CHAPTER 8: DISORDERS OF HEMOGLOBIN

Hemoglobin is critical for normal oxygen delivery to tissues; it is also present in erythrocytes in such high concentrations that it can alter red cell shape, deformability, and viscosity. Hemoglobinopathies are disorders affecting the structure, function, or production of hemoglobin. These conditions are usually inherited and range in severity from asymptomatic laboratory abnormalities to death in utero. Different forms may present as hemolytic anemia, erythrocytosis, cyanosis, or vasoocclusive stigmata.

HEMOGLOBIN STRUCTURE

Different hemoglobins are produced during embryonic, fetal, and adult life (Fig. 8-1). Each consists of a tetramer of globin polypeptide chains: a pair of α-like chains 141 amino acids long and a pair of β-like chains 146 amino acids long. The major adult hemoglobin, HbA, has the structure α₂β₂. HbF (α₂γ₂) predominates during most of gestation, and HbA₂ (α₂δ₂) is minor adult hemoglobin. Embryonic hemoglobins need not be considered here.

Each globin chain enfolds a single heme moiety, consisting of a protoporphyrin IX ring complexed with a single iron atom in the ferrous state (Fe²⁺). Each heme moiety can bind a single oxygen molecule; a molecule of hemoglobin can transport up to four oxygen molecules.

The amino acid sequences of the various globins are highly homologous to one another. Each has a highly helical secondary structure. Their globular tertiary structures cause the exterior surfaces to be rich in polar (hydrophilic) amino acids that enhance solubility and the interior to be lined with nonpolar groups, forming a hydrophobic pocket into which heme is inserted. The tetrameric quaternary structure of HbA contains two αβ dimers. Numerous tight interactions (i.e., α₁β₁ contacts) hold the α and β chains together. The complete tetramer is held together by interfaces (i.e., α₁β₂ contacts) between the α-like chain of one dimer and the non-α chain of the other dimer.

The hemoglobin tetramer is highly soluble, but individual globin chains are insoluble. Unpaired globin precipitates, forming inclusions that damage the cell. Normal globin chain synthesis is balanced so that each newly synthesized α or non-α globin chain will have an available partner with which to pair.

Solubility and reversible oxygen binding are the key properties deranged in hemoglobinopathies. Both depend most on the hydrophilic surface amino acids, the hydrophobic amino acids lining the heme pocket, a key histidine in the F helix, and the amino acids forming the α₁β₁ and α₁β₂ contact points. Mutations in these strategic regions tend to be the ones that alter oxygen affinity or solubility.

FUNCTION OF HEMOGLOBIN

To support oxygen transport, hemoglobin must bind O₂ efficiently at the partial pressure of oxygen (PO₂) of the alveolus, retain it, and release it to tissues at the PO₂ of tissue capillary beds. Oxygen acquisition and delivery over a relatively narrow range of oxygen tensions depend on a property inherent in the tetrameric arrangement of heme and globin subunits within the hemoglobin molecule called cooperativity or heme–heme interaction.

At low oxygen tensions, the hemoglobin tetramer is fully deoxygenated (Fig. 8-2). Oxygen binding begins slowly as O₂ tension rises. However, as soon as some oxygen has been bound by the tetramer, an abrupt increase occurs in the slope of the curve. Thus, hemoglobin molecules that have bound some oxygen develop a higher oxygen affinity, greatly accelerating their ability to combine with more oxygen. This S-shaped oxygen equilibrium curve (Fig. 8-2), along which substantial amounts of oxygen loading and unloading can occur over a narrow range of oxygen tensions, is physiologically more useful than the high-affinity hyperbolic curve of individual monomers.

Oxygen affinity is modulated by several factors. The Bohr effect is the ability of hemoglobin to deliver more oxygen to tissues at low pH. It arises from the stabilizing action of protons on deoxyhemoglobin, which binds protons more readily than oxyhemoglobin because the latter is a weaker acid (Fig. 8-2). Thus, hemoglobin has a lower oxygen affinity at low pH. The major small molecule that alters oxygen affinity in humans is 2,3-bisphosphoglycerate (2,3-BPG, formerly 2,3-DPG), which lowers oxygen affinity when bound to hemoglobin. HbA has a reasonably high affinity for 2,3-BPG. HbF does not bind 2,3-BPG, so it tends to have a higher oxygen affinity in vivo. Hemoglobin also binds nitric oxide reversibly; this interaction influences vascular tone, but its clinical relevance remains controversial.

Proper oxygen transport depends on the tetrameric structure of the proteins, the proper arrangement of the charged amino acids, and interaction with protons or 2,3-BPG.

DEVELOPMENTAL BIOLOGY OF HUMAN HEMOGLOBINS

Red cells first appearing at about 6 weeks after conception contain the embryonic hemoglobins Hb Portland (ζ₂γ₂), Hb Gower I (ζ₂ε₂), and Hb Gower II (α₂ε₂). At 10–11 weeks, fetal hemoglobin (HbF; α₂γ₂) becomes predominant. The switch to nearly exclusive synthesis of adult hemoglobin (HbA; α₂β₂) occurs at about 38 weeks (Fig. 8-1). Fetuses and newborns therefore require α-globin but not β-globin for normal gestation. A major advance in understanding the HbF-to-HbA transition has been the demonstration that the transcription factor Bcl11a plays a pivotal role in its regulation. Small amounts of HbF are produced during postnatal life. A few red cell clones called F cells are progeny of a small pool of immature committed erythroid precursors (BFU-e) that retain the ability to produce HbF. Profound erythroid stresses, such as severe hemolytic anemias, bone marrow transplantation, or cancer chemotherapy, cause more of the F-potent BFU-e to be recruited. HbF levels thus tend to rise in some patients with sickle cell anemia or thalassemia. This phenomenon probably explains the ability of hydroxyurea to increase levels of HbF in adults. Agents such as butyrate and histone deacetylase inhibitors can also activate fetal globin genes partially after birth.

