5% to 20% range,¹⁸ and another mutant found in a Sardinian family in which the Hb A₂ level is normal.¹⁹

FIGURE 28-5 Red blood cells (RBCs) from a patient with β-thalassemia minor, showing microcytic, hypochromic RBCs with target cells, other poikilocytes, and basophilic stippling (arrow).

β-thalassemia major

β-Thalassemia major is characterized by a severe anemia that requires regular transfusion therapy. It is usually diagnosed between 6 months and 2 years of age (after completion of the γ to β switch) when the child’s Hb A level does not increase as expected.³^,¹³

In untreated β-thalassemia major, the hemoglobin level can fall as low as 3 to 4 g/dL.³^,¹³ The MCV ranges from 50 to 70 fL.¹¹^,¹⁵ The peripheral blood film shows marked microcytosis, hypochromia, anisocytosis, and poikilocytosis, including target cells, teardrop cells, and elliptocytes. Polychromasia and nucleated red blood cells may be observed (Figure 28-6). RBC inclusions are commonly found, including basophilic stippling, Howell-Jolly bodies, and Pappenheimer bodies, the latter as a result of the excess nonheme iron in the RBCs. The reticulocyte count is only mildly to moderately elevated and is inappropriately lower in relation to the amount of RBC hyperplasia and hemolysis present.³ The inappropriate reticulocytosis results from the apoptosis of RBC precursors in the bone marrow (ineffective erythropoiesis).

FIGURE 28-6 Red blood cells from a patient with β-thalassemia major. Note basophilic stippling, microcytosis, hypochromia, target cells, nucleated red blood cells, and red cell fragments. A, ×500. B, ×1000. Source: (Adapted from Carr JH, Rodak BF: Clinical hematology atlas, ed 4, Philadelphia, 2013, Saunders.)

Hb A is absent or decreased, depending on the specific genotype, which determines whether none (β⁰/β⁰) or a decreased amount (β⁺/β⁺ or β⁰/β⁺) of β chains are produced. Hb A is produced only if a β⁺ mutation is present, and usually ranges from 10 to 30%.³^,¹⁵ Hb F ranges from 70% to greater than 90%, depending on the genotype and amount of Hb A.¹³^,¹⁵ The level of Hb A₂ is variable and can be within or above the reference interval.¹¹ The bone marrow shows marked erythroid hyperplasia, with a myeloid-to-erythroid (M:E) ratio of 1:20 (reference interval is 1.5:1 to 3.3:1). As a result of the massive destruction of erythroid cells and release of free hemoglobin, the serum haptoglobin level is reduced or absent, and the serum lactate dehydrogenase activity is markedly elevated (Chapter 23).

Transfusion therapy is the major therapeutic option for patients with thalassemia major and typically is initiated when the hemoglobin drops to less than 7 g/dL and the patient has clinical symptoms.³^,²⁰ Typically, 10 to 15 mL/kg of RBCs are transfused every 2 to 5 weeks.³ RBCs that are less than 7 to 10 days old are used for transfusion to allow for maximum donor RBC survival in the patient.³ Typing the patient for the major blood group antigens and transfusing antigen-negative donor RBCs are recommended to reduce the risk of alloimmunization.¹¹^,¹⁵

Administration of RBC transfusions at regular intervals began in the mid-1970s. The pretransfusion hemoglobin level is usually maintained between 9 and 10.5 g/dL.³^,²¹ Such transfusion regimens are termed hypertransfusion and are used not only to correct the anemia but to also suppress the marked erythropoiesis. With erythropoiesis suppressed, the marked marrow expansion does not occur, and therefore the bone changes do not take place. In addition, the reduction in erythropoiesis decreases the amount of iron absorbed in the intestinal enterocytes.¹⁶ Children receiving this therapy do not develop hepatosplenomegaly and have much-improved growth and development.³ The transfusion regimens, however, lead to an excess iron burden. Because there is no effective physiologic pathway for iron excretion in the body, the iron contained in the transfused RBCs accumulates in the body. This iron is stored in organs outside the bone marrow (e.g., liver, heart, pancreas), which results in organ damage. The accumulation of iron in the liver leads to cirrhosis, and the deposition of iron in the heart leads to cardiac dysfunction and arrhythmias. In the past, with transfusion therapy alone, thalassemic patients died in their teens, typically from cardiac failure. Now patients undergo iron chelation therapy with the transfusion therapy. Iron chelating agents bind excess iron so that it can be excreted in the urine and stool. The standard chelation therapy is a daily infusion of deferoxamine, usually administered subcutaneously with an infusion pump over 8 to 12 hours.³^,¹¹ Owing to the cost, inconvenience, and side effects, patients may have poor compliance with the regimen.³^,¹¹ Two oral iron chelators, deferasirox and deferiprone, have been approved by the U.S. Food and Drug Administration, which may improve compliance, but their long-term efficacy compared to deferoxamine is still being evaluated.³^,¹³^,²² Additional oral iron chelating drugs are in development. Iron chelation treatment has been able to prevent iron accumulation and the subsequent complications of iron overload, helping to extend life expectancy of patients with β-thalassemia major into the fourth and fifth decade and beyond.³^,¹⁵^,²²

Hematopoietic stem cell transplantation (HSCT) is the only curative therapy for thalassemia major.³^,²³ In patients with a good risk profile (on a regular chelation therapy regimen, with no hepatomegaly or liver fibrosis), the average overall and thalassemia-free survival rates are greater than 90% and 80%, respectively.¹⁵^,²³ The highest survival rates occur in young patients with an HLA identical sibling donor.²³ Because there is only a 25% chance that a sibling will have the identical HLA genotype, this option is not available to all patients. A well-matched unrelated donor can be used, but survival is not as high, and finding an immunologic match in an unrelated donor is less likely.

Hemoglobin F induction agents, such as hydroxyurea, 5-azacytidine, short chain fatty acids, erythropoietic-stimulating agents, and thalidomide derivatives have been evaluated for therapy in thalassemia major because of their ability to “switch on” the γ-globin gene to produce more γ chains.²⁴ The γ chains then combine with the excess α chains to form Hb F, thus partially correcting the α/β chain imbalance. Hydroxyurea therapy has benefited a few β-thalassemia major patients, allowing them to become transfusion-independent, but has not been beneficial in the majority of patients.³^,²⁴ Studies with the other Hb F-inducing agents have shown initial promising results.²⁴ However, larger and better-designed studies are needed to determine the efficacy of these agents in thalassemia major.²⁴

In 2010, a successful lentilviral β-globin gene transfer was reported in an adult with severe Hb E-β⁰-thalassemia who became transfusion-independent.²⁵ Based on this success, clinical trials have opened at multiple sites using lentalviral vectors for β- and γ-globin gene transfer.²⁶ In addition, research on the use of gene therapy to increase expression of the patient’s own γ-globin genes has also intensified.²⁶ Ideally, in the future, gene therapy will be able to correct the genetic defect.

