Primary Structure: The α- and non–α-globin chains of Hb are 141 and 146 amino acid residues in length, respectively. Some sequence homology has been noted, with 64 individual amino acid residues in identical positions in both α- and β-chains. The β-chain differs from the δ- and γ-chains by 39 and 10 residues, respectively. The amino terminal of the β-globin chain is the site of attachment of glucose (Hb A_1c), urea, and salicylate.^H13 The carboxy terminal amino acid of the β-chain is tyrosine and can function as a part of salt bridges. Although no disulfide bonds are present, six SH groups are noted (from cysteine at positions α104, β93, and β112). The γ-chain has a glycine amino terminal, and the alkali resistance of Hb F is attributed to the presence of threonine and tryptophan at positions 112 and 130 of the γ-chain, respectively. The γ-chain is unique in that it is the only globin chain to be susceptible to acetylation, and acetylated Hb F is a prominent feature in cord and neonatal blood and may form as much as 25% of the total Hb.

Secondary Structure: Approximately 75 to 80% of polypeptide chains of the α- and non–α-chains are arranged in helices, with the remainder forming nonhelical turns. The β-chain of Hb A is arranged into eight helices identified as A through H. In contrast, the α-chain is missing an equivalent of the D helix and so has only seven helices. Nomenclature within the helices identifies the helix and the position within the helix of the amino acid residue (e.g., F3 is the third amino residue in the F helix). Amino acid residues in the peptide chains that join adjacent helices are described by the identification of two adjacent helices and the position of residues within the joining peptide. For example, EF3 would be the third residue in the peptide joining the E and F helices.

Tertiary Structure: The tertiary structure of Hb refers to the arrangement of helices into a three-dimensional, pretzel-like structure. The heme group, located in a crevice between the E and F helices, is attached to histidine residues in each globin chain. This attachment is essential to maintaining the secondary and tertiary structure of the globin chains.

Quaternary Structure: The quaternary structure of Hb results from the attachment of the four globin chains to each other. Strong α₁β₁ and α₂β₂ dimeric bonds hold the molecule in a stable form. The tetrameric α₁β₂ and α₂β₁ bonds make significant contributions to the stability of the structure. Shifting, rotation, and sliding in the quaternary structure result in a number of physiologic effects, including the sigmoid-oxygen dissociation curve and the Bohr effect (described in greater detail later in this chapter).

Hb Bart’s: Hb Bart’s results from deletion of all four α-globin genes with subsequent inability to produce any α-globin chains, leading to failure of synthesis of Hb A, F, or A₂. In the fetus, an excess number of γ-globin chains join together to form unstable tetramers known as Hb Bart’s (γ⁴). Mothers who carry a fetus with Hb Bart’s usually present clinically between 20 and 26 weeks’ gestation with pregnancy-induced hypertension and polyhydramnios. Ultrasound of the fetus shows hydrops. Severe anemia (Hb usually <80 g/L) is noted on a fetal blood sample obtained by cordocentesis. It is important to rule out other causes for the hydropic fetus by performing serologic testing for toxoplasmosis, rubella, cytomegalovirus, and herpes simplex testing.

High-performance liquid chromatography (HPLC) analysis of a cordocentesis blood sample shows one or two very sharp and narrow peaks at the injection point on the chromatogram (Figure 32-7, a). The major band is Hb Bart’s with a smaller band attributed to Hb Portland. Complete absence of Hb F is noted. Alkaline electrophoresis shows a band migrating at or close to the solvent front (Hb Bart’s) with another band in the Hb A position (Hb Portland).

Hb Bart’s hydrops fetalis is almost invariably fatal^H18 with some fetuses dying in utero and others surviving a few hours after birth. Treatment using intrauterine transfusion has had very limited success, with potential complications in the children of growth retardation and severe brain damage, which may be related to long-standing intrauterine anemia.

Laboratory investigation of the parents of fetuses with Hb Bart’s shows a normal HPLC pattern with normal Hb F and A₂ quantification. Parental analysis typically shows a decreased concentration of Hb and decreased MCH and MCV with the blood smear showing hypochromic, microcytic red cells. The Hb H test may be positive in one or both parents. A two α-gene cis-deletion (-/αα) or a three gene deletion (-/-α) is seen in genetic testing of both parents. This requirement restricts the incidence of Hb Bart’s to a much smaller population than would be expected based on the worldwide distribution of two α-gene deletions, as the presence of trans deletions in both parents would not give rise to a four gene deletion in the offspring. Hb Bart’s is relatively common in South East Asia, particularly in Thailand, the Philippines, and Hong Kong, where there is a high prevalence of the –SEA deletion.