GENETICS AND BIOSYNTHESIS OF HUMAN HEMOGLOBIN

The human hemoglobins are encoded in two tightly linked gene clusters; the α-like globin genes are clustered on chromosome 16, and the β-like genes on chromosome 11 (Fig. 8-1). The α-like cluster consists of two α-globin genes and a single copy of the ζ gene. The non-α gene cluster consists of a single ε gene, the Gγ and Aγ fetal globin genes, and the adult δ and β genes.

Important regulatory sequences flank each gene. Immediately upstream are typical promoter elements needed for the assembly of the transcription initiation complex. Sequences in the 5′ flanking region of the γ and the β genes appear to be crucial for the correct developmental regulation of these genes, while elements that function like classic enhancers and silencers are in the 3′ flanking regions. The locus control region (LCR) elements located far upstream appear to control the overall level of expression of each cluster. These elements achieve their regulatory effects by interacting with trans-acting transcription factors. Some of these factors are ubiquitous (e.g., Sp1 and YY1), while others are more or less limited to erythroid cells or hematopoietic cells (e.g., GATA-1, NFE-2, and EKLF). The LCR controlling the α-globin gene cluster is modulated by a SWI/SNF-like protein called ATRX; this protein appears to influence chromatin remodeling and DNA methylation. The association of α thalassemia with mental retardation and myelodysplasia in some families appears to be related to mutations in the ATRX pathway. This pathway also modulates genes specifically expressed during erythropoiesis, such as those that encode the enzymes for heme biosynthesis. Normal red blood cell (RBC) differentiation requires the coordinated expression of the globin genes with the genes responsible for heme and iron metabolism. RBC precursors contain a protein, α-hemoglobin-stabilizing protein (AHSP), that enhances the folding and solubility of α globin, which is otherwise easily denatured, leading to insoluble precipitates. These precipitates play an important role in the thalassemia syndromes and certain unstable hemoglobin disorders. Polymorphic variation in the amounts and/or functional capacity of AHSP might explain some of the clinical variability seen in patients inheriting identical thalassemia mutations.

There are five major classes of hemoglobinopathies (Table 8-1). Structural hemoglobinopathies occur when mutations alter the amino acid sequence of a globin chain, altering the physiologic properties of the variant hemoglobins and producing the characteristic clinical abnormalities. The most clinically relevant variant hemoglobins polymerize abnormally, as in sickle cell anemia, or exhibit altered solubility or oxygen-binding affinity. Thalassemia syndromes arise from mutations that impair production or translation of globin mRNA, leading to deficient globin chain biosynthesis. Clinical abnormalities are attributable to the inadequate supply of hemoglobin and the imbalances in the production of individual globin chains, leading to premature destruction of erythroblasts and RBC. Thalassemic hemoglobin variants combine features of thalassemia (e.g., abnormal globin biosynthesis) and of structural hemoglobinopathies (e.g., an abnormal amino acid sequence). Hereditary persistence of fetal hemoglobin (HPFH) is characterized by synthesis of high levels of fetal hemoglobin in adult life. Acquired hemoglobinopathies include modifications of the hemoglobin molecule by toxins (e.g., acquired methemoglobinemia) and clonal abnormalities of hemoglobin synthesis (e.g., high levels of HbF production in preleukemia and α thalassemia in myeloproliferative disorders).

EPIDEMIOLOGY

Hemoglobinopathies are especially common in areas in which malaria is endemic. This clustering of hemoglobinopathies is assumed to reflect a selective survival advantage for the abnormal RBC, which presumably provide a less hospitable environment during the obligate RBC stages of the parasitic life cycle. Very young children with α thalassemia are more susceptible to infection with the nonlethal Plasmodium vivax. Thalassemia might then favor a natural protection against infection with the more lethal P. falciparum.

Thalassemias are the most common genetic disorders in the world, affecting nearly 200 million people worldwide. About 15% of American blacks are silent carriers for α thalassemia; α-thalassemia trait (minor) occurs in 3% of American blacks and in 1–15% of persons of Mediterranean origin. β-Thalassemia has a 10–15% incidence in individuals from the Mediterranean and Southeast Asia and 0.8% in American blacks. The number of severe cases of thalassemia in the United States is about 1000. Sickle cell disease is the most common structural hemoglobinopathy, occurring in heterozygous form in ~8% of American blacks and in homozygous form in 1 in 400. Between 2 and 3% of American blacks carry a hemoglobin C allele.

