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Laboratory Diagnosis

The comprehensive examination and typing of any of the hemoglobinopathies is a complex and skilled procedure. Reference should be made to the appropriate textbooks for full information. Briefly, there are simple routine procedures which can be used for screening. The first step is to examine a peripheral blood smear, which is diagnostic in the majority of cases. Prenatal diagnosis may be made by genetic examination of fetal cells obtained by amniocentesis or (more recently) from fetal cells obtained from maternal peripheral blood samples.

In all hemoglobinopathies, the red cells will be abnormal: target cells are a consistent finding and their absence makes any of the hemoglobinopathies unlikely. If target cells are frequent (more than 20% of all cells), hemoglobin C or E should be suspected. If elongated and sickle-shaped red cells are seen, with marked polychromasia or with nucleated red cells, then sickle cell anemia is probable.

Tests for Sickle Cell Hemoglobin

Hemoglobin S is distinguished from all other hemoglobins because, when it is deoxygenated, it precipitates at 25° C in 2.4 M. phosphate buffer (pH 6.8). This is the basis of a standard, commercially available test. "Sickling" can be recognized by adding one drop of a fresh 2% sodium metabisulfite solution to one drop of a 2% suspension of red cells on a glass slide. This must be covered with a coverslip and left for 30 minutes before microscopy. A negative test excludes sickle cell hemoglobin, but a positive test does not differentiate between the homozygous and heterozygous states.

The various hemoglobins were originally detected by the fact that they have differing electrical charges, and thus will move differently in an electrical field. Electrophoresis will readily separate hemoglobins A, S, C, E and A2 from one another. To distinguish hemoglobin D from S requires an acid rather than alkaline buffer.

Hemoglobin F can be extracted and quantitated because it is resistant to alkaline solutions, which denature all other hemoglobins. When sophisticated laboratory support is available, the method of "fingerprinting" can be utilized. If the result of these investigations suggests a homozygous state, the family should be studied.

In addition to the specific examination of the blood, patients with an abnormal hemoglobin will have evidence of a hemolytic anemia (a raised unconjugated serum bilirubin, an increase in urinary and fecal urobilinogen, raised serum iron, negative Coombs' test, decrease in osmotic fragility, and a decrease in red cell survival). An increase in the amount of fetal hemoglobin is also significant (Table 31.1). Higher levels can provide some protection against the complication of sickle cell disease.

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