Hb H Disease: This disorder is usually caused by a three α-globin gene deletion (-/-α) and is characterized by a chronic anemia of variable severity. Individuals with nondeletional Hb H disease (α^Tα/-) are usually more severely affected and are more likely to require transfusion therapy than those with deletional Hb H disease. Significant underproduction of α-globin chains occurs with subsequent joining of free β-globin chains to form the insoluble β-globin chain tetramer Hb H. HPLC analysis of a hemolysate from an individual with Hb H disease shows two bands with low retention times forming a doublet together with a normal Hb A band. Hb F and Hb A₂ concentrations are within the reference interval (see Figure 32-7, d). Electrophoresis at alkaline pH shows a fast moving band together with a band in the Hb A position that possibly has reduced staining when compared with other samples run concurrently. The complete blood count (CBC) (Table 32-1) shows a moderately reduced concentration of Hb and markedly reduced mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH), increased red cell distribution width (RDW), and slightly raised red blood cell (RBC) count. Iron studies are normal, although the ferritin concentration may be elevated. The Hb H preparation is positive, with many cells having typical punctate inclusions (Figure 32-8).

Iron therapy is not indicated, and transfusion therapy is usually unnecessary except in acute illness, in pregnancy, and with exposure to oxidant drugs, which destabilizes Hb H, causing precipitation of the somewhat insoluble protein. Genetic counseling is recommended to prospective parents who have Hb H disease.

α-Thalassemia Minor: α-Thalassemia minor is the result of a two α-chain gene deletions. These deletions may be seen on the same gene (-/αα,α⁰-thalassemia) described as a cis deletion or on different genes (-α/-α, α⁺-thalassemia) described as a trans deletion. The CBC of affected individuals shows a mildly reduced Hb with low MCV and MCH. HPLC analysis shows no abnormal Hb peaks, and Hb F and Hb A₂ concentrations are within the reference interval. The Hb H preparation may show a rare cell with punctate inclusions. Iron studies are normal. In the routine clinical laboratory, the diagnosis of α-thalassemia major is based on exclusion criteria rather than definitive tests. The presence of thalassemic indices in a patient with normal Hb A₂ and Hb F quantifications is very often the only basis for many diagnoses of α-thalassemia major, particularly in the setting of a positive family history. A photometric enzyme-linked immunosorbent assay (ELISA) for the identification of adult carriers of the (–SEA) α⁰-thalassemia deletion has been described.^H51 This method is based on the measurement of minuscule quantities of embryonic ζ-cells present in these adult carriers. Not all α-thalassemia deletions have continued ζ-chain production, and so a negative test is not diagnostic of α-thalassemia major. This test could be useful in screening individuals at risk for giving birth to a fetus with Hb Bart’s hydrops fetalis.

α-Thalassemia Silent: α-Thalassemia trait describes a single α-globin chain gene deletion (-α/αα). A single α-globin gene deletion is frequently clinically and hematologically silent. A CBC of an individual with this trait shows a normal or marginally decreased Hb concentration, MCV, and MCH. Iron studies are normal, and no abnormal Hb peaks are seen on HPLC analysis.

In Table 32-2, the effects of various α-thalassemias on the percentage of β-chain hemoglobin variants are shown.^H52 In general, the percentage of hemoglobin variant decreases as a percentage of total hemoglobin as the number of α-chain deletions increases.

β⁰-Thalassemia (β-Thalassemia Major): This is sometimes called Cooley’s anemia, after the physician who in 1925 first described the condition in the children of Italian and Greek immigrants in New York by noting that these children (1) failed to grow, (2) had frequent infections, (3) appeared pale and malnourished, (4) had splenomegaly, and (5) had facial bone changes.

β-Thalassemia major results from mutations that interfere with translation or are involved in the initiation, elongation, or termination of globin chain synthesis. Mutations that interfere with translation account for almost 50% of all β-thalassemia mutations. Included in this are frame shift or nonsense mutations that produce premature termination codons, resulting in incomplete translation of the β-globin gene and nonproduction of the β-globin chain, leading to β⁰-thalassemia.

Clinical presentation usually occurs at younger than 1 year of age with features such as small size for age, abdominal girth expansion, and failure to thrive. Physical examination of the subject may reveal frontal bossing^H63 (an unusually prominent forehead) caused by thickening of the cranial bones, pallor, and prominence of the cheek bones, which, in older children, obscures the base of the nose and exposes the teeth. These features are a result of marrow expansion (up to a 30-fold increase) caused by ineffective erythropoiesis with production of highly unstable α-globin tetramers leading to increased plasma volume and the formation of extramedullary erythropoietic tissue, especially in the thorax and the paraspinal region. The spleen, liver, and heart may be enlarged also because of extramedullary hematopoiesis.

Typical CBC results include severe anemia with Hb concentration between 30 and 65 g/L, MCV 48 to 72 fL, and MCH 230 to 320 g/L. On the peripheral blood smear, a characteristic markedly abnormal RBC morphology is noted; this includes a large number of microcytes, numerous target cells, which may have a bridge joining the central and peripheral pigment zones, polychromasia, and occasional spherocytes, schistocytes, and nucleated red cells. When a patient’s red blood cells are of unequal size (anisocytosis), the diameter of the RBC varies from 3 to 15 µm with little pigment, and shape distortion is noted, along with prominent basophilic stippling. RBC osmotic fragility is frequently observed. Typical peripheral blood on a patient with β⁰ thalassemia is shown in Figure 32-9.

