Hemoglobin Variants Associated With Hemolytic Anemia, Altered Oxygen Affinity, and Methemoglobinemias

Pathophysiology of Unstable Hemoglobin Disorders

The mechanisms by which unstable hemoglobin mutations produce hemoglobin precipitation remain incompletely understood; however, the major outlines of the process have been described ( Fig. 44.2 ). The fundamental step in pathogenesis appears to be derangement of the normal linkages between heme and globin. Loss of appropriate globin chain folding and interaction ultimately destabilizes the heme–globin linkage and likely leads to partial proteolysis of the chain, thereby releasing heme from that linkage. Once freed from its cleft, heme probably binds nonspecifically to other regions of the globin molecule, forming precipitated hemichromes, these lead to further denaturation and aggregation of the globin subunits. These form a precipitate containing α- and β-globin chains, globin fragments, and heme, called the Heinz body .

Heinz bodies interact with delicate red blood cell membrane components (see Chapter 46 Chapter 45 ), thereby reducing red blood cell deformability. These rigid cells tend to be detained in the splenic microcirculation and “pitted,” reflecting attempts by the splenic macrophages to remove the Heinz bodies. Red blood cell damage can be aggravated by the release of free heme into the red blood cell. Several biochemical perturbations correlate with the presence of free heme, such as generation of reactive oxidants (i.e., hydrogen peroxide, superoxide, and hydroxyl radicals). The end result of this process is premature destruction of the red blood cell, producing hemolytic anemia.

Individual unstable hemoglobins vary in their propensity to generate Heinz bodies and hemolysis. For example, Hb Zurich exhibits relatively mild insolubility. Hemolysis is minimal in nonstressed patients with this variant and becomes clinically apparent only in the presence of additional oxidant stresses, such as infection, fever, or the ingestion of oxidant agents. Because of the propensity of unstable hemoglobins to be hypersensitive to oxidation, some patients with unstable hemoglobins can exhibit episodic appearance or aggravation hemolysis in response to many of the same oxidative stressors as those exacerbating the clinical phenotype of glucose-6-phosphate dehydrogenase (G6PD)-deficient patients (see Chapter 45, Chapter 48 ).

Patterns of Inheritance and Clinical Manifestations

Unstable hemoglobins are usually inherited as autosomal dominant disorders. However, the rate of spontaneous mutation appears to be high, so the absence of affected parents or siblings does not rule out the presence of an unstable hemoglobin in an individual family. Nonetheless, the presence of a positive family history can be a useful adjunct to diagnosis and should provoke consideration of an unstable hemoglobin as the cause of the familial hemolytic diathesis.

The clinical syndrome associated with unstable hemoglobin disorders is often called congenital Heinz body hemolytic anemia . This term derives from the fact that only the most severe cases were detected before the widespread availability of sophisticated methods for detecting and characterizing abnormal hemoglobins. Clinical manifestations are highly variable, ranging from a virtually asymptomatic state in the absence of environmental stressors to severe hemolytic anemia manifesting at birth. Patients with chronic hemolysis present with variable degrees of typical symptoms, including anemia, reticulocytosis, hepatosplenomegaly, jaundice, leg ulcers, and a propensity toward premature biliary tract disease.

For Hb variants with a given degree of reduced solubility, the degree of anemia may fluctuate because some of these variants also exhibit altered oxygen affinity. Thus Hb Köln has increased oxygen affinity, resulting in relatively higher levels of tissue hypoxia and erythropoietin stimulation at any given level of hematocrit (see Diagnosis); therefore patients with Hb Köln tend to have higher hematocrit levels than expected on the basis of hemolytic severity because of increased erythropoietin stimulation. By contrast, Hb Hammersmith exhibits decreased oxygen affinity, improving oxygen delivery and allowing patients to function at a lower hematocrit level (see low affinity variants later in this chapter). Hb Zurich possesses, for complex molecular reasons, a higher-than-normal affinity for carbon monoxide. A high carbon monoxy–Hb level develops in patients with Hb Zurich who also smoke. Binding of carbon monoxide protects Hb Zurich from denaturation, thus reducing hemolysis, so these people tend to exhibit lesser degrees of hemolytic anemia than do nonsmoking relatives.

Diagnosis

The presence of an unstable hemoglobin should be suspected in patients with one or more stigmata of accelerated red blood cell destruction: chronic or intermittent hemolytic anemia or jaundice, premature development of bilirubin gallstones or biliary tract disease (as a result of accelerated red blood cell turnover), unexplained reticulocytosis, or bouts of intermittent symptoms that can be related to exposure to oxidant drugs or infections. Other suggestive symptoms include dark urine, transient jaundice, and leg ulcers. It is important to remember that significant anemia may or may not be present despite hemolysis brisk enough to produce the other signs and symptoms of accelerated red cell destruction, because of compensatory erythroid hyperplasia, especially in younger otherwise healthy patients.