β-thalassemia intermedia

Thalassemia intermedia is a term used to describe anemia that is more severe than β-thalassemia minor but does not require regular transfusions to maintain hemoglobin level and quality of life (transfusion-independent).³^,¹¹ Although patients with thalassemia intermedia typically maintain a hemoglobin level greater than 7 g/dL, it is the clinical features rather than the hemoglobin level that determine the diagnosis.³^,¹¹ In these patients, the α/β chain imbalance falls between that observed in β-thalassemia minor and β-thalassemia major but without the need for regular transfusion therapy. The genotypes of thalassemia intermedia show great heterogeneity. Patients can be homozygous for mutations that cause a mild decrease in β-globin expression. Conversely, they may be compound heterozygous, with one gene causing a mild decrease in β chain production and the other causing a marked reduction in β chain production.³^,¹¹ In rare instances, only one of the β-globin genes carries a mutation, but it is severe enough to cause a significant anemia. These cases are sometimes called dominantly inherited β-thalassemia.¹³ Many of the thalassemia intermedia phenotypes are generated from the coinheritance of one or two abnormal β-globin genes with another hemoglobin defect, such as abnormal α-globin genes or unstable hemoglobins.³^,¹¹ The coinheritance of α-thalassemia may permit homozygotes with more severe β-thalassemia mutations to remain transfusion independent because the α/β chain ratio is more balanced and fewer free α chains are available to precipitate and cause hemolysis.³ Less severe clinical manifestations also occur when a β-thalassemia mutation is combined with a mutation that increases the expression of the γ-globin gene.³ The increase in Hb F production (α₂γ₂) helps to compensate for the reduction in Hb A, while helping to correct the α/β balance. Examples of these situations are the deletional forms of δβ⁰-thalassemia. Individuals homozygous for these mutations, or compound heterozygotes for δβ⁰-thalassemia and a β-thalassemia mutation, have thalassemia intermedia with increased γ chain and Hb F synthesis.³^,¹³ Conversely, coinheritance of a triplicated α-globin gene locus (ααα) (see section on α-thalassemia) is also a cause of thalassemia intermedia in some individuals heterozygous for β-thalassemia due to the production of more α chains and greater imbalance of the α/β chain ratio.³^,²⁷

Because of the genetic heterogeneity of β-thalassemia intermedia, the laboratory and clinical features vary. The degree of anemia and jaundice varies, depending on the extent to the α/β chain imbalance. Because of the presence of splenomegaly, the platelet and neutrophil counts may be low. The clinical course varies from minimal symptoms (despite moderately severe anemia) to severe exercise intolerance and pathologic fractures.¹¹ Patients with thalassemia intermedia also have iron overload even though they do not receive transfusions.³ The markedly accelerated ineffective erythropoiesis suppresses hepcidin production by the liver, which results in more iron absorption by the intestinal enterocytes.¹⁶ Cardiac, liver, and endocrine complications, however, present 10 to 20 years later in thalassemia intermedia patients than in patients who receive regular transfusions.³

Other thalassemias caused by defects in the β-globin gene cluster

Other thalassemias may be caused by deletion, inactivation, or fusion of a combination of genes of the β-globin gene cluster, such as hereditary persistence of fetal hemoglobin (HPFH), δβ⁰-thalassemia, and Hb Lepore thalassemia.³^,¹¹^,²⁸

Thalassemias with increased levels of fetal hemoglobin

HPFH and δβ⁰-thalassemia are closely related, heterogeneous conditions in which Hb F is expressed at increase levels beyond infancy into adulthood. These conditions have similarities but can be differentiated by the clinical presentation, hemoglobin level, MCV, and amount of Hb F produced.¹³

In HPFH, the β-globin gene cluster typically contains a deletion in the δβ region that leads to the increased production of Hb F. However, there are also HPFH conditions that have intact β-globin gene clusters with non-deletional mutations in the promoter region of the γ-globin genes that lead to the increased Hb F production.¹¹^,¹³^,²⁹ Because individuals with these mutations are characteristically asymptomatic, this condition is of little significance except when it interacts with other forms of thalassemia or structural hemoglobin variants, such as Hb S. The additional γ chains produced are able to replace the missing β chains and help to restore the balance of α and non-α chains (γ or β). Significant variation is seen in heterozygotes for deletional-type HPFH, but these patients typically are asymptomatic with a normal MCV and Hb F levels of 10% to 35%, depending on the mutation.³^,¹¹ Homozygotes for deletional-type HPFH are also asymptomatic. They have a normal to slightly increased hemoglobin level, 100% Hb F, with slightly hypochromic and microcytic RBCs.¹³ The increase in hemoglobin observed in some patients is likely a response to the slight hypoxia induced by the higher oxygen affinity of Hb F compared with Hb A.³ When assessed using the Kleihauer-Betke acid elution stain (discussed later), the distribution of Hb F in HPFH is usually pancellular (deletional-types), but it can be heterocellular (non-deletional-types). In contrast, the Hb F distribution in the other β-globin gene cluster thalassemias is always heterocellular.³^,³⁰

The δβ⁰-thalassemias are also characterized by deletions in the δ- and β-globin genes and an increase in Hb F in adult life. Non-deletional types have also been described.¹¹ In this condition, however, the increase in production of the γ chains is not sufficient to completely restore the balance between the α and non-α chains. Heterozygous δβ⁰-thalassemia individuals (δβ⁰/β) have a decreased level of Hb A, normal or decreased level of Hb A₂, and 5% to 20% Hb F.¹¹^,¹³ They have a β-thalassemia minor phenotype, with a slight decrease in hemoglobin level and hypochromic, microcytic RBCs. Homozygous δβ⁰-thalassemia individuals (δβ⁰/δβ⁰) have hypochromic, microcytic RBCs, 100% Hb F, and a β-thalassemia intermedia phenotype¹¹^,¹³ (Table 28-3).

Hemoglobin lepore thalassemia

Hemoglobin Lepore (δβ^Lepore) is structural variant and rare type of δβ-thalassemia caused by a fusion of the δβ-globin genes.¹² This mutation occurs during meiosis due to nonhomologous crossover between the δ-globin locus on one chromosome and the β-globin locus on the other chromosome. The Lepore globin chain expressed by the δβ fusion gene contains the first 22 to 87 amino acids of the N-terminus of the δ chain and the last 31 to 97 amino acids of the C-terminus of the β chain, depending on the variant.¹² The δβ fusion gene produces a reduced level of the Lepore globin chain because its transcription is under the control of the δ-globin gene promoter, which is much less active than the β-globin gene promoter.³ Conversely, in the reciprocal fusion on the other chromosome (called anti-Lepore), the β-globin gene locus is intact, so normal production of the β chain occurs.³^,¹³ In heterozygotes (δβ^Lepore/β), there is a decreased level of Hb A and Hb A₂, an increase in Hb F, and approximately 5% to 15% Hb Lepore.³^,¹³ The clinical manifestations are similar to β-thalassemia minor. In homozygotes (δβ^Lepore/ δβ^Lepore), there are no normal δ- or β-globin genes, no production of Hb A and Hb A₂, and approximately 80% Hb F and 20% Hb Lepore.¹³ The clinical manifestations are similar to β-thalassemia major³ (Table 28-3).