White blood cell (WBC) and platelet counts are usually normal. Ferritin is usually within the upper half of the reference interval, and total bilirubin is mildly elevated with a borderline elevation in the conjugated fraction. Urinalysis frequently shows increased urobilinogen or urobilin concentration, and urine is often dark brown to black because of the presence of dipyroles and mesobilifuscin. The latter features reflect ineffective hematopoiesis with intramedullary red cell destruction. HPLC analysis (see Figure 32-7, b) shows a major Hb F peak with absence of an Hb A peak and variable Hb A₂ (interval, 1 to 5.9%; mean, 1.7%) peak. Electrophoresis at alkaline and acid pH shows a dominant band in the F position on both gels.^H2

Family studies on both parents and siblings should be performed, and the classical β-thalassemia minor pattern described later in the chapter should be found in the parents. Siblings may be normal or may have β-thalassemia minor. A family case history is seen in Figure 32-10.

Transfusion together with iron chelation is the only therapy, and splenectomy is frequently performed. Following splenectomy inclusion, bodies consisting of denatured α-chains can be observed in the blood smear after staining with methyl violet. Puberty is often delayed, incomplete, or completely absent. In boys, active spermatogenesis may occur and Leydig cell function is normal. In the older, chronically transfused patient, iron overload is a common feature. Diabetes mellitus and hypoparathyroidism are frequent sequelae. Numerous cardiopulmonary conditions, including pericarditis and myocardial hemosiderosis, are the leading causes of death in transfused patients, and are frequently associated with β⁰-thalassemia.

β⁺-Thalassemia (β-Thalassemia Intermedia): With this disorder, a significant reduction in the production of β-globin chain occurs with subsequent reduction in the quantity of Hb A present; this is attributed to a wide variety of genotypes. Many different causes of β⁺-thalassemia have been identified, with variations in one or two β-globin genes. Clinical severity in individuals with variations in two β-globin chains is much less than when variation is seen in only one gene, a condition sometimes called dominant β-thalassemia. The severity of clinical features is reduced with coinheritance of α-thalassemia. β⁺-Thalassemia is found in Mediterranean countries, especially in the Eastern Mediterranean.

Clinical presentation varies from symptoms similar to β⁰-thalassemia to those associated with β-thalassemia trait. Transfusions usually are not necessary, and hydroxyurea therapy is frequently used to increase the production of Hb F and to mitigate disease symptoms.

HPLC analysis shows a large Hb F peak with a reduced Hb A peak. Hb A₂ is above the reference interval at concentrations greater than those associated with β-thalassemia minor. Bands in the A and F positions are seen on electrophoresis at both alkaline and acid pH. Hb is significantly reduced (60 to 100 g/L). The peripheral blood smear shows the same features seen in β⁰-thalassemia, including anisocytosis, hypochromia, target cells, basophilic stippling, and nucleated RBCs.

β-Thalassemia Minor (β-Thalassemia Trait): Patients with β-thalassemia minor are very often asymptomatic, except at times of hematopoietic stress, such as infection or pregnancy, when they may require, in extreme situations, blood transfusions because of the development of anemia. The CBC on patients with β-thalassemia trait shows low normal or decreased Hb concentration and hematocrit, decreased MCV (<72 fL) and MCH (<27 pg), and normal RDW. The discriminant factor is <60 pg (see following “Analytical Methods” section for definition). However, for patients with liver disease and β-thalassemia, MCV and MCH may be at the low end of the reference interval.

The peripheral blood smear shows microcytic RBCs with occasional hypochromia, poikilocytosis, and target cells.

The diagnosis of β-thalassemia minor, with appropriate indices in the CBC, is dependent on the finding of a raised Hb A₂ (>3.5%). Hb A₂ and Hb A₂′ should be added together to obtain an accurate Hb A₂ value for the investigation of β-thalassemia minor. Hb A₂ may be elevated in HIV-positive women without hypochromic microcytic indices,^H39 hyperthyroidism, and megoblastic anemia, and with some unstable hemoglobins.^H70 Iron-depleted individuals should become iron replete before a definitive diagnosis of β-thalassemia is made, because Hb A₂ may be falsely low. HPLC is the preferred method for this quantification; densitometric scanning of the Hb A₂ band on an alkaline electrophoresis gel is not recommended because of poor precision and accuracy.^H70 In 30 to 40% of all cases of β-thalassemia minor, Hb F will also be elevated (>1.0%). The life span of the RBC may be reduced, and diabetic individuals may show a lower Hb A_1c compared with normal individuals with equivalent glycemic control. The β-thalassemia mutation may be identified by Southern blot using mutation-specific probes or by gap–polymerase chain reaction (PCR).