INHERITANCE AND ONTOGENY

Hemoglobinopathies are autosomal codominant traits—compound heterozygotes who inherit a different abnormal mutant allele from each parent exhibit composite features of each. For example, patients inheriting sickle β thalassemia exhibit features of β thalassemia and sickle cell anemia. The α chain is present in HbA, HbA₂, and HbF; α-chain mutations thus cause abnormalities in all three. The α-globin hemoglobinopathies are symptomatic in utero and after birth because normal function of the α-globin gene is required throughout gestation and adult life. In contrast, infants with β-globin hemoglobinopathies tend to be asymptomatic until 3–9 months of age, when HbA has largely replaced HbF. Prevention or partial reversion of the switch should thus be an effective therapeutic strategy for β-chain hemoglobinopathies.

Electrophoretic techniques are widely used for hemoglobin analysis. Electrophoresis at pH 8.6 on cellulose acetate membranes is especially simple, inexpensive, and reliable for initial screening. Agar gel electrophoresis at pH 6.1 in citrate buffer is often used as a complementary method because each method detects different variants. Some important variants are electrophoretically silent. These mutant hemoglobins can usually be characterized by more specialized techniques such as isoelectric focusing and/or high-pressure liquid chromatography (HPLC), which is rapidly replacing electrophoresis for initial analysis.

Quantitation of the hemoglobin profile is often desirable. HbA₂ is frequently elevated in β-thalassemia trait and depressed in iron deficiency. HbF is elevated in HPFH, some β-thalassemia syndromes, and occasional periods of erythroid stress or marrow dysplasia. For characterization of sickle cell trait, sickle thalassemia syndromes, or HbSC disease, and for monitoring the progress of exchange transfusion therapy to lower the percentage of circulating HbS, quantitation of individual hemoglobins is also required. In most laboratories, quantitation is performed only if the test is specifically ordered. Complete characterization, including amino acid sequencing or gene cloning and sequencing, is readily available from several reference laboratories.

Because some variants can comigrate with HbA or HbS (sickle hemoglobin), electrophoretic assessment should always be regarded as incomplete unless functional assays for hemoglobin sickling, solubility, or oxygen affinity are also performed, as dictated by the clinical presentation. The best sickling assays involve measurement of the degree to which the hemoglobin sample becomes insoluble, or gelated, as it is deoxygenated (i.e., sickle solubility test). Unstable hemoglobins are detected by their precipitation in isopropanol or after heating to 50°C. High-O₂ affinity and low-O₂ affinity variants are detected by quantitating the P₅₀, the partial pressure of oxygen at which the hemoglobin sample becomes 50% saturated with oxygen. Direct tests for the percentage carboxyhemoglobin and methemoglobin, employing spectrophotometric techniques, can readily be obtained from most clinical laboratories on an urgent basis.

Laboratory evaluation remains an adjunct rather than the primary diagnostic aid. Diagnosis is best established by recognition of a characteristic history, physical findings, peripheral blood smear morphology, and abnormalities of the complete blood cell count (e.g., profound microcytosis with minimal anemia in thalassemia trait).

SICKLE CELL SYNDROMES

The sickle cell syndromes are caused by a mutation in the β-globin gene that changes the sixth amino acid from glutamic acid to valine. HbS (α₂β₂^{6 Glu→Va1}) polymerizes reversibly when deoxygenated to form a gelatinous network of fibrous polymers that stiffen the RBC membrane, increase viscosity, and cause dehydration due to potassium leakage and calcium influx (Fig. 8-3). These changes also produce the sickle shape. Sickled cells lose the pliability needed to traverse small capillaries. They possess altered "sticky" membranes that are abnormally adherent to the endothelium of small venules. These abnormalities provoke unpredictable episodes of microvascular vasoocclusion and premature RBC destruction (hemolytic anemia). Hemolysis occurs because the spleen destroys the abnormal RBC. The rigid adherent cells clog small capillaries and venules, causing tissue ischemia, acute pain, and gradual end-organ damage. This venoocclusive component usually dominates the clinical course. Prominent manifestations include episodes of ischemic pain (i.e., painful crises) and ischemic malfunction or frank infarction in the spleen, central nervous system, bones, liver, kidneys, and lungs (Fig. 8-3).

Several sickle syndromes occur as the result of inheritance of HbS from one parent and another hemoglobinopathy, such as β thalassemia or HbC (α₂β₂^{6 Glu→Lys}), from the other parent. The prototype disease, sickle cell anemia, is the homozygous state for HbS (Table 8-2).

Clinical manifestations of sickle cell anemia

Most patients with sickling syndromes have hemolytic anemia, with hematocrits from 15 to 30%, and significant reticulocytosis. Anemia was once thought to exert protective effects against vasoocclusion by reducing blood viscosity. However, natural history and drug therapy trials suggest that an increase in the hematocrit and feedback inhibition of reticulocytosis might be beneficial, even at the expense of increased blood viscosity. The role of adhesive reticulocytes in vasoocclusion might account for these paradoxical effects.

Granulocytosis is common. The white count can fluctuate substantially and unpredictably during and between painful crises, infectious episodes, and other intercurrent illnesses.

Vasoocclusion causes protean manifestations. Intermittent episodes of vasoocclusion in connective and musculoskeletal structures produce ischemia manifested by acute pain and tenderness, fever, tachycardia, and anxiety. These recurrent episodes, called painful crises, are the most common clinical manifestation. Their frequency and severity vary greatly. Pain can develop almost anywhere in the body and may last from a few hours to 2 weeks. Repeated crises requiring hospitalization (>3 per year) correlate with reduced survival in adult life, suggesting that these episodes are associated with accumulation of chronic end-organ damage. Provocative factors include infection, fever, excessive exercise, anxiety, abrupt changes in temperature, hypoxia, and hypertonic dyes.