Screening for β-thalassemia minor

Because of the high carrier frequency of β-thalassemia mutations worldwide, screening has become an important global health issue.⁵^,⁶ Mass-screening programs in Italy and Greece combined with prenatal diagnosis have led to a significant reduction in the number of children born with β-thalassemia major.¹³ Carrier parents have a 25% risk of having a child with thalassemia major or thalassemia intermedia, depending on the particular β globin gene mutations.¹² Potential carriers of these disorders can be initially identified by measuring the hemoglobin level, MCV, and the Hb A₂ and Hb F levels.¹³^,³¹ Other causes of microcytic anemias, such as iron deficiency, need to be ruled out. Molecular genetic testing of the HBB gene is performed for carrier detection in couples seeking preconception counseling and prenatal testing.¹⁵

α-thalassemias

In contrast to β-thalassemia, in which point mutations in the β-globin gene cluster are the most common type of mutation, in α-thalassemia large deletions involving the α₁- and/or α₂-globin genes are the predominant genetic defect. Non-deletional mutations (mostly point mutations) also occur in α-thalassemia but are uncommon.¹²^,¹³^,³² The extent of decreased production of the α chain depends on the specific mutation, the number of α-globin genes affected, and whether the affected α-globin gene is α₂ or α₁.¹¹ The α₂-globin gene produces approximately 75% of the α chains in normal RBCs, so mutations in the α₂-globin gene generally cause more severe anemia than mutations affecting the α₁-globin gene.¹¹^,¹³^,³² The notation for the normal α-globin gene complex or haplotype is αα, which signifies the two normal genes (α₂ and α₁) on one chromosome 16. A normal genotype is αα/αα.

The α-thalassemias are divided into two haplotypes: α⁰-thalassemia and α⁺-thalassemia. In the α⁰-thalassemia haplotype (originally named α-thal-1), a deletion of both α-globin genes on chromosome 16 results in no α chain production from that chromosome. The designation, – –, is used for the α⁰-thalassemia haplotype.¹¹^,¹³ There are 21 known mutations that produce the α⁰-thalassemia haplotype and involve deletion of both of the α-globin genes or the entire α-globin gene cluster (including the ζ-globin gene) on one chromosome.¹² The α⁰ haplotype (– –) is found in approximately 4% of the population in Southeast Asia, is found less frequently in the Mediterranean region, and occurs infrequently in other parts of the world.⁶

In the α⁺-thalassemia haplotype (originally named α-thal-2), a deletional or non-deletional mutation in one of the two α-globin genes on chromosome 16 results in decreased α chain production from that chromosome.¹¹ The designation, – α, is used for the deletional mutations, while the designation, α^Tα, is used for the non-deletional mutations. The deletional α⁺haplotype (– α) is by far the most common of the α-thalassemia haplotypes. It is widely distributed throughout the thalassemia belt and central Africa (Figure 27-3), with a carrier frequency reaching 50% to 80% in some regions of Saudi Arabia, India, Southeast Asia, and Africa.⁶ The deletional α⁺ haplotype (– α) is also found in about 30% of African Americans.³²The non-deletional α⁺ haplotype (α^Tα) is relatively uncommon.¹³^,³² More than 40 different mutations are known, the majority of which are point mutations that affect the predominant α₂gene.¹² The α^Tα haplotype produces unstable α chains or fewer α chains than in the – α haplotype and generally results in a more severe anemia.¹³^,³²

One of the most common non-deletional α-globin gene mutations is Constant Spring (α₂^142Stop^→^Gln), also called α^CS, haplotype, α^CSα.¹²^,¹³ It is the result of a point mutation in the α₂-globin gene that changes the stop codon at 142 to a glutamine codon.¹²^,¹³^,³² As a result, additional bases are added to the end of the mRNA during transcription until the next stop codon is reached. The elongated mRNA is very unstable and produces only a small amount of the α^CS chain.³^,³³^,³⁴ The α^CS chains (with an additional 31 amino acids added to the C-terminal end) combine with β chains to form Hb Constant Spring, but the incorporation of a longer α chain makes the tetramer unstable.³⁴ Because of the instability of both the mRNA and the Hb tetramer, the circulating level of Hb Constant Spring is very low.¹³^,³²^,³⁴ Consequently, hemoglobin Constant Spring is difficult to detect by alkaline hemoglobin electrophoresis, and when present, is visualized as a faint, slow-moving band near the point of origin.¹³

Clinical syndromes of α-thalassemia

Four clinical syndromes are present in α-thalassemia, depending on the gene number, cis or trans pairing, and the amount of α chains produced.¹¹^,¹³ The four syndromes are¹¹^,¹³ (Table 28-4):

Silent carrier state
α-thalassemia minor
Hb H disease
Hb Bart hydrops fetalis syndrome

TABLE 28-4

Clinical Syndromes of α-Thalassemia

Genotype	Hb A	Hb Bart (in Newborn)	Hb H (in Adult)	Hb Constant Spring
Normal (Normal Hematologic Parameters)
αα/αα	N	0	0	0
Silent Carrier State (Asymptomatic; Normal Hematologic Parameters)
– α/αα	N	1%–2%	0	0
α^CSα/αα	N	1%–3%	0	< 1%
α-Thalassemia Minor (Asymptomatic; Mild Hemolytic Anemia; Microcytic, Hypochromic)
– –/αα	Sl ↓	5%–15%	0	0
– α/– α	Sl ↓	5%–15%	0	0
α^CSα/α^CSα*	Sl ↓	5%–15%	0	< 6%
Hb H Disease (Mild to Moderate Hemolytic Anemia; Transfusion-Independent**; Microcytic, Hypochromic)
– –/– α		10%–40%	1%–40%	0
– –/α^CSα^†		↑	↑	< 1%
Hb Bart Hydrops Fetalis Syndrome (Severe Anemia; Usually Infants are Stillborn or Die Shortly after Birth)
– –/– –	0	80%–90% (remainder Hb Portland)	NA	0

* αCSα/αCSα genotype results in mild to moderate hemolytic anemia with jaundice and hepatosplenomegaly.

* *Patients who are transfusion-independent do not require regular transfusions for survival, but may need transfusions occasionally, such as during pregnancy or infections.

† – –/αCSα genotype and other non-deletional genotypes (– –/αTα) result in Hb H disease that is moderate to severe and may require more frequent transfusions than the deletional – –/– α genotype.

↓, Decreased; ↑↑ increased more than – –/– α; 0, absent; < , less than; CS, Constant Spring; Hb, hemoglobin; N, normal; NA, not applicable.

Silent carrier state

The deletion of one α-globin gene, leaving three functional α-globin genes (– α/αα), is the major cause of the silent carrier state. The α/β chain ratio is nearly normal, and no hematologic abnormalities are present.¹¹^,¹³ Because one α-globin gene is absent, there is a slight decrease in α chain production. There is a slight excess of γ chains at birth that form tetramers of Hb Bart (γ₄) in the range of 1% to 2%.¹¹^,¹³ There is no reliable way to diagnose silent carrier state other than genetic analysis. A non-deletional α⁺ mutation in one α-globin gene (α^Tα/αα) also results in the silent carrier state. In the heterozygous mutation, α^CSα/αα, Hb Constant Spring is less than 1% of the total hemoglobin.¹³

