Chapter 18: 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 γ chains (hemoglobin Bart's) are also unstable. These hemoglobins are found in α-thalassemia (see Chap. 16).

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

Amino acid substitutions or deletions that weaken noncovalent forces, 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 and ultimately destruction of red cells, and a hemolytic anemia.

An autosomal dominant disorder. 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 because they are thought to be lethal.

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

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

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

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

Physical findings may include pallor, jaundice, 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 MCV 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 abundant.

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

Diagnosis is confirmed by demonstration of an unstable hemoglobin. This may be done by:

— Isopropanol precipitation test: a simple screening test that involves the incubation of the hemolysate with a 17 percent of isopropanol; hemolysates containing unstable hemoglobin variants form a precipitate, whereas a normal hemolysate remains clear.

— Heat denaturation test: cumbersome and usually not used.

— Heinz body detection: requires the incubation of erythrocytes with a supravital stain (see Fig. 18–1).

— Hemoglobin electrophoresis: may be useful, but a normal pattern does not rule out an unstable hemoglobin and thus electrophoresis is not a screening or reliable test for unstable hemoglobins. Some unstable globin variants can be identified by reverse phase high performance liquid chromatography (HPLC), due to changes in their hydrophobicity.

Determination of the P₅₀O₂, a measure of hemoglobin oxygen affinity, may be helpful to detect unstable hemoglobins with altered oxygen-hemoglobin affinity.

Unstable hemoglobins can be detected and identified by DNA analysis.

Consider the possibility of an unstable hemoglobin in all patients with a hereditary nonspherocytic hemolytic anemia (see Chap. 15), 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 levels of 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 infection or administration of oxidative drugs.

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 in patients with high oxygen-affinity hemoglobins.