Repeated micro-infarction can destroy tissues having microvascular beds prone to sickling. Thus, the spleen is frequently lost within the first 18–36 months of life, causing susceptibility to infection, particularly by pneumococci. Acute venous obstruction of the spleen (splenic sequestration crisis), a rare occurrence in early childhood, may require emergency transfusion and/or splenectomy to prevent trapping of the entire arterial output in the obstructed spleen. Occlusion of retinal vessels can produce hemorrhage, neovascularization, and eventual detachments. Renal papillary necrosis invariably produces isosthenuria. More widespread renal necrosis leads to renal failure in adults, a common late cause of death. Bone and joint ischemia can lead to aseptic necrosis, especially of the femoral or humeral heads; chronic arthropathy; and unusual susceptibility to osteomyelitis, which may be caused by organisms, such as Salmonella, rarely encountered in other settings. The hand–foot syndrome is caused by painful infarcts of the digits and dactylitis. Stroke is especially common in children; a small subset tend to experience repeated episodes. Stroke is less common in adults and is often hemorrhagic. A particularly painful complication in males is priapism, due to infarction of the penile venous outflow tracts; permanent impotence is a frequent consequence. Chronic lower leg ulcers probably arise from ischemia and superinfection in the distal circulation.

Acute chest syndrome is a distinctive manifestation characterized by chest pain, tachypnea, fever, cough, and arterial oxygen desaturation. It can mimic pneumonia, pulmonary emboli, bone marrow infarction and embolism, myocardial ischemia, or in situ lung infarction. Acute chest syndrome is thought to reflect in situ sickling within the lung, producing pain and temporary pulmonary dysfunction. Often it is difficult or impossible to distinguish among other possibilities. Pulmonary infarction and pneumonia are the most frequent underlying or concomitant conditions in patients with this syndrome. Repeated episodes of acute chest pain correlate with reduced survival. Acutely, reduction in arterial oxygen saturation is especially ominous because it promotes sickling on a massive scale. Chronic acute or subacute pulmonary crises lead to pulmonary hypertension and cor pulmonale, an increasingly common cause of death as patients survive longer. Considerable controversy exists about the possible role played by free plasma HbS in scavenging NO₂, thus raising pulmonary vascular tone. Trials of sildenafil to restore NO₂ levels were terminated because of adverse effects.

Sickle cell syndromes are remarkable for their clinical heterogeneity. Some patients remain virtually asymptomatic into or even through adult life, while others have repeated crises requiring hospitalization from early childhood. Patients with sickle thalassemia and sickle-HbE tend to have similar, slightly milder, symptoms, perhaps because of the ameliorating effects of production of other hemoglobins within the RBC. Hemoglobin SC disease, one of the more common variants of sickle cell anemia, is frequently marked by lesser degrees of hemolytic anemia and a greater propensity for the development of retinopathy and aseptic necrosis of bones. In most respects, however, the clinical manifestations resemble sickle cell anemia. Some rare hemoglobin variants actually aggravate the sickling phenomenon.

The clinical variability in different patients inheriting the same disease-causing mutation (sickle hemoglobin) has made sickle cell disease the focus of efforts to identify modifying genetic polymorphisms in other genes that might account for the heterogeneity. The complexity of the data obtained thus far has dampened the expectation that genomewide analysis will yield individualized profiles that predict a patient's clinical course. Nevertheless, a number of interesting patterns have emerged from these modifying gene analyses. For example, genes affecting the inflammatory response or cytokine expression appear to be modifying candidates. Genes that affect transcriptional regulation of lymphocytes may be involved. Thus, it appears likely that key polymorphic changes in the patient's inflammatory response to the damages provoked by sickle red cells or in the response to chronic or recurrent infections may prove to be important for prognosticating the clinical severity of disease.

Clinical manifestations of sickle cell trait

Sickle cell trait is usually asymptomatic. Anemia and painful crises are rare. An uncommon but highly distinctive symptom is painless hematuria often occurring in adolescent males, probably due to papillary necrosis. Isosthenuria is a more common manifestation of the same process. Sloughing of papillae with urethral obstruction has been reported, as have isolated cases of massive sickling or sudden death due to exposure to high altitudes or extremes of exercise and dehydration. Avoidance of dehydration and extreme physical stress should be advised.

Diagnosis

Sickle cell syndromes are suspected on the basis of hemolytic anemia, RBC morphology (Fig. 8-4), and intermittent episodes of ischemic pain. Diagnosis is confirmed by hemoglobin electrophoresis and the sickling tests already discussed. Thorough characterization of the exact hemoglobin profile of the patient is important because sickle thalassemia and hemoglobin SC disease have distinct prognoses and clinical features. Diagnosis is usually established in childhood, but occasional patients, often with compound heterozygous states, do not develop symptoms until the onset of puberty, pregnancy, or early adult life. Genotyping of family members and potential parental partners is critical for genetic counseling. Details of the childhood history establish prognosis and need for aggressive or experimental therapies. Factors associated with increased morbidity and reduced survival are more than three crises requiring hospitalization per year, chronic neutrophilia, a history of splenic sequestration or hand–foot syndrome, and second episodes of acute chest syndrome. Patients with a history of cerebrovascular accidents are at higher risk for repeated episodes and require partial exchange transfusion and especially close monitoring using Doppler carotid flow measurements. Patients with severe or repeated episodes of acute chest syndrome may need lifelong transfusion support, utilizing partial exchange transfusion, if possible.