α-thalassemia minor

Deletion of two α-globin genes is the major cause of α-thalassemia minor. It exists in two forms: homozygous α⁺ (– α/– α) or heterozygous α⁰ (– –/αα).¹¹^,¹³ This syndrome is asymptomatic and characterized by a mild anemia (typical hemoglobin concentration is 12 to 13 g/dL) with microcytic, hypochromic RBCs. At birth, the proportion of Hb Bart is in the range of 5% to 15%.¹³ In adults, the production of α and β chains is balanced, so Hb H (β₄) is not usually present. Homozygosity for non-deletional mutations in both α₂-globin genes (α^Tα/α^Tα) produces a mild to moderate hemolytic anemia, often with jaundice and hepatosplenomegaly.¹³^,³⁴ In the homozygous mutation, α^CSα/α^CSα, Hb Constant Spring is 5% to 6% of the total hemoglobin and the hemoglobin concentration is 9 to 11 gm/dL.¹¹^,¹³^,³⁴

Hemoglobin h disease

Deletion of three α-globin genes is the major cause of Hb H disease in which only one α-globin gene remains to produce α chains (– –/– α).¹¹^,¹³ This genetic abnormality is particularly common in Asians because of the prevalence of the α⁰ gene haplotype (– –). It is characterized by the accumulation of excess unpaired β chains that form tetramers of Hb H in adults. In the newborn, Hb Bart comprises 10% to 40% of the hemoglobin, with the remainder being Hb F and Hb A. After the γ to β switch, Hb H replaces most of the Hb Bart, so Hb H is in the range of 1% to 40%, with a reduced amount of Hb A₂, traces of Hb Bart, and the remainder Hb A.¹¹^,¹³^,³²^,³³ The non-deletional α⁺ haplotype, when combined with the α⁰ haplotype (– –/α^Tα), generally produces a more severe Hb H disease with a higher level of Hb H than the α⁰ interaction with the deletional α⁺ haplotype (– –/– α).¹³^,³²^,³⁴ Hb H-Hb Constant Spring (– –/α^CSα) is an example.¹³^,

Hb H disease is characterized by a mild to moderate, chronic hemolytic anemia with hemoglobin concentrations averaging 7 to 10 g/dL, and reticulocyte counts of 5% to 10%, although a wide variability in clinical and laboratory findings exists.¹¹^,³⁶ The bone marrow exhibits erythroid hyperplasia, and the spleen is usually enlarged. Patients with deletional Hb H disease are transfusion-independent, that is, they do not require regular transfusions. However, infection, pregnancy, or exposure to oxidative drugs may cause a hemolytic crisis requiring transfusions on a temporary basis.

Hemolytic crises often lead to the detection of the disease because individuals with Hb H disease may otherwise be asymptomatic. The RBCs are microcytic and hypochromic, with marked poikilocytosis, including target cells and bizarre shapes. Hb H is vulnerable to oxidation and gradually precipitates in the circulating RBCs to form inclusion bodies of denatured hemoglobin.¹¹ These inclusions alter the shape and viscoelastic properties of the RBCs, contributing to the decreased RBC survival. Splenectomy is beneficial in patients with markedly enlarged spleens.³² When incubated with brilliant cresyl blue or new methylene blue, RBCs with Hb H display fine, evenly distributed, granular inclusions. These inclusions are typically removed as the RBC passes through the spleen. Before splenectomy, only a portion of the cells have this characteristic, but after the spleen is removed, most of the RBCs are full of these inclusions. These cells are often described as “golf balls” or “raspberries” (Figure 28-7).

FIGURE 28-7 Red blood cells from a patient with hemoglobin H disease, incubated with brilliant cresyl blue, which have acquired fine, evenly dispersed granular inclusions and “golf ball” appearance. Source: (From the American Society for Hematology slide bank.)

Two distinct conditions are associated with Hb H disease and congenital physical and intellectual abnormalities: alpha-thalassemia retardation-16 (ATR-16) syndrome and alpha-thalassemia X-linked intellectual disability (ATRX) syndrome. Patients with the ATR-16 syndrome inherit or acquire a large deletion in the short arm of chromosome 16, which removes the ζ- and α-globin genes as well as all the flanking genes to the terminus of the chromosome.³³ Patients have physical deformities, intellectual disabilities, and Hb H disease.³²^,³³^,³⁵ The ATRX syndrome is due to mutations of the ATRX gene located on the X chromosome.³⁷^,³⁸ The ATRX protein is a component of a large complex that regulates expression of various genes, including the α-globin genes.³³^,³⁸ The regulation is accomplished by DNA remodeling and/or methylation, thus affecting the transcription, replication, and repair of the target genes.³²^,³³^,³⁶^,³⁸ Therefore, when the ATRX gene is mutated, patients have decreased α chain production.³²^,³³^,³⁵^,³⁸ Affected males with ATRX syndrome have pronounced intellectual disability, physical deformities, developmental delay, and Hb H disease. An acquired Hb H disease with mutations in the ATRX gene has been found in myelodysplastic syndrome.³³^,³⁸^,³⁹

Hb bart hydrops fetalis syndrome

Homozygous α⁰-thalassemia (– –/– –) results in the absence of all α chain production and usually results in death in utero or shortly after birth.³^,¹¹ The fetus is severely anemic, which leads to cardiac failure and edema in the fetal subcutaneous tissues (hydrops fetalis). Hb Bart (γ₄) is the predominant hemoglobin, along with a small amount of Hb Portland (ζ₂γ₂) and traces of Hb H.³^,¹¹ Hb Bart has a very high oxygen affinity; it does not deliver oxygen to the tissues.¹¹^,¹³ The fetus can survive until the third trimester because of Hb Portland, but this hemoglobin cannot support the later stages of fetal growth, and the affected fetus is severely anoxic.¹¹ The fetus is delivered prematurely and is usually stillborn or dies shortly after birth. In addition to anemia, edema, and ascites, the fetus has gross hepatosplenomegaly and cardiomegaly.³^,¹¹ At delivery, there is a severe microcytic, hypochromic anemia (hemoglobin concentration of 3 to 8 gm/dL) with numerous nucleated RBCs in the peripheral blood.³³ The bone marrow cavity is expanded, and marked erythroid hyperplasia is present, along with foci of extramedullary erythropoiesis.

Hydropic pregnancies are hazardous to the mother, resulting in toxemia and severe postpartum hemorrhage.¹¹ Hydropic changes are detected in midgestation by means of ultrasound testing.⁴⁰ If both parents carry one α⁰-thalassemia haplotype (– –/αα), prenatal diagnosis of homozygosity can be made by molecular genetic testing of fetal cells from chorionic villus sampling or amniotic fluid.³³ Absence of the α-globin genes establishes the diagnosis. Early termination of the pregnancy prevents the serious maternal complications.¹¹

Thalassemia associated with structural hemoglobin variants

Hemoglobin s-thalassemia

Sickle cell anemia (Hb SS)- α-thalassemia is a genetic abnormality due to the coinheritance of two abnormal β-globin genes for Hb S and an α-thalassemia haplotype. Hb SS-α⁺-thalassemia is fairly common because the genes for Hb S and the α⁺-thalassemia haplotype, – α, are common in populations of African ancestry. Individuals with Hb SS–α⁺-thalassemia have a milder anemia with higher hemoglobin levels and lower reticulocyte counts than those with sickle cell anemia alone.⁴¹ In one study, Hb SS individuals with the genotypes, αα/αα, – α/αα, and – α/– α, had average hemoglobin concentrations of 8.4, 9.0, and 9.5 g/dL, respectively, and reticulocyte counts of 10.8%, 8.8%, and 6.9%, respectively.⁴¹

Hb S-β-thalassemia is a compound heterozygous condition that results from the inheritance of a β-thalassemia gene from one parent and an Hb S gene from the other. This syndrome has been reported in the populations of Africa, the Mediterranean area, the Middle East, and India.¹³ The clinical expression of Hb S-β-thalassemia depends on the type of β-thalassemia mutation inherited.¹¹^,¹³ Individuals with Hb S-β⁺-thalassemia produce variable amounts of normal β chains. Patients have mostly Hb S with slightly elevated Hb A₂ and variable amounts of Hb F and Hb A, depending on the specific abnormal β⁺ gene inherited. These patients can be distinguished from those with sickle cell anemia by the presence of microcytosis, splenomegaly, an elevated Hb A₂ level, and an Hb A level that is less than the Hb S level.