δβ-Thalassemia: Deletion of both δ- and β-genes results in δβ-thalassemia. Both heterozygous and homozygous conditions have been described. It is found in a variety of ethnic groups but is most prevalent in countries of the Eastern Mediterranean, especially Greece and Italy, and is the result of one of eight mutations that have been described to date. CBC analysis shows a reduced concentration of Hb (80 to 135 g/L) with reduced MCV and MCH and sometimes an increased RDW. HPLC analysis shows an Hb A peak with a normal or reduced Hb A₂ concentration and a raised Hb F concentration (between 5 and 20%), with the highest HbF concentration seen in the Sardinian type of δβ-thalassemia. Hb Lepore sometimes is incorrectly classified as a δβ-thalassemia because of a reduction in the production of both δ- and β-globin chains, or as an Hb variant because of the presence of an abnormal globin chain.

Hereditary Persistence of Fetal Hemoglobin (HPFH): The term hereditary persistence of Hb F is used to describe a group of genetic conditions in which the concentration of Hb F is increased above the upper limit of the reference interval because of a reduction in β-globin synthesis and a compensatory increase in δ-globin synthesis. Two major classes of HPFH have been described: heterocellular and deletional. Several deletional variants of HPFH have been described, including Greek, Indian, Italian, Corfu, and black.

In black HPFH, the Hb F is raised to between 10 and 36% of the total Hb with normal Hb A₂ concentrations. Hb, MCV, and MCH are within the reference intervals. This condition is clinically innocuous and asymptomatic. Similarly, no clinical abnormalities are associated with Greek HPFH, although the concentration of Hb F is in the range of 15 to 25%.

Nondeletional HPFH, sometimes named heterocellular HPFH, describes a group of presentations in which the increase in Hb F is distributed heterogeneously among the red cells in otherwise normal individuals. The Hb F concentration varies between 1 and 13% of total Hb heterozygotes and between 19 and 21% of homozygotes. No clinical or hematologic abnormalities are noted.

Hemoglobin S [β6(A3)^Glu→Val]: Hb S in the heterozygous or homozygous state is the most widespread of the Hb variants and arises from a substitution of valine for glutamic acid at position 6 in the A helix of the β-globin chain. Hb S is found in high frequency in West and North Africa, the Middle East (especially Saudi Arabia), and the Indian subcontinent. Approximately 8% of African Americans are heterozygous for Hb S, and homozygous Hb S is found in 1 in 500 newborns in this group. Four haplotypes originating from different geographic locations have been described. The widespread distribution of the single point gene mutation responsible for the synthesis of Hb S in areas where P. falciparum malaria is endemic is due to protection of Hb S heterozygotes from the worst manifestations of the malaria.

Homozygous Hemoglobin S (Hb SS): In homozygous Hb S, a valine-for-glutamic acid substitution occurs on both β-globin chains because of the inheritance of mutated β-globin chain genes from both parents. This condition is described as “sickle cell anemia” or “sickle cell disease” because of the sickle-shaped RBCs that occur when a “sickle cell crisis” occurs. It sometimes is written as β^Sβ^S.^H65

HPLC analysis (see Figure 32-7, e) of a hemolysate of an individual homozygous for Hb S shows no Hb A peak and a small Hb A₂ peak. The apparent Hb A₂ concentration may be falsely increased because of the presence of glycated Hb S. Hemoglobin S forms 85 to 90% of the total Hb. The Hb F concentration is variable, with females having higher concentrations than men, and is somewhat, although not exclusively, haplotype dependent. The highest Hb F concentrations (10 to 25%) are found in individuals from the Middle East and the Indian subcontinent with the Arab-Indian haplotype. Low Hb F concentrations (5 to 6%) are found in the West African Cameroon (sometimes called Senegal) haplotype. The remaining haplotypes, the Benin and Bantu, have Hb F concentrations in the range of 6 to 7%. Increased concentrations of Hb F mitigate to some extent the clinical manifestations of sickle cell anemia. Electrophoresis (Figure 32-11) at both alkaline and acid pH shows a single large band in the Hb S position with small bands at the Hb A₂ and Hb F positions. The sickle cell screen test is positive.

CBC analysis of an individual homozygous for Hb S indicates a moderate to a major decrease in Hb concentration (60 to 100 g/L) with normal to increased MCV and MCH. In individuals with a concurrent thalassemia, the Hb is further decreased and both MCV and MCH are lowered. In the neonate the peripheral blood smear shows occasional sickle and target cells and Howell-Jolly bodies. As a patient’s age increases, these features of hyposplenism become increasingly evident. In the adult the percentage of sickle cells observed can be as great as 30 to 40%. In the setting of a sickle cell crisis, fewer sickle cells may be present than when individuals are clinically well. Howell-Jolly bodies, target cells, Pappenheimer bodies, boat-shaped cells, and nucleated RBCs are noted. The platelet count and neutrophil counts are elevated. Sometimes blister cells in which the Hb appears to be present in only one half of the cell are observed.