UNSTABLE HEMOGLOBINS

Amino acid substitutions that reduce solubility or increase susceptibility to oxidation produce unstable hemoglobins that precipitate, forming inclusion bodies injurious to the RBC membrane. Representative mutations are those that interfere with contact points between the α and β subunits (e.g., Hb Philly [β^35Tyr→Phe]), alter the helical segments (e.g., Hb Genova [β^28Leu→Pro]), or disrupt interactions of the hydrophobic pockets of the globin subunits with heme (e.g., Hb Koln [β^98Val→Met]) (Table 8-3). The inclusions, called Heinz bodies, are clinically detectable by staining with supravital dyes such as crystal violet. Removal of these inclusions by the spleen generates pitted, rigid cells that have shortened life spans, producing hemolytic anemia of variable severity, sometimes requiring chronic transfusion support. Splenectomy may be needed to correct the anemia. Leg ulcers and premature gallbladder disease due to bilirubin loading are frequent stigmata.

Unstable hemoglobins occur sporadically, often by spontaneous new mutations. Heterozygotes are often symptomatic because a significant Heinz body burden can develop even when the unstable variant accounts for only a portion of the total hemoglobin. Symptomatic unstable hemoglobins tend to be β-globin variants because sporadic mutations affecting only one of the four α globins would generate only 20–30% abnormal hemoglobin.

HEMOGLOBINS WITH ALTERED OXYGEN AFFINITY

High-affinity hemoglobins (e.g., Hb Yakima [β^99Asp→His]) bind oxygen more readily but deliver less O₂ to tissues at normal capillary PO₂ levels (Fig. 8-2). Mild tissue hypoxia ensues, stimulating RBC production and erythrocytosis (Table 8-3). In extreme cases, the hematocrits can rise to 60–65%, increasing blood viscosity and producing typical symptoms (headache, somnolence, or dizziness). Phlebotomy may be required. Typical mutations alter interactions within the heme pocket or disrupt the Bohr effect or salt-bond site. Mutations that impair the interaction of HbA with 2,3-BPG can increase O₂ affinity because 2,3-BPG binding lowers O₂ affinity.

Low-affinity hemoglobins (e.g., Hb Kansas [β^102Asn→Lys]) bind sufficient oxygen in the lungs, despite their lower oxygen affinity, to achieve nearly full saturation. At capillary oxygen tensions, they lose sufficient amounts of oxygen to maintain homeostasis at a low hematocrit (Fig. 8-2) (pseudoanemia). Capillary hemoglobin desaturation can also be sufficient to produce clinically apparent cyanosis. Despite these findings, patients usually require no specific treatment.

METHEMOGLOBINEMIAS

Methemoglobin is generated by oxidation of the heme iron moieties to the ferric state, causing a characteristic bluish-brown muddy color resembling cyanosis. Methemoglobin has such high oxygen affinity that virtually no oxygen is delivered. Levels >50–60% are often fatal.

Congenital methemoglobinemia arises from globin mutations that stabilize iron in the ferric state (e.g., HbM Iwata [α^87His→Tyr], Table 8-3) or from mutations that impair the enzymes that reduce methemoglobin to hemoglobin (e.g., methemoglobin reductase, NADP diaphorase). Acquired methemoglobinemia is caused by toxins that oxidize heme iron, notably nitrate and nitrite-containing compounds, including drugs commonly used in cardiology and anesthesiology.

DIAGNOSIS AND MANAGEMENT OF PATIENTS WITH UNSTABLE HEMOGLOBINS, HIGH-AFFINITY HEMOGLOBINS, AND METHEMOGLOBINEMIA

Unstable hemoglobin variants should be suspected in patients with nonimmune hemolytic anemia, jaundice, splenomegaly, or premature biliary tract disease. Severe hemolysis usually presents during infancy as neonatal jaundice or anemia. Milder cases may present in adult life with anemia or only as unexplained reticulocytosis, hepatosplenomegaly, premature biliary tract disease, or leg ulcers. Because spontaneous mutation is common, family history of anemia may be absent. The peripheral blood smear often shows anisocytosis, abundant cells with punctate inclusions, and irregular shapes (i.e., poikilocytosis).

The two best tests for diagnosing unstable hemoglobins are the Heinz body preparation and the isopropanol or heat stability test. Many unstable Hb variants are electrophoretically silent. A normal electrophoresis does not rule out the diagnosis.

Severely affected patients may require transfusion support for the first 3 years of life because splenectomy before age 3 years is associated with a significantly higher immune deficit. Splenectomy is usually effective thereafter, but occasional patients may require lifelong transfusion support. After splenectomy, patients can develop cholelithiasis and leg ulcers, hypercoagulable states, and susceptibility to overwhelming sepsis. Splenectomy should be avoided or delayed unless it is the only alternative. Precipitation of unstable hemoglobins is aggravated by oxidative stress, e.g., infection and antimalarial drugs, which should be avoided when possible.