The interaction of β^silent-thalassemia (in which β chains are produced at mildly reduced levels) and Hb S results in a condition that may be slightly more severe than sickle cell trait. Typically, there is mild hemolytic anemia with splenomegaly. These patients can be distinguished from patients with sickle cell trait by the presence of microcytosis and splenomegaly. Hemoglobin electrophoresis or HPLC confirms this condition when the quantity of Hb S exceeds that of Hb A. In sickle cell trait, Hb A is the predominant hemoglobin.

The combination of β⁰-thalassemia and Hb S produces a phenotype similar to sickle cell anemia with a similar incidence of stroke and a similar life expectancy.⁴² Both conditions lack Hb A and produce severe painful crises as the predominant symptom. Typically, the microcytosis and elevated Hb A₂ level in Hb S-β⁰-thalassemia distinguish it from sickle cell anemia.

Hemoglobin c-thalassemia

Hb C-β-thalassemia produces moderately severe hemolysis, splenomegaly, hypochromia, microcytosis, and numerous target cells. The hemoglobin electrophoresis pattern varies, depending on the type of β-thalassemia gene defect, with higher Hb C concentrations in patients when there is minimal or no β chain production.¹³

Hemoglobin e-thalassemia

Hb E-β-thalassemia is a significant concern in Southeast Asia and Eastern India owing to the high prevalence of both genetic mutations.¹³ Hb E is due to a point mutation that inserts a splice site in the β-globin gene, and results in decreased production of Hb E.³ In the homozygous state (Hb EE) the clinical symptoms are similar to a mild β-thalassemia. (Chapter 27) When the mutations are coinherited in the compound heterozygous state, there is a marked reduction of β chain production. The clinical symptoms are similar to β-thalassemia intermedia or β-thalassemia major, depending on the particular β-globin gene mutation.¹³ Table 28-5 summarizes some compound heterozygous states of β-thalassemia combined with a structural β-globin defect.

TABLE 28-5

β-Thalassemia Associated with Structural β-Globin Variants (Compound Heterozygotes)

Genotype	Hb A	Hb A2	Hb F	Other Hb	RBC Morphology	Clinical Manifestations*	Treatment
Hb S-β⁺-thalassemia Hb S-β⁰-thalassemia	↓ 0		N to ↑ N to ↑	Hb S > Hb A Hb S	Microcytes, sickle cells, target cells	Ranges from mild to severe anemia with recurrent vasoocclusive crises	Ranges from no treatment to transfusion support and pain control
Hb C-β⁺-thalassemia Hb C-β⁰-thalassemia	↓ 0	^† ^†		Hb C > Hb A Hb C	Microcytes, Hb C crystals, target cells	Ranges from moderate to severe anemia	Usually no treatment needed
Hb E-β⁺-thalassemia Hb E-β⁰-thalassemia	↓ 0	^† ^†	↑ ↑	Hb E > Hb A Hb E	Microcytes, target cells	Ranges from mild to severe anemia with transfusion dependency	Ranges from no treatment to transfusion support

* Clinical manifestations depend on the amount of Hb A produced; compound heterozygotes with the β0 gene have more severe symptoms.

† Not all methods can quantitate Hb A2 in the presence of the abnormal hemoglobin. High-performance liquid chromatography can separate Hb A2 from Hb C; capillary zone electrophoresis can separate Hb A2 from Hb E.

↑, Increased; ↓, decreased; 0, absent; Hb, hemoglobin; N, normal; RBC, red blood cell.

Diagnosis of thalassemia

History and physical examination

Individual and family histories are paramount in the diagnosis of thalassemia. The ethnic background of the individual should be investigated because of the increased prevalence of specific gene mutations in certain populations. In the clinical examination, findings that suggest thalassemia include pallor (due to the anemia); jaundice (due to the hemolysis); splenomegaly (caused by sequestration of the abnormal RBCs, excessive extravascular hemolysis, and some extramedullary erythropoiesis); and skeletal deformities (due to the massive expansion of the bone marrow cavities). These findings are particularly prominent in untreated or partially treated β-thalassemia major.¹³ Table 28-6 contains a summary of tests for the diagnosis of thalassemia.

TABLE 28-6

Laboratory Diagnosis of Thalassemias¹⁵^,³¹^,³³^,⁴³

Screening tests	Complete blood count Peripheral blood film review Iron studies (to rule out IDA)	HGB, HCT, MCV, MCH, MCHC: ↓ RETIC: sl to mod ↑ Varying degrees of microcytosis, hypochromia, target cells, anisocytosis, poikiloctosis, RBC inclusions, NRBCs Serum ferritin and serum iron: N or ↑; TIBC: N
Presumptive diagnosis	Supravital stain Hemoglobin fraction quantification by electrophoresis, HPLC, and/or CZE	α-thal: Hb H inclusions β-thal: Hb A ↓ or 0; Hb A_2↑ (carriers); Hb F: usually ↑; Hb Lepore; other mutants α-thal: Hb A: ↓ or 0 (hydrops fetalis); Hb A2 ↓; Hb Bart, Hb H, Hb Constant Spring, other mutants
Definitive* diagnosis	Molecular genetic tests: β-thal: > 250 mutations in HBB α-thal: > 100 mutations in HBA1 and/or HBA2	Step 1: Targeted mutation analysis: β-thal: initial screen for four to six most common mutations if specific ethnic group known α-thal: initial screen for seven most common deletions If negative, Step 2: DNA sequence analysis If negative, Step 3: Deletion/duplication analysis (e.g., MLPA, aCGH)

* Required for prenatal diagnosis, preconception risk assessment/carrier detection in couples, diagnosis of rare or complex mutations, determining prognosis in young children

** From reference 12 (HbVar database, accessed May 10, 2014).

aCGH, Array comparative genomic hybridization; CZE, capillary zone electrophoresis; Hb, hemoglobin; HCT, hematocrit; HGB, hemoglobin level (g/dL); IDA, iron deficiency anemia; MCH, mean cell hemoglobin; MCHC, mean cell hemoglobin concentration; MCV, mean cell volume; MLPA, multiplex ligation-dependent probe amplification; mod, moderate; RETIC, reticulocyte count; RBCs, red blood cells; NRBCs, nucleated red blood cells; sl, slight; thal, thalassemia.

