Chapter 17: Hemoglobinopathies Associated with Unstable Hemoglobin

The unstable hemoglobins discussed here result from a mutation that changes the amino acid sequence of one of the globin chains, leading to instability and precipitation of the hemoglobin molecule.

Homotetramers of normal β chains (hemoglobin H) or, less often, γ chains (hemoglobin Bart’s) are also unstable. These hemoglobins are found in α-thalassemia (see Chap. 15).

The tetrameric hemoglobin molecule has numerous noncovalent interactions that maintain the structure of each subunit and bind the subunits to each other.

Amino acid substitutions or indels that weaken noncovalent interactions allow hemoglobin to denature and precipitate as insoluble globins, which may attach to the cell membrane, forming Heinz bodies.

Heinz bodies impair erythrocyte deformability, impeding the ability to negotiate the splenic sinuses; “pitting” of Heinz bodies causes loss of membrane area and ultimately destruction of red cells, reflected in a hemolytic anemia.

These are autosomal dominant disorders. The patients are heterozygotes and have a combination of hemoglobin A and unstable hemoglobin in their red cells. Homozygous and compound heterozygotes are not observed; the conditions are thought to be lethal.

Sometimes patients develop an unstable hemoglobin as a de novo mutation. More than 80% of patients have a defect in the β globin chain. Defects in the α globin chain are less likely to cause a clinical disorder because genome has four α-globin genes, and a mutation in one gene results in a minor proportion of abnormal globin in the cell.

Decreased hemoglobin concentration is variable from virtually normal to severely decreased. Patients may have reticulocytosis, elevated indirect bilirubin, elevated lactic dehydrogenase, and decreased to absent haptoglobin. The blood film shows polychromasia, anisocytosis, poikilocytosis, and, sometimes, basophilic stippling (Heinz bodies).

Hemolysis is usually compensated. A patient with an unstable hemoglobin with high oxygen affinity may have a hemoglobin level in the upper normal range.

The treatment with oxidant drugs may precipitate hemolytic episodes, making the diagnosis apparent.

In β-chain mutations, chronic hemolytic anemia becomes evident after neonatal period, because during the first year of life γ chains (fetal hemoglobin) are replaced by mutant β chains.

Physical findings may include pallor, jaundice, and splenomegaly.

Some patients have dark urine, probably from the catabolism of free heme groups or Heinz bodies.

Hemoglobin concentration may be normal or decreased. The mean corpuscular hemoglobin may be decreased because of loss of hemoglobin from denaturation and pitting.

Blood film may show hypochromia, poikilocytosis, polychromasia, anisocytosis, and basophilic stippling.

Heinz bodies are commonly found in circulating red cells; after splenectomy they become more frequent.

Reticulocytosis is often disproportionate to the severity of the anemia, particularly when the abnormal hemoglobin has a high oxygen affinity.

Diagnosis of an unstable hemoglobin is confirmed by one of the following:

— Isopropanol precipitation test: a simple screening test that involves the incubation of the hemolysate with a 17% solution of isopropanol. Hemolysates containing unstable hemoglobin variants form a precipitate, whereas a normal hemolysate remains clear. This is a very sensitive but not entirely specific test for unstable hemoglobins.

— Heat denaturation test: more cumbersome, time-consuming, but as specific as isopropanol test. However, it is less available.

— Heinz body detection: requires the incubation of erythrocytes with a supravital stain. However, Heinz bodies are not specific for unstable hemoglobins (Figure 17–1).

— Hemoglobin electrophoresis: may be useful but insensitive because a normal pattern does not rule out an unstable hemoglobin. Thus, electrophoresis is not a screening or reliable test for unstable hemoglobins.

Some unstable globin variants can be better identified by reverse phase high performance liquid chromatography, because of changes in their hydrophobicity but again with not perfect specificity.

Determination of hemoglobin oxygen affinity (P₅₀O₂) is the correct test to detect unstable hemoglobins with altered oxygen-hemoglobin affinity.

Specific mutation of unstable hemoglobins can only be identified by DNA analysis.

Consider the possibility of an unstable hemoglobin in all patients with a hereditary nonspherocytic hemolytic anemia (see Chap. 14), especially with hypochromic red cells and reticulocytosis out of proportion to the degree of anemia.

Not all patients with a positive test for unstable hemoglobin have this disorder; a false-positive isopropanol stability test may be seen in patients with sickle hemoglobin, elevated methemoglobin, or hemoglobin F.

Hemoglobin H and hemoglobin Bart’s are also unstable. These can be detected by electrophoresis and are found in patients with α-thalassemia.

Most patients have a relatively benign course.

Gallstones are common, often requiring cholecystectomy.

Hemolytic episodes may be precipitated by administration of oxidative drugs or less often during infections.

Treatment is usually not required. Folic acid is often given, although benefit is not proven. Splenectomy may be useful in some patients but may cause serious complications.