High-O₂ affinity hemoglobin variants should be suspected in patients with erythrocytosis. The best test for confirmation is measurement of the P₅₀. A high-O₂ affinity Hb causes a significant left shift (i.e., lower numeric value of the P₅₀); confounding conditions, e.g., tobacco smoking or carbon monoxide exposure, can also lower the P₅₀.

High-affinity hemoglobins are often asymptomatic; rubor or plethora may be telltale signs. When the hematocrit reaches to 55–60%, symptoms of high blood viscosity and sluggish flow (e.g., headache, lethargy, dizziness) may be present. These persons may benefit from judicious phlebotomy. Erythrocytosis represents an appropriate attempt to compensate for the impaired oxygen delivery by the abnormal variant. Overzealous phlebotomy may stimulate increased erythropoiesis or aggravate symptoms by thwarting this compensatory mechanism. The guiding principle of phlebotomy should be to improve oxygen delivery by reducing blood viscosity and increasing blood flow rather than restoration of a normal hematocrit. Modest iron deficiency may aid in control.

Low-affinity hemoglobins should be considered in patients with cyanosis or a low hematocrit with no other reason apparent after thorough evaluation. The P₅₀ test confirms the diagnosis. Counseling and reassurance are the interventions of choice.

Methemoglobin should be suspected in patients with hypoxic symptoms who appear cyanotic but have a PaO₂ sufficiently high that hemoglobin should be fully saturated with oxygen. A history of nitrite or other oxidant ingestions may not always be available; some exposures may be inapparent to the patient, and others may result from suicide attempts. The characteristic muddy appearance of freshly drawn blood can be a critical clue. The best diagnostic test is methemoglobin assay, which is usually available on an emergency basis.

Methemoglobinemia often causes symptoms of cerebral ischemia at levels >15%; levels >60% are usually lethal. Intravenous injection of 1 mg/kg of methylene blue is effective emergency therapy. Milder cases and follow-up of severe cases can be treated orally with methylene blue (60 mg three to four times each day) or ascorbic acid (300–600 mg/d).

The thalassemia syndromes are inherited disorders of α- or β-globin biosynthesis. The reduced supply of globin diminishes production of hemoglobin tetramers, causing hypochromia and microcytosis. Unbalanced accumulation of α and β subunits occurs because the synthesis of the unaffected globins proceeds at a normal rate. Unbalanced chain accumulation dominates the clinical phenotype. Clinical severity varies widely, depending on the degree to which the synthesis of the affected globin is impaired, altered synthesis of other globin chains, and coinheritance of other abnormal globin alleles.

CLINICAL MANIFESTATIONS OF β-THALASSEMIA SYNDROMES

Mutations causing thalassemia can affect any step in the pathway of globin gene expression: transcription, processing of the mRNA precursor, translation, and posttranslational metabolism of the β-globin polypeptide chain. The most common forms arise from mutations that derange splicing of the mRNA precursor or prematurely terminate translation of the mRNA.

Hypochromia and microcytosis characterize all forms of β thalassemia because of the reduced amounts of hemoglobin tetramers (Fig. 8-5). In heterozygotes (β-thalassemia trait), this is the only abnormality seen. Anemia is minimal. In more severe homozygous states, unbalanced α- and β-globin accumulation causes accumulation of highly insoluble unpaired α chains. They form toxic inclusion bodies that kill developing erythroblasts in the marrow. Few of the proerythroblasts beginning erythroid maturation survive. The surviving RBCs bear a burden of inclusion bodies that are detected in the spleen, shortening the RBC life span and producing severe hemolytic anemia. The resulting profound anemia stimulates erythropoietin release and compensatory erythroid hyperplasia, but the marrow response is sabotaged by the ineffective erythropoiesis. Anemia persists. Erythroid hyperplasia can become exuberant and produce masses of extramedullary erythropoietic tissue in the liver and spleen.

Massive bone marrow expansion deranges growth and development. Children develop characteristic "chipmunk" facies due to maxillary marrow hyperplasia and frontal bossing. Thinning and pathologic fracture of long bones and vertebrae may occur due to cortical invasion by erythroid elements and profound growth retardation. Hemolytic anemia causes hepatosplenomegaly, leg ulcers, gallstones, and high-output congestive heart failure. The conscription of caloric resources to support erythropoiesis leads to inanition; susceptibility to infection; endocrine dysfunction; and in the most severe cases, death during the first decade of life. Chronic transfusions with RBCs improve oxygen delivery, suppress the excessive ineffective erythropoiesis, and prolong life, but the inevitable side effects, notably iron overload, usually prove fatal by age 30 years.

Severity is highly variable. Known modulating factors are those that ameliorate the burden of unpaired α-globin inclusions. Alleles associated with milder synthetic defects and coinheritance of α-thalassemia trait reduce clinical severity by reducing accumulation of excess α globin. HbF persists to various degrees in β thalassemias. γ-Globin gene chains can substitute for β chains, generating more hemoglobin and reducing the burden of α-globin inclusions. The terms β-thalassemia major and β-thalassemia intermedia are used to reflect the clinical heterogeneity. Patients with β-thalassemia major require intensive transfusion support to survive. Patients with β-thalassemia intermedia have a somewhat milder phenotype and can survive without transfusion. The terms β-thalassemia minor and β-thalassemia trait describe asymptomatic heterozygotes for β thalassemia.