Laboratory methods

Complete blood count with peripheral blood film review

Although most thalassemias result in a microcytic and hypochromic anemia, laboratory results can vary from borderline abnormal to markedly abnormal; this depends on the type and number of globin gene mutations. The hemoglobin and hematocrit are decreased, but the RBC count can be disproportionately high relative to the degree of anemia, which can generate a very low MCV and mean cell hemoglobin (MCH). The mean cell hemoglobin concentration (MCHC) is also decreased. The RBC distribution width (RDW) is elevated (reflecting anisocytosis) in untreated β-thalassemia major, but it is often normal in β-thalassemia minor. On a Wright-stained peripheral blood film, the RBCs are typically microcytic and hypochromic, except in the silent carrier phenotypes, in which the RBCs appear normal. In β-thalassemia minor, α-thalassemia minor, and Hb H disease, the cells are microcytic with target cells and slight to moderate poikilocytosis. In homozygous and compound heterozygous β-thalassemia, extreme poikilocytosis may be present, including target cells and elliptocytes, in addition to polychromasia, basophilic stippling, Howell-Jolly bodies, Pappenheimer bodies, and nucleated RBCs.

Reticulocyte count

The reticulocyte count is elevated, which indicates that the bone marrow is responding to a hemolytic process. In Hb H disease, the typical reticulocyte count is 5% to 10%.¹³ In homozygous β-thalassemia, it is typically 2% to 8%, disproportionately low relative to the degree of anemia.¹³ An inadequate reticulocytosis reflects the ineffective erythropoiesis.

Supravital staining

In α-thalassemia minor, Hb H disease, and silent carrier α-thalassemia, brilliant cresyl blue or new methylene blue stain may be used to induce precipitation of the intrinsically unstable Hb H.³⁵^,⁴³ Hb H inclusions (denatured β-globin chains) typically appear as small, multiple, irregularly shaped greenish-blue bodies that are uniformly distributed throughout the RBC. They produce a pitted pattern on the RBCs similar to the pattern of a golf ball or raspberry (Figure 28-7). In Hb H disease, almost all RBCs contain Hb H inclusions.³⁵ In α-thalassemia minor, only a few cells may contain these inclusions, and in silent carrier α-thalassemia, only a rare cell does. These inclusions appear different from Heinz bodies, which are larger and fewer in number and most often appear eccentrically along the inner membrane of the RBC. This test is very sensitive in detecting Hb H in the α-thalassemia conditions.⁴³

Assessment of normal and variant hemoglobins

The major clinical laboratory methods used to identify and quantify normal and variant hemoglobins include hemoglobin electrophoresis, cation-exchange high-performance liquid chromatography (HPLC), and capillary zone electrophoresis (CZE).⁴⁴ Each of these methods has advantages and limitations, and no one method is able to identify and quantify all hemoglobins. Therefore, a combination of at least two of the above methods is used for confirmation of a hemoglobin variant.⁴⁵ Molecular genetic testing is required to detect specific mutations in globin genes and definitively identify the type of thalassemia or hemoglobinopathy. Molecular genetic testing is not usually required in adults with typical findings on the CBC, electrophoresis, and/or HPLC, but it is required for prenatal diagnosis, preconception risk assessment/carrier detection in couples, diagnosis of rare or complex mutations, and determining prognosis in young children.¹⁵^,³¹^,³³^,⁴⁴

Hemoglobin electrophoresis at alkaline pH has been the traditional tool for thalassemia and hemoglobinopathy diagnosis. In this method, patient RBC lysate is spotted on a solid support (such as agarose) and subjected to an electrical current in an alkaline buffer. Normal and variant hemoglobins will migrate and separate on the support according to their charge. The support is stained, and each hemoglobin band is quantified by scanning densitometry and reported as a percentage of the total hemoglobin.⁴⁵ This technique is able to distinguish the common hemoglobins, such as Hb A, Hb F, Hb S, Hb C, and the fast-moving hemoglobins, Hb H and Hb Bart.^44-46 Electrophoresis, however, has several limitations: it is labor-intensive and cannot accurately quantify Hb A₂ and Hb F. In addition, Hb S and Hb C must be confirmed by another method because Hb D and Hb G comigrate with Hb S, and Hb E and Hb O^Arabcomigrate with Hb C.⁴⁵ Methods used for confirmation usually include agar electrophoresis at acid pH, HPLC, or CZE, or in the case of Hb S, the solubility test (Figures 27-6, 27-7, 27-8,27-9). Figure 28-8 shows the relative hemoglobin mobilities in alkaline electrophoresis for various thalassemias and hemoglobinopathies.

FIGURE 28-8 Relative electrophoretic mobilities on cellulose acetate (pH 8.4) of various hemoglobins (Hbs) important in the diagnosis of thalassemia syndromes and hemoglobinopathies. β0T, β0-thalassemia major, β0/β0 (no Hb A, increased Hb F, slight increase in Hb A₂); β+T, β+-thalassemia major, β⁺/β⁺ (decreased Hb A, increased Hb F, slight increase in Hb A₂); βTT, β-thalassemia minor (slight decrease in Hb A, increased Hb A₂, some Hb F); δβ0T, δβ0-thalassemia, homozygous, δβ0/δβ0 (100% Hb F); HPFH, hereditary persistence of fetal hemoglobin, heterozygous (mostly Hb A, some Hb F, no Hb A₂); N, normal; SCA, sickle cell anemia (no Hb A, mostly Hb S, increased Hb F, normal Hb A₂); SCT, sickle cell trait (Hb A > Hb S, normal Hb A₂ and F); S-β0T, sickle cell-β0-thalassemia (no Hb A, increased Hb A₂ and F, mostly Hb S); S-β+T, sickle cell-β+-thalassemia (Hb A < Hb S, increased Hb A₂ and F).

In HPLC, patient RBC lysate in buffer is injected into a cation-exchange column. Both normal and variant hemoglobins will bind to the column. An elution buffer is injected and forms a gradient of varying ionic strength.⁴⁵ The various hemoglobin types will be differentially eluted from the column, each having a specific column retention time. As each hemoglobin fraction passes near the end of the column, a detector measures the absorbance of the fraction at 415 nm, which is recorded as a peak on a chromatogram.⁴⁵ The area under the peak is used to quantify the hemoglobin fraction, which is reported as a percentage of total hemoglobin. With the availability of fully automated instruments, HPLC has replaced hemoglobin electrophoresis in many laboratories as the routine screening method for analysis of hemoglobins.⁴⁴ The method is ideal for thalassemia screening because it can accurately and quickly quantitate Hb A, Hb A₂, and Hb F with 100% sensitivity and 90% specificity if no hemoglobin variants are present (Figure 28-9).⁴⁶ The precise and accurate quantification of Hb A₂ is particularly important in screening individuals for β-thalassemia minor (trait). HPLC can also presumptively identify and quantify hemoglobin variants even in low concentration.⁴⁴^,⁴⁵HPLC, however, requires specialized instrumentation and extensive experience and training to accurately interpret the complex chromatograms.⁴⁴^,⁴⁵^,⁴⁷ Additional limitations of HPLC include the following: Hb A₂ and Hb E have the same retention time and therefore cannot be accurately quantified by this method; Hb A₂ can be overestimated in the presence of Hb S due to overlapping peaks and underestimated in the presence of Hb D^Punjab; and it is not able to identify all variants.^44-47 A manual microcolumn method is also available for the measurement of Hb A₂.⁴⁵

FIGURE 28-9 Separation and quantification of hemoglobin fractions by high performance liquid chromatography (Bio-Rad, left) and capillary electrophoresis (Sebia, right). A, Healthy adult with Hb F < 1% and Hb A₂ < 3.5%; B, Adult with β-thalassemia minor with increased Hb F and Hb A₂. Source: (Modified from Giordano PC: Strategies for basic laboratory diagnostics of the hemoglobinopathies in multiethnic societies: interpretation of results and pitfalls. Int Jnl Lab Hem 35: 465-479, 2013, Figure 3, p. 472.)

