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In the 1976 hemoglobinopathy proficiency testing survey of the Center for Disease Control (CDC), whole-blood samples from hematologically normal adults and from individuals heterozygous for beta-thalassemia were shipped to survey participants. The object of this survey was to determine the state of the art for technics used to quantitate hemoglobin A2 (Hb A2) and to test the ability of laboratories to differentiate between blood samples having normal Hb A2 levels and those having elevated levels (i.e., those from individuals with beta-thalassemia trait). The results of Hb A2 quantitation obtained from 183 volunteer participant laboratories were compared with those obtained from 24 reference laboratories. Individual values varied greatly among laboratories and among methods for both normal and elevated Hb A2 samples. The results returned by many laboratories were not within 2 SD of the reference laboratory mean and also were not sufficiently accurate to differentiate between the normal blood samples and those with beta-thalassemia trait. The results suggest that methods for quantitating Hb A2 need to be standardized and a suitable method for determining laboratory performance found.

Although the isopropanol precipitation test is a commonly used and sensitive test for detecting unstable hemoglobins, false-positive reactions are frequently observed. In this study, the storage temperature of the test samples, type of stored sample amount of fetal hemoglobin (Hb F) in the sample, and presence of potassium cyanide (KCN) in the hemolysate preparation all affected the accuracy of the test. The addition of 2% KCN to samples before testing eliminated or greatly reduced false-positive reactions. Hb F levels greater than 4% caused false-positive results. When specimens with Hb F levels less than 4% were stored at 4C and as whole blood, they showed no false-positive reaction for as long as two weeks. However, duplicate specimens stored as whole blood but unrefrigerated and those stored as hemolysates showed false-positive reactions after three days.

Compared with other methods for determining hemoglobin A2 (Hb A2) levels, the new microchromatography procedures are fast, easy laboratory technics useful for quantitating Hb A2 that can be readily monitored for quality control of results. A reliable method for preparing a stable hemolysate control for monitoring the accuracy and precision of Hb A2 microchromatographic procedures is described. The recombination of purified hemoglobin fractions in known concentrations and the addition of potassium cyanide and carbon monoxide produce a control solution that gives stable hemoglobin A2 values for at least 40 weeks. Variability among six reference laboratories was small.