Treatment of children with homozygous HbS includes the use of hydroxyurea with an increase in the quantity of HbF sometimes to 25%. In adults, transfusion is the usual treatment, and the patient is retransfused when the Hb A concentration falls to 20% of the total hemoglobin.

Heterozygous Hemoglobin S (Hb S Trait): HPLC analysis (see Figure 32-7, f) of a hemolysate of a blood sample from an individual who is heterozygous for Hb S shows peaks in the Hb A and S positions, with 40% of the total Hb found in the Hb S peak. Hb S concentrations less than 30% are suggestive of coinheritance of α-thalassemia. Hb F concentration is variable. Electrophoresis (see Figure 32-11) at both alkaline and acid pH shows bands in the A and S positions.

CBC analysis from an individual who is heterozygous for Hb S shows a slightly decreased concentration of Hb, and sickle cells are not typically seen on the peripheral blood film. Patients are often asymptomatic, and the first time an individual is diagnosed as heterozygous for Hb S (sickle cell trait) is often when an Hb A_1c analysis is requested on the individual, or when a family study is initiated for genetic counseling. In the United States, neonatal screening programs are designed specifically to detect both heterozygous and homozygous Hb S in newborns. Although individuals with sickle cell trait are clinically asymptomatic, genetic counseling should be considered because coinheritance of two β-globin gene abnormalities may contribute to a sickle cell disorder. α- and β-Thalassemia can be coinherited with heterozygous Hb S.

Hemoglobin SC (Hb SC Disease): SC disease arises when both β-globin chains are substituted at position 6 with valine (Hb S) or lysine (Hb C). On HPLC and capillary electrophoresis, analysis peaks are noted in the S and C positions, with the S peak forming the majority of the Hb present.

Electrophoresis (see Figure 32-11) at both alkaline and acid pH shows bands in the S and C positions, and the sickling test is positive.

Hemoglobin SD: Hb S may be coinherited with Hb D (SD disease). Individuals with this disease have similar but milder clinical presentation when compared with that of sickle cell disease (Hb SS). HPLC analysis shows two peaks—one in the Hb S position forming approximately 38 to 42% of the total Hb, and the other in the Hb D position forming 43 to 45% of the total Hb. The Hb F concentration is usually within the reference interval, although concentrations as high as 14% have been observed in some individuals with SD disease. Alkaline electrophoresis shows a band in the S position. Acid electrophoresis shows bands in the S and A positions. The sickling test is positive. CBC analysis shows a greatly decreased concentration of Hb with normal to slightly elevated MCV. Target, boat-shaped, nucleated, red, and sickle cells—together with anisocytosis and poikilocytosis—are noted on the peripheral blood smear.

Hemoglobin S/O Arab: Coinheritance of Hb S and Hb O Arab presents a similar or somewhat milder clinical presentation to sickle cell disease and is found in the Middle East and North Africa.

Hemoglobin S/G Philadelphia: One or more abnormal α-globin chains can combine with Hb S. In African Americans and West Africans, the combination of Hb G Philadelphia [α68(E17)^Asn→Lys] with Hb S is prevalent. HPLC analysis (see Figure 32-7, i) on blood samples from these individuals shows at least two major peaks and two smaller peaks. The two major peaks are due to combinations of the normal α-chain with the normal β-chain and the abnormal α-chain with the normal β-chain. The two smaller peaks are due to combinations of the normal α-chain with the abnormal β-chain and the abnormal α-chain with the abnormal β-chain. Electrophoresis at alkaline pH shows major bands in the A and S positions with a minor band in the C position. At acid pH, bands are seen in the A and S positions. CBC analysis gives a slightly decreased Hb concentration with normal MCV and MCH. α- or β-Thalassemia can be coinherited with Hb G Philadelphia and Hb S. In these cases, CBC analysis results in markedly decreased MCV and MCH with reduced Hb concentration.

Double heterozygosity Hb G Philadelphia/Hb S occurs in 1 in 125,500 African Americans. On HPLC of patients with Hb G Philadelphia/Hb S trait, four bands are noted, representing four different hemoglobin species, namely, normal α-chain with normal β-chain, abnormal α-chain with normal β-chain, normal α-chain with abnormal β-chain, and abnormal α-chain with abnormal β-chain. Hemoglobin electrophoresis at alkaline pH shows bands in the A, S, and C positions. At acid, pH bands are noted in the A and S positions. In African Americans, the Hb G Philadelphia mutation occurs with an α-chain deletion, which is cis to the mutated alle (α^G/αα). This results in a change in the amount of mutated α-chain (α^G) from the usual 25 to around 30%. No clinical or hematologic manifestations are noted with Hb G Philadelphia/Hb S.