In capillary zone electrophoresis (CZE), patient RBC lysate is introduced into a thin silica glass capillary tube in an alkaline buffer. When a current is applied, the various hemoglobin fractions migrate to the cathode at different velocities due to electro-endoosmotic flow.⁴⁵ As each hemoglobin fraction passes near the end of the capillary, a detector measures the absorbance of the fraction at 415 nm, which is recorded as a peak on a electrophoretogram. The instrument calculates the percentage of each hemoglobin fraction using an integration of the area under the peak and the migration time.⁴⁵ Fully automated systems are available that provide rapid and accurate identification and quantification. The peaks are placed into zones in the electrophoretogram for easier identification, and it can presumptively identify hemoglobin variants, including those in low concentration (Figure 28-9).⁴⁵ An advantage of CZE over HPLC is that it can separate and quantify Hb A₂ in the presence of Hb E.⁴⁴ However, because there is overlap in the peaks for Hb A₂ and Hb C, it cannot quantify Hb A₂ in the presence of Hb C.⁴⁴ As with HPLC, it also cannot detect all variants.⁴⁴^,⁴⁵ Complementing electrophoresis, HPLC, and/or CZE results, however, have minimized the limitations of all these methods.⁴⁷Other technologies such as isoelectric focusing and mass spectrometry are used in newborn screening programs for detection of common hemoglobin variants.^44-45

Molecular genetic testing

For mutations in the HBB gene, targeted mutation analysis using polymerase chain reaction (PCR)-based methods can be initially performed for detection and quantification of the four to six most common mutations if an individual’s ethnicity is known.³^,¹⁵ This strategy allows a mutation detection rate of 91% to 95% in Mediterranean, Middle East, Thai, and Chinese populations, and 75% to 80% in African and African-American populations.¹⁵ In multiethnic individuals or if the ethnicity is unknown, DNA sequencing of the HBB gene is performed including exons, intervening sequences, splice sites, and 5’ and 3’ untranslated regulatory regions.¹⁵^,⁴⁶ This strategy enables detection of approximately 95% of known mutants.¹⁵^,³¹ If sequencing is not successful, testing can reflex to deletion/duplication analysis (such as multiplex ligation-dependent probe amplification or array-based comparative genomic hybridization) (Chapter 31).¹⁵^,³¹^,⁴⁶

For mutations in the HBA1 or HBA2 genes, PCR-based targeted mutation analysis can also be initially performed for the seven most common deletional mutations.³³ This strategy detects approximately 90% of all alleles, but the detection rate varies by method.³³^,³⁶ If the above screening is not successful, DNA sequencing of the HBA1 and HBA2 genes or deletion/duplication analysis can be performed as described above.³³

When the parents’ mutation is known, analysis for the specific mutation in fetal cells can be done on specimens from amniocentesis (at 15 to 18 weeks’ gestation), chorionic villus sampling (at 10 to 12 weeks’ gestation), or with preimplantation genetic diagnosis using a cell from a 3-day-old embryo after in vitro fertilization.¹⁵^,³³

Other procedures

The classic alkali denaturation test is accurate and precise to quantify Hb F in the 0.2% to 50% range.⁴⁸ Most human hemoglobins are denatured on exposure to a strong alkali, but Hb F is not. The Hb F can be separated and its concentration compared with that of other hemoglobins. Consistent methodology is required to ensure accurate results.⁴⁸ However, automated HPLC is now often used to quantify Hb F.⁴⁵^,⁴⁶

In the Kleihauer-Betke acid elution slide test, peripheral blood films are ethanol-fixed and immersed in a citrate-acid buffer (pH 3.3). Adult hemoglobins are eluted from the RBCs, whereas Hb F resists acid elution and remains in the cell. When the cells are subsequently stained, RBCs containing Hb F will take up the stain, whereas RBCs containing only adult hemoglobin will appear as “ghosts.” This test determines if the Hb F distribution in RBCs is pancellular (found in all RBCs in deletional HPFH cases) or heterocellular (found in some but not all RBCs in β-globin gene cluster thalassemias and non-deletional HPFH cases).³⁵^,⁴⁵ The Kleihauer-Betke slide test is also used to estimate the volume of fetal-maternal hemorrhage to determine if an increased dose of Rh immune globulin is needed for an Rh-negative mother who delivers an Rh-positive baby. Because the Kleihauer-Betke slide test is cumbersome to perform and results are difficult to replicate, flow cytometry is becoming the standard test to measure fetal-maternal hemorrhage quickly and accurately.⁴⁹

In underdeveloped countries with limited technology, a single-tube osmotic fragility test has been used to screen populations for thalassemia carriers.¹¹ This is based on the fact that carriers have hypochromic RBCs, resulting in decreased osmotic fragility.⁴⁶^,⁵⁰ An aliquot of anticoagulated blood is incubated in 0.375% saline for 5 minutes.⁴⁴ Because the solution is hypotonic, normal RBCs will lyse and the solution will clear. However, patients with thalassemia have hypochromic RBCs that will not lyse in 0.375% saline, and the solution will remain turbid. This test is not specific for thalassemia and will be positive for any condition causing hypochromia, including iron deficiency anemia.

Differential diagnosis of thalassemia minor and iron deficiency anemia

The RBCs in thalassemia minor are microcytic and hypochromic, and this disease must be differentiated from iron deficiency anemia and other microcytic, hypochromic anemias. The differential diagnosis for microcytic, hypochromic anemias is relatively limited (Table 20-1). Differentiating thalassemia minor from iron deficiency is important to avoid unnecessary tests or treatments. An incorrect presumption that a patient has iron deficiency may lead to inappropriate iron therapy or to unnecessary diagnostic procedures, such as a colonoscopy, to identify a source of blood loss.

Clinical history is crucial. A family history of thalassemia raises the suspicion for this diagnosis. A history of previously normal hemoglobin levels and RBC indices, significant bleeding, or pica leads to the diagnosis of iron deficiency.⁵¹ Pica means cravings for nonfood items such as clay, dirt, or starch. The most common pica symptom in the United States is pagophagia, the craving to chew on ice.⁵¹

Iron deficiency and β-thalassemia minor are best differentiated using serum ferritin level, serum iron level, total iron-binding capacity, transferrin saturation, and Hb A₂ level, along with a complete blood count (CBC) and examination of a peripheral blood film.⁵¹^,⁵² Additional testing may also include soluble transferrin receptor and zinc protoporphyrin levels (Chapter 20).⁵¹

Before evaluating Hb A₂ levels for β-thalassemia minor, iron deficiency should be ruled out. Low iron levels in patients with β-thalassemia minor decrease the Hb A₂ levels.⁵² The iron stores need to be replenished before the laboratory analysis for thalassemia is undertaken.