THALASSEMIA SYNDROMES

The four classic α thalassemias, most common in Asians, are α-thalassemia-2 trait, in which one of the four α-globin loci is deleted; α-thalassemia-1 trait, with two deleted loci; HbH disease, with three loci deleted; and hydrops fetalis with Hb Barts, with all four loci deleted (Table 8-4). Nondeletion forms of α thalassemia also exist.

α-Thalassemia-2 trait is an asymptomatic, silent carrier state. α-Thalassemia-1 trait resembles β-thalassemia minor. Offspring doubly heterozygous for α-thalassemia-2 and α-thalassemia-1 exhibit a more severe phenotype called HbH disease. Heterozygosity for a deletion that removes both genes from the same chromosome (cis deletion) is common in Asians and in those from the Mediterranean region, as is homozygosity for α-thalassemia-2 (trans deletion). Both produce asymptomatic hypochromia and microcytosis.

In HbH disease, HbA production is only 25–30% normal. Fetuses accumulate some unpaired γ chains (Hb Barts; γ-chain tetramers). In adults, unpaired β chains accumulate and are soluble enough to form β₄ tetramers called HbH. HbH forms few inclusions in erythroblasts and precipitates in circulating RBC. Patients with HbH disease have thalassemia intermedia characterized by moderately severe hemolytic anemia but milder ineffective erythropoiesis. Survival into midadult life without transfusions is common.

The homozygous state for the α-thalassemia-1 cis deletion (hydrops fetalis) causes total absence of α-globin synthesis. No physiologically useful hemoglobin is produced beyond the embryonic stage. Excess γ globin forms tetramers called Hb Barts (γ₄), which has a very high oxygen affinity. It delivers almost no O₂ to fetal tissues, causing tissue asphyxia, edema (hydrops fetalis), congestive heart failure, and death in utero. α-Thalassemia-2 trait is common (15–20%) among people of African descent. The cis α-thalassemia-1 deletion is almost never seen, however. Thus, α-thalassemia-2 and the trans form of α-thalassemia-1 are very common, but HbH disease and hydrops fetalis are rare.

It has been known for some time that some patients with myelodysplasia or erythroleukemia produce RBC clones containing HbH. This phenomenon is due to mutations in the ATRX pathway that affect the LCR of the α-globin gene cluster.

DIAGNOSIS AND MANAGEMENT OF THALASSEMIAS

The diagnosis of β-thalassemia major is readily made during childhood on the basis of severe anemia accompanied by the characteristic signs of massive ineffective erythropoiesis: hepatosplenomegaly; profound microcytosis; a characteristic blood smear (Fig. 8-5); and elevated levels of HbF, HbA₂, or both. Many patients require chronic hypertransfusion therapy designed to maintain a hematocrit of at least 27–30% so that erythropoiesis is suppressed. Splenectomy is required if the annual transfusion requirement (volume of RBCs per kilogram of body weight per year) increases by >50%. Folic acid supplements may be useful. Vaccination with Pneumovax in anticipation of eventual splenectomy is advised, as is close monitoring for infection, leg ulcers, and biliary tract disease. Many patients develop endocrine deficiencies as a result of iron overload. Early endocrine evaluation is required for glucose intolerance, thyroid dysfunction, and delayed onset of puberty or secondary sexual characteristics.

Patients with β-thalassemia intermedia exhibit similar stigmata but can survive without chronic hypertransfusion. Management is particularly challenging because a number of factors can aggravate the anemia, including infection, onset of puberty, and development of splenomegaly and hypersplenism. Some patients may eventually benefit from splenectomy. The expanded erythron can cause absorption of excessive dietary iron and hemosiderosis, even without transfusion.

β-Thalassemia minor (i.e., thalassemia trait) usually presents as profound microcytosis and hypochromia with target cells but only minimal or mild anemia. The mean corpuscular volume is rarely >75 fL; the hematocrit is rarely <30–33%. Hemoglobin analysis classically reveals an elevated HbA₂ (3.5–7.5%), but some forms are associated with normal HbA₂ and/or elevated HbF. Genetic counseling and patient education are essential. Patients with β-thalassemia trait should be warned that their blood picture resembles iron deficiency and can be misdiagnosed. They should eschew empirical use of iron, yet iron deficiency can develop during pregnancy or from chronic bleeding.

Persons with α-thalassemia trait may exhibit mild hypochromia and microcytosis usually without anemia. HbA₂ and HbF levels are normal. Affected individuals usually require only genetic counseling. HbH disease resembles β-thalassemia intermedia, with the added complication that the HbH molecule behaves like moderately unstable hemoglobin. Patients with HbH disease should undergo splenectomy if excessive anemia or a transfusion requirement develops. Oxidative drugs should be avoided. Iron overload leading to death can occur in more severely affected patients.

PREVENTION

Antenatal diagnosis of thalassemia syndromes is now widely available. DNA diagnosis is based on polymerase chain reaction amplification of fetal DNA, obtained by amniocentesis or chorionic villus biopsy followed by hybridization to allele-specific oligonucleotides probes or direct DNA sequencing.

Thalassemic structural variants are characterized by both defective synthesis and abnormal structure.

