NEW ULTRA-MICRO HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC METHOD FOR DETERMINING THE GAMMA-CHAIN COMPOSITION OF HEMOGLOBIN-F IN NORMAL ADULTS

The difficulty in isolating the minute quantity of Hb F (< 1%) present in the red blood cells of normal adults greatly complicates the deter mination of its gamma chain composition. We have developed a rapid ult ra-micro high-performance liquid chromatographic (HPLC) method and use d