Hemoglobin C [β6(A3)^Glu→Lys]: Hb C arises from a substitution of lysine for glutamic acid at position 6 of the β-globin chain. Hb C may be found in the homozygous (Hb C disease, β^Cβ^C) or heterozygous (Hb C trait) state. Hb C is commonly found in West Africa and the Caribbean. It is the second most commonly studied, after Hb S, of all Hb variants. HPLC analysis (see Figure 32-7, g) on samples from individuals with homozygous Hb C shows a large peak in the C position, with Hb C forming 90 to 95% of the total Hb. Hb F concentrations are variable. Glycated Hb C is found as a small peak eluting before the Hb C peak. The ratio of the elution times of glycated Hb C to Hb C is the same as that of Hb A_1c and Hb A. Electrophoresis at alkaline and acid pH shows a single band in the C position.

Mild to moderate anemia is the most common clinical presentation. CBC analysis shows normal or slightly decreased Hb concentrations with a normochromic and normocytic red cell morphology. An increase in polychromasia may be present, and the reticulocytes may contribute to an increase in the MCV. The peripheral blood smear shows numerous target cells with occasional nucleated RBCs and characteristic irregular contracted red cells (sometimes called pyknocytes). Hb C crystals may be seen, and bilirubin concentrations may be slightly elevated. Red cell survival and osmotic fragility are decreased.

Heterozygous Hemoglobin C (Hb C Trait): HPLC analysis (see Figure 32-7, h), capillary electrophoresis, and electrophoresis at both alkaline and acid pH (see Figure 32-11) on blood samples from individuals who are heterozygous for Hb C reveal bands in the A and C positions, with Hb C forming 38 to 45% of the total Hb. CBC analysis may show target cells and generally is normochromic with the MCV near the lower limit of the reference interval.

Heterozygous Hb C individuals are usually asymptomatic. Genetic counseling may be useful when prospective parents have abnormalities in the β-globin gene.

Hb C may be coinherited with both α- and β-thalassemia, and the concentration of Hb C is related to the number of functioning α-genes. With only two functioning α-genes, the Hb C concentration can fall to 32% of the total Hb. Coinheritance of Hb C with β⁰– or β⁺-thalassemia results in moderately severe anemia with splenomegaly. The Hb F concentration is often increased.

Hemoglobin D Punjab [β121(GH4)^Glu→Gln]: Hb D Punjab is an Hb variant in which glutamic acid at position 121 of the β-globin chain is replaced with glutamine. The names Hb D Los Angeles and Hb D Punjab are used to describe this variant, with the former name used more often in North America and the latter in the United Kingdom. Hb D Punjab is found in the Punjab region of the Indian subcontinent, especially in Sikhs of the Lycus Valley. Large-scale immigration from this area to the United Kingdom, the United States, and Canada has widened the distribution of Hb D Punjab. Hb D Punjab is also found in Caucasians whose foreparents lived in the Indian subcontinent at the time of the British Raj. Hemoglobin D Punjab is found in both heterozygous (Hb D Punjab trait) and homozygous (Hb D Punjab disease, β^Dβ^D) states.

HPLC analysis of blood from an individual with homozygous Hb D Punjab shows normal or marginally raised Hb F and Hb A₂ peaks with a large peak in the Hb D position forming more than 90% of the total Hb. Electrophoresis at alkaline pH shows a band in the S position, which migrates to the A position in acid electrophoresis. CBC analysis shows a mild decrease in Hb concentrations, MCV, and MCH with target cells observed in the blood smear. Patients present clinically with mild anemia.

HPLC analysis on individuals with Hb D Punjab trait shows two peaks—one at the A position and the other at the D position—with Hb D forming 30 to 40% of the total Hb. The Hb F and Hb A₂ concentrations are within or slightly above the reference intervals. Electrophoresis at alkaline pH shows two bands—one in the A position and the other in the S position. On electrophoresis at acid pH, a single band in the A position is noted. HPLC is the preferred method for identification of Hb D because a similar electrophoretic pattern, on both alkaline and acid pH, is seen with Hb G. CBC analysis is unremarkable except for the presence of target cells on the blood smear.

Individuals with Hb D Punjab trait are clinically asymptomatic.

Coinheritance of Hb D Punjab (both heterozygous and homozygous states) with β-thalassemia is common. CBC analysis on these patients shows decreased concentrations of Hb with markedly decreased MCV and MCH. Target and irregular contracted cells together with hypochromia and anisocytosis are seen on the blood smear. Quantification of Hb A₂ in individuals with coinheritance of Hb D Punjab and β-thalassemia presents a challenge to the laboratory in that HPLC analysis underestimates the Hb A₂ concentration because of an unstable rising baseline in these individuals. Although not normally recommended, quantification of Hb A₂ by densitometry on alkaline electrophoresis may be the only method available to many laboratories. CBC analysis shows a greatly decreased concentration of Hb with low MCV and MCH. The blood smear shows target cells (erythrocytes with increased surface area-to-volume ratio that appear as a target with a bull’s eye) and contracted cells with hypochromia and anisocytosis.

