Indications for Testing
Testing for unstable hemoglobinopathies is useful in cases of unexplained hemolytic anemia or when there is a known familial pathogenic hemoglobin variant.
The first step in the evaluation of a suspected unstable hemoglobinopathy is a CBC with peripheral smear and reticulocyte count. The initial evaluation usually reveals normocytic anemia (ranging from mild to severe) with nonspecific findings of hemolysis. Hemolysis may be chronic or may be induced by oxidative stress, such as exposure to sulfonamide drugs. Brisk reticulocytosis is generally observed. “Bite” or “blister” cells may be observed in the peripheral smear. In severe cases, particularly when hyperunstable hemoglobins are present, the peripheral smear may reveal anisocytosis, basophilic stippling, Howell-Jolly bodies, and microspherocytes.
If a hemolytic anemia workup is performed, characteristic findings include decreased hemoglobin, elevated unconjugated bilirubin, elevated lactate dehydrogenase, decreased haptoglobin, and disproportionately elevated aspartate aminotransferase (compared with alanine aminotransferase).
Stability testing is performed by incubating with isopropanol (isopropanol stability test) or treating with heat at 50° Celsius (heat stability test). A precipitate will form if unstable hemoglobins are present. A false-positive result may occur if there is much hemoglobin F present (as is typical in neonates) or in cases of methemoglobinemia.
Heinz bodies may be detected by supravital staining of erythrocytes in peripheral blood. However, the absence of Heinz bodies does not rule out unstable hemoglobinopathy. Furthermore, Heinz bodies are not specific to the unstable hemoglobinopathies, and may be observed in other conditions that lead to hemoglobin precipitates (eg, glucose-6-phosphate dehydrogenase [G6PD] deficiency). Results of this test are unreliable in infants younger than 6 months of age.
Hemoglobin electrophoresis (eg, isoelectric focusing [IEF]) or high-performance liquid chromatography (HPLC) may reveal increased hemoglobin A2 and F. However, unstable hemoglobin variants, particularly hyperunstable variants, undergo rapid denaturation and degradation within the erythrocyte, and remaining hemoglobins may appear relatively normal. Therefore, a normal hemoglobin electrophoresis or HPLC result does not rule out an unstable hemoglobinopathy. Hemoglobin electrophoresis should not be repeated in patients with a previous result who do not require intervention or monitoring.
Globin gene sequencing is the only technique that may detect some of the rare unstable hemoglobins. Therefore, sequencing of the globin genes, including the gamma globin gene in affected neonates, is often needed for a definitive diagnosis.