HEMOGLOBIN LEPORE

Hb Lepore (α₂[δβ]₂) arises by an unequal crossover and recombination event that fuses the proximal end of the δ-gene with the distal end of the closely linked β-gene. It is common in the Mediterranean basin. The resulting chromosome contains only the fused δβ gene. The Lepore (δβ) globin is synthesized poorly because the fused gene is under the control of the weak δ-globin promoter. Hb Lepore alleles have a phenotype like β thalassemia, except for the added presence of 2–20% Hb Lepore. Compound heterozygotes for Hb Lepore and a classic β-thalassemia allele may also have severe thalassemia.

HEMOGLOBIN E

HbE (i.e., α₂β₂^26Glu→Lys) is extremely common in Cambodia, Thailand, and Vietnam. The gene has become far more prevalent in the United States as a result of immigration of Asian persons, especially in California, where HbE is the most common variant detected. HbE is mildly unstable but not enough to affect RBC life span significantly. Heterozygotes resemble individuals with a mild β-thalassemia trait. Homozygotes have somewhat more marked abnormalities but are asymptomatic. Compound heterozygotes for HbE and a β-thalassemia gene can have β-thalassemia intermedia or β-thalassemia major, depending on the severity of the coinherited thalassemic gene.

The β^E allele contains a single base change in codon 26 that causes the amino acid substitution. However, this mutation activates a cryptic RNA splice site, generating a structurally abnormal globin mRNA that cannot be translated from about 50% of the initial pre-mRNA molecules. The remaining 40–50% are normally spliced and generate functional mRNA that is translated into β^E-globin because the mature mRNA carries the base change that alters codon 26.

Genetic counseling of the persons at risk for HbE should focus on the interaction of HbE with β thalassemia rather than HbE homozygosity, a condition associated with asymptomatic microcytosis, hypochromia, and hemoglobin levels rarely <100 g/L (<10 g/dL).

HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN

HPFH is characterized by continued synthesis of high levels of HbF in adult life. No deleterious effects are apparent, even when all of the hemoglobin produced is HbF. These rare patients demonstrate convincingly that prevention or reversal of the fetal to adult hemoglobin switch would provide effective therapy for sickle cell anemia and β thalassemia.

ACQUIRED HEMOGLOBINOPATHIES

The two most important acquired hemoglobinopathies are carbon monoxide poisoning and methemoglobinemia (discussed earlier). Carbon monoxide has a higher affinity for hemoglobin than does oxygen; it can replace oxygen and diminish O₂ delivery. Chronic elevation of carboxyhemoglobin levels to 10 or 15%, as occurs in smokers, can lead to secondary polycythemia. Carboxyhemoglobin is cherry red in color and masks the development of cyanosis usually associated with poor O₂ delivery to tissues.

Abnormalities of hemoglobin biosynthesis have also been described in blood dyscrasias. In some patients with myelodysplasia, erythroleukemia, or myeloproliferative disorders, elevated HbF or a mild form of HbH disease may also be seen. The abnormalities are not severe enough to alter the course of the underlying disease.

BONE MARROW TRANSPLANTATION, GENE THERAPY, AND MANIPULATION OF HBF

Bone marrow transplantation provides stem cells able to express normal hemoglobin; it has been used in a large number of patients with β thalassemia and a smaller number of patients with sickle cell anemia. Early in the course of disease, before end-organ damage occurs, transplantation is curative in 80–90% of patients. In highly experienced centers, the treatment-related mortality rate is <10%. Since survival into adult life is possible with conventional therapy, the decision to transplant is best made in consultation with specialized centers.

Gene therapy of thalassemia and sickle cell disease has proved to be an elusive goal. Uptake of gene vectors into the nondividing hematopoietic stem cells has been inefficient. Lentiviral-type vectors that can transduce nondividing cells may solve this problem.

Reestablishing high levels of fetal hemoglobin synthesis should ameliorate the symptoms of β chain hemoglobinopathies. Cytotoxic agents such as hydroxyurea and cytarabine promote high levels of HbF synthesis, probably by stimulating proliferation of the primitive HbF-producing progenitor cell population (i.e., F cell progenitors). Unfortunately, this regimen has not yet been effective in β thalassemia. Butyrates stimulate HbF production but only transiently. Pulsed or intermittent administration has been found to sustain HbF induction in the majority of patients with sickle cell disease. It is unclear whether butyrates will have similar activity in patients with β thalassemia.

Patients with hemolytic anemias sometimes exhibit an alarming decline in hematocrit during and immediately after acute illnesses. Bone marrow suppression occurs in almost everyone during acute inflammatory illnesses. In patients with short RBC life spans, suppression can affect RBC counts more dramatically. These hypoplastic crises are usually transient and self-correcting before intervention is required.

Aplastic crisis refers to a profound cessation of erythroid activity in patients with chronic hemolytic anemias. It is associated with a rapidly falling hematocrit. Episodes are usually self-limited. Aplastic crises are caused by infection with a particular strain of parvovirus, B19A. Children infected with this virus usually develop permanent immunity. Aplastic crises do not often recur and are rarely seen in adults. Management requires close monitoring of the hematocrit and reticulocyte count. If anemia becomes symptomatic, transfusion support is indicated. Most crises resolve spontaneously within 1–2 weeks.