Individuals with coinheritance of Hb D Punjab and β-thalassemia present with a notable compensated anemia.

Hemoglobin D Iran [b22(B4)^Glu→Gln]: Hb D Iran is a β-globin chain variant in which glutamine replaces glutamic acid at position 22 of the β-globin chain.

On HPLC analysis, peaks are seen in the A and A₂ positions with quantification for Hb A₂ far above that normally expected. Alkaline electrophoresis shows two bands—one in the A position and the other in the S position. On acid electrophoresis, a single band in the A position is noted. Individuals with Hb D Iran are asymptomatic.

Hemoglobin E [b26(B8)^Glu→Lys]: Hb E is a β-chain variant with lysine replacing glutamic acid at position 26 of the β-globin chain. More individuals have the Hb variant Hb E than any other variant. Hb E is found in both homozygous and heterozygous states and may be combined with β-thalassemia. It is widespread in the Far East, including Southern China, Cambodia, Thailand, and Laos. Hb E is increasingly found in the United States and Canada and is caused by emigration from this area. It may be thought of as the “thalassemic variant,” as some of the features of the CBC resemble thalassemia especially in the homozygous state.

HPLC analysis of blood from individuals with homozygous Hb E (Hb E disease, β^Eβ^E) shows a single peak (>90% of the total Hb) coeluting with Hb A₂. Hemoglobin F is within or marginally above the reference interval. On alkaline electrophoresis, a single band is noted in the C position, which migrates to the A position in acid electrophoresis. CBC analysis shows normal to marginally decreased Hb concentrations with low MCV and MCH. Target cells are noted in the peripheral blood smear. Iron studies are normal.

Homozygous Hb E individuals are usually asymptomatic, although slight anemia may be present.

HPLC analysis of blood from individuals with heterozygous Hb E reveals two peaks—one in the A position and the other in the A₂ position. Hb E forms approximately 30% of the total Hb. Capillary electrophoresis resolves Hb E from Hb A₂. Hb A₂ is higher in patients with Hb E than in patients without a hemoglobin variant or thalassemia as the result of decreased synthesis of the abnormal β-globin chain, allowing for increased binding between the excess α-globin and δ-globin chains producing Hb A.^H36-H37 CBC analysis shows normal Hb concentrations and occasionally low MCV. Target cells are noted in the peripheral blood smear. Iron studies are normal.

Heterozygous Hb E individuals are usually asymptomatic, although slight anemia may be present.

Coinheritance of Hb E and thalassemia produces an anemia of variable severity. HPLC on a patient with coinheritance of β-thalassemia and homozygous E is shown in Figure 32-7, C. Coinheritance of homozygous Hb E and β-thalassemia leads to a severe anemia with greatly reduced Hb concentrations, MCV, and MCH with increased Hb F concentration. Numerous target cells are noted on the peripheral blood smear, together with microcytosis, anisocytosis, hypochromia, and a few nucleated red cells. Iron studies are normal. In the most severe cases, the clinical presentation is similar to that of β⁰-thalassemia, and transfusion may be the only therapy. Coinheritance of heterozygous Hb E with α-thalassemia produces a less severe anemia with low Hb and MCV and MCH. Target cells are noted on the peripheral blood smear, together with microcytosis and hypochromia. Patients who are pregnant may need to be monitored closely, although transfusion is not usually required.

Quantification of Hb A₂, which is important in the diagnosis of possible coinheritance of β-thalassemia with Hb E, provides a challenge to the laboratory in that Hb E and Hb A₂ coelute. Molecular studies are the only satisfactory method by which to establish coinheritance of Hb E and β-thalassemia, although the severity of the disease, family studies, and increased Hb F may lead to suspicion of coinheritance.

Hemoglobin O Arab [b121(GH4)^Glu→Lys]: Hb O Arab is a β-chain variant with lysine replacing glutamic acid at position 121 of the β-globin chain. Hb O Arab is found in a wide variety of ethnic groups in North Africa and Eastern Europe and is not confined, nor is it even common, among Arab populations. Hb O Arab has been found in both heterozygous and homozygous states.

HPLC analysis of blood from an individual with homozygous Hb O Arab shows a single band between the S and C positions, with Hb O Arab forming more than 90% of the total Hb. Electrophoresis at alkaline pH shows a band close to the C position. On electrophoresis at acid pH, a band is seen between the A and S positions (but closer to A). CBC analysis shows a normal or marginally low Hb concentration, MCV, and MCH. The peripheral blood smear shows slight microcytosis.

No unusual hematologic features are noted in individuals with heterozygous Hb O Arab. HPLC analysis on blood from these individuals shows two peaks—one in the A position and the other eluting close to the C position and forming 30 to 40% of the total Hb. Electrophoresis at alkaline electrophoresis shows bands in the A and close to the C position. On acid electrophoresis, two bands are noted—one in the A position and the other in a position between the A and S positions.