A mild erythrocytosis (high RBC count) and marked microcytosis (low MCV) are found more commonly in β-thalassemia minor. In iron deficiency anemia, the RBC count and MCV may be normal or decreased, depending on whether the deficiency is developing or long-standing.^53-55 The RDW can be normal or increased in both β-thalassemia minor and iron deficiency anemia, with a significant overlap of values; therefore, the RDW alone cannot distinguish these conditions.^52-54 Various discrimination indices have been proposed to distinguish β-thalassemia minor from iron deficiency anemia using a calculation based on the RBC count, hemoglobin level, MCV, MCH, and/or RDW (such as the Mentzer, Green and King, England and Fraser, Shine and Lal, and Srivastava indices).^52-54 Unfortunately, the sensitivity of these indices in discriminating β-thalassemia minor and iron deficiency anemia ranged from 60% to 96% in various studies, which leads to a high number of false-negative results. Thus their use for screening is not appropriate.³^,¹¹^,^51-53 The peripheral blood film may demonstrate basophilic stippling in β-thalassemia minor, which can distinguish it from iron deficiency. Because target cells can be found in both conditions, however, their presence does not help discriminate between the two disorders.

Summary

Thalassemias are a group of heterogeneous disorders in which one or more globin chains are reduced or absent.
Thalassemias result in a hypochromic, microcytic anemia due to decreased production of hemoglobin. The imbalance of globin chain synthesis causes an excess of the normally produced globin chain that damages the RBCs or their precursors and results in hemolysis.
β-Thalassemia is caused by mutations that affect the β-globin gene complex. It is clinically manifested as silent carrier state, thalassemia minor, thalassemia intermedia, or thalassemia major.
In the silent carrier state (β^silent/β), the blood picture is completely normal. β-thalassemia minor (β⁰/β or β⁺/β) is a mild, asymptomatic, microcytic, hypochromic anemia; it is usually characterized by an elevated Hb A₂level, which aids in diagnosis. β-thalassemia major is a severe anemia leading to transfusion dependence. β-thalassemia intermedia manifests abnormalities with a severity between those of β-thalassemia major and β-thalassemia minor, and does not require regular transfusions.
The α-thalassemias are usually caused by a deletion of one, two, three, or all four of the α-globin genes, resulting in reduced or absent production of α chains.
In α-thalassemias, tetramers of γ chains form Hb Bart in the fetus and newborn, and tetramers of β chains form Hb H in the adult.
The α-thalassemias are divided clinically into silent carrier state, α-thalassemia minor, Hb H disease, and Hb Bart hydrops fetalis syndrome.
Silent carrier α-thalassemia is a result of the deletion, or rarely a non-deletional mutation, of one of four α-globin genes (– α/αα) or (α^Tα/αα); it is associated with a normal RBC profile and is asymptomatic. α-Thalassemia minor is a result of the deletion of two α-globin genes (– α/– α or – –/αα) and is clinically similar to β-thalassemia minor except that Hb A₂is not increased.
Hb H disease is a result of the deletion of three of the four α-globin genes (– –/– α); Hb H inclusions (β_{4) precipitate in older circulating RBCs, causing a hemolytic anemia. The RBCs are microcytic and hypochromic, and the disease is clinically similar to}β-thalassemia intermedia. In Hb Bart hydrops fetalis syndrome, all four of the α-globin genes are deleted (– –/– –). There is severe anemia, and fetal death usually occurs in utero or shortly after birth. The predominant hemoglobin is Hb Bart (γ₄).
The preliminary diagnosis of thalassemia is made from the complete blood count results and RBC morphology, hemoglobin electrophoresis, high-performance liquid chromatography, or capillary zone electrophoresis. Molecular genetic testing is required for definitive diagnosis.
Thalassemia trait must be differentiated from other microcytic, hypochromic anemias, especially iron deficiency anemia. Iron studies are important for this differentiation.

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.

The thalassemias are caused by:
Structurally abnormal hemoglobins
Absent or defective synthesis of a polypeptide chain in hemoglobin
Excessive absorption of iron
Abnormal or defective protoporphyrin synthesis
Thalassemia is more prevalent in individuals from areas along the tropics because it confers:
Heat resistance to those heterozygous for a thalassemia gene
Selective advantage against tuberculosis
Selective advantage against malaria
Resistance to mosquito bites
The hemolytic anemia associated with the thalassemias is due to:
Imbalance of globin chain synthesis
Microcytic, hypochromic cells
Ineffective erythropoiesis caused by immune factors
Structurally abnormal hemoglobin
β-Thalassemia minor (heterozygous) usually exhibits:
Increased Hb Constant Spring
50% Hb F
No Hb A
Increased Hb A₂
RBC morphologic features in β-thalassemia would most likely include:
Microcytes, hypochromia, target cells, elliptocytes, stippled cells
Macrocytes, acanthocytes, target cells, stippled cells
Microcytes, sickle cells
Macrocytes, hypochromia, target cells, stippled cells
The predominant hemoglobin present in β⁰-thalassemia major is:
Hb A
Hb A₂
Hb F
Hb C
Heterozygous HPFH is characterized by:
10% to 35% Hb F with normal RBC morphology
100% Hb F with slightly hypochromic, microcytic cells
A decreased amount of Hb F with normal RBC morphology
5% to 15% Hb F with hypochromic, macrocytic cells
Hb H is composed of:
Two α and two β chains
Two ε and two γ chains
Four β chains
Four γ chains
Hb Bart is composed of:
Two α and two β chains
Two ε and two γ chains
Four β chains
Four γ chains
When one α gene is deleted (α–/αα), a patient has:
Normal hemoglobin levels
Mild anemia (hemoglobin range 9 to 11 g/dL)
Moderate anemia (hemoglobin range 7 to 9 gm/dL)
Marked anemia requiring regular transfusions
In which part of the world is the α gene mutation causing Hb Bart hydrops fetalis (– –/– –) most common?
Northern Africa
Mediterranean
Middle East
Southeast Asia
The condition Hb S-β⁰-thalassemia has a clinical course that resembles:
Sickle cell trait
Sickle cell anemia
β-Thalassemia minor
β-Thalassemia major
Hb H inclusions in a supravital stain preparation appear as:
A few large, blue, round bodies in the RBCs with aggregated reticulum
Uniformly stained blue cytoplasm in the RBC
Small, evenly distributed, greenish-blue granules that pit the surface of RBCs
Uniform round bodies that adhere to the RBC membrane
Which of the following laboratory findings is inconsistentwith β-thalassemia minor?
A slightly elevated RBC count and marked microcytosis
Target cells and basophilic stippling on the peripheral blood film
Hemoglobin level of 10 to 13 g/dL
Elevated MCHC and spherocytic RBCs
A 4-month-old infant of Asian heritage is seen for a well-baby check. Because of pallor, the physician suspects anemia and orders a CBC. The RBC count is 4.5 × 10⁹/L, Hb concentration is 10 g/dL, and MCV is 77 fL, with microcytosis, hypochromia, poikilocytosis, and mild polychromasia noted on the peripheral blood film. These findings should lead the physician to suspect:
β-Thalassemia major
α-Thalassemia silent carrier state
Iron deficiency anemia
Homozygous α-thalassemia (– –/– –)

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^*The author extends appreciation to Martha Payne, who provided the foundation of this chapter, and Rakesh P. Mehta, who authored this chapter in previous editions.

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