Laboratory diagnosis depends on identification of a mutant hemoglobin that precipitates more easily than normal hemoglobin. The peripheral blood smear may or may not show evidence of hemolysis (i.e., poikilocytosis, polychromasia, or shift cells; Fig. 44.3A ). The morphologic evidence for precipitated hemoglobin is the Heinz body, the intraerythrocytic inclusion body detected by staining the peripheral blood smear with a supravital dye, such as brilliant cresyl blue or new methylene blue (see Fig. 44.3B and C ). The spleen removes Heinz bodies efficiently, especially if hemolysis is not particularly acute or brisk. Thus Heinz bodies may not be demonstrable at all times. Two provocative laboratory maneuvers are used to aid detection, both of which unmask the tendency of unstable hemoglobins to precipitate: the heat instability test (heating of a hemoglobin solution to 50°C) or the isopropanol instability test (insolubility in 17% isopropanol).

Hemoglobin electrophoresis should be performed but should not be relied on as the major diagnostic criterion for ruling in or ruling out a hemoglobinopathy . Many amino acid substitutions that have a profound effect on solubility do not change the overall charge on the hemoglobin molecule. For example, Hb Köln, the most common of the unstable hemoglobin mutations, arises from a mutation changing the valine at position 98 to a methionine. This mutation is electrically neutral; it does not alter electrophoretic mobility. Therefore these variants do not form an abnormal band on an electrophoresis gel. Demonstration of an abnormal band would clearly add strong evidence in support of the diagnosis. However, a normal electrophoretogram should never be regarded as strong evidence against the presence of a mutant hemoglobin, especially if the clinical picture or family history otherwise supports the diagnosis. Mass spectrometry analysis of hemoglobin and direct globin gene sequencing are supplanting electrophoresis as diagnostic strategies. They usually provide unambiguous identification of a sequence abnormality. However, electrophoresis is still in use in many clinical settings. Thus the aforementioned precautions in interpretation are still worth noting.

Additional sophisticated analyses of hemoglobin can be obtained from reference laboratories if detailed characterization seems warranted. For example, abnormal hemoglobin or globin bands migrating to novel positions on an isoelectric focusing gel can result from hemoglobin or globin moieties lacking heme in groups. When heme is added to the sample and the proteins are reanalyzed, these bands disappear. This behavior is nearly diagnostic of an unstable variant.

Detection of unstable hemoglobins is occasionally compromised by the selective precipitation of the unstable variant into Heinz bodies. Because most patients are heterozygotes, this phenomenon greatly reduces the apparent percentage of the variant in soluble form. Thus even a variant possessing altered electrophoretic mobility may be very difficult to detect. Indeed, some unstable hemoglobins, such as Hb Geneva or Hb Terre Haute, are so unstable that no mutant gene product can be detected in the steady state. These abnormal hemoglobins actually produce a thalassemic phenotype. They may be detectable only by isotope labeling studies or direct analysis of the globin genes.

The amino acid sequence predicted from genetic sequencing may rarely be inaccurate because of posttranslational conversion of the substituted residue into one causing an unstable hemoglobin. For example, in the first reported case of congenital Heinz body hemolytic anemia due to Hb Bristol, the DNA sequence predicts a valine-to-methionine substitution at β67. Through posttranslational modification, the methionine is altered to aspartate, a hydrophilic residue that disrupts the heme pocket.

The differential diagnosis of unstable hemoglobin variants is usually straightforward if this general category of hemolytic disorders is suspected. The most common form of G6PD deficiency can also manifest with bouts of intermittent or chronic hemolysis exacerbated by oxidant drugs or infection (see Chapter 45 ). This diagnosis should be considered, as should other causes of chronic or intermittent hemolytic anemia, including red blood cell membrane disorders (e.g., hereditary spherocytosis) or immune hemolytic anemias (see Chapter 46, Chapter 47 ). Spherocytes are relatively rare in patients with unstable hemoglobin disorders; this is sometimes a useful discriminant.

Management

The severity of the clinical complications of unstable hemoglobins varies enormously. Many patients can be managed adequately by observation and education to avoid agents that provoke hemolysis. Some patients require transfusions during bouts of severe acute hemolytic anemia. Patients who have significant morbidity because of chronic anemia or repeated episodes of severe hemolysis should be considered candidates for splenectomy, especially if hypersplenism has developed. Children with severe hemolysis may require transfusion support until they are old enough (at least 3 or 4 years of age) to undergo splenectomy without unacceptable immunologic compromise. Splenectomy is usually effective for abolition or reduction of anemia. However, splenectomy should be used only as a last resort because of the long-term risks of overwhelming sepsis and thrombosis. Infection often exacerbates hemolysis. Fever should therefore prompt close monitoring of patients for evidence of hemolysis or infection. Postsplenectomy patients with a hemolytic diathesis are also afflicted by a hypercoagulable state, probably due to the deranged membrane architecture resulting from oxidative damage to red cells no longer being cleared by the spleen. They thus require monitoring for thrombotic events and may need intermittent or long-term anticoagulant therapy.