Hybrid Hemoglobins: Hybrid Hbs, or crossover Hbs, describe a group of Hb variants in which one of the globin chains is a hybrid of amino acid sequences of two other globin chains. The term crossover Hb is sometimes used because there is a point in the amino acid sequence at which there is crossover from the amino acid sequence of one globin chain to another globin chain. Individuals with these hybrid Hb variants present with clinical features and laboratory findings, particularly in their CBC, that are similar to those of thalassemia. Production of the hybrid globin chain is reduced. Hb Lepore is the prototypical hybrid Hb.

Hemoglobin Lepore: Hb Lepore is classified as a δβ-hybrid Hb variant on the basis that the non–α-chain is a hybrid of δ- and β-globin chains. It is unique in that it is the only hemoglobinopathy named after the family name of the index case. δβ-Hybrid Hbs arise because there are deletions of part of the 3′ portion of the δ-globin gene and in the 5′ portion of the β-globin chain with resultant formation of a δβ-fusion gene. Three distinct variations of Hb Lepore have been described. In Hb Lepore-Hollandia [δβ-hybrid (δ through 22; β from 50)], a variant found in Canada and Papua New Guinea, fusion occurs of the first 22 amino acid residues of the δ-globin chain with the amino acids from position 50 onward of the β-globin chain. In Hb Lepore-Baltimore [δβ-hybrid (δ through 50: β from 86)], found mainly in individuals of Spanish ancestry, the first 50 amino acid residues of the δ-globin chain are fused with amino acid residues from position 86 of the β-globin chain. In Hb Lepore-Boston-Washington [δβ-hybrid (δ through 87; β from 116)], the most common Hb Lepore, the first 87 amino acid residues of the δ-globin chain are fused with amino acid residues from position 116 onward of the β-globin chain. Hb Lepore-Boston-Washington, sometimes called Hb Lepore-Boston, is found mainly in individuals of Italian descent, although it has been found in individuals from Eastern Europe.

HPLC analysis of blood from individuals with Hb Lepore shows greatly elevated Hb A₂ concentration with marginally reduced Hb A. The Hb A₂ concentration is usually greater than 10% of the total Hb and is falsely increased because of the coelution of Hb A₂ and Hb Lepore. Electrophoresis at alkaline pH shows a band in the S position for Hb Lepore-Boston-Washington and in a position between the A and S positions for the other Hb Lepore variants. At acid pH, a single band is present in the A position for all Hb Lepore variants. Hb A₂ and Hb Lepore are resolved on capillary electrophoresis.

CBC analysis shows greatly reduced concentrations of Hb, MCV, and MCH in Hb Lepore homozygotes. Hematologic findings are very similar to those of β-thalassemia major or intermedia. CBC analysis on heterozygotes shows slightly reduced MCV and MCH. Hematologic findings are very similar to those of β-thalassemia trait. Iron studies are normal in both heterozygotes and homozygotes. The similarity of the hematology in Hb Lepore and in β-thalassemia makes careful review of the HPLC and electrophoretic analysis essential. A greatly elevated Hb A₂ by HPLC analysis and a small band in the S position on electrophoresis at alkaline pH suggest Hb Lepore.

Elongation Hemoglobins: Elongation Hbs, of which there are 13, including seven α-chain and six β-chain variants, result from lengthening of the C or N terminus of either globin chain. The most important, from a clinical perspective, are the five C terminal α-chain variants in which the terminal codon TAA is changed and an amino acid sequence is added. The prototypical elongation Hb is Hb Constant Spring. In this variant, the C terminal TAA codon is changed to CAA in the α₂-gene, and a 31 amino acid sequence is added at the C terminal end to give an α-globin chain length of 173 amino acid residues, rather than the normal 142 residue length. This increase in length results in instability of the Hb variant, and synthesis of this elongated globin chain is reduced. Hemoglobin Constant Spring is found in South East Asia, especially in Vietnam, Cambodia, and Laos, and is found in both heterozygous and homozygous states.

Patients with Hb Constant Spring present with slightly reduced Hb, MCV, and MCH with hypochromia and microcytosis in the peripheral blood smear. Iron studies are often normal.

The instability of Hb Constant Spring presents a challenge to the laboratory diagnosis of this variant. The blood used for analytical procedures should be as fresh as possible. Samples older than 24 hours should not be used. HPLC analysis of blood from individuals with Hb Constant Spring demonstrates a small peak in the C position, which forms approximately 4 to 6% of the total Hb in the homozygote and 1 to 3% in the heterozygote. On electrophoresis at alkaline pH, a small band migrating cathodally to the application point may be seen. This electrophoretic mobility is unique in that it is the only Hb variant that moves toward the cathode rather than the anode.

Hemoglobin Constant Spring is commonly found in combination with α⁰-thalassemia, especially the –^SEA mutation. The clinical presentation of this combination results in a severe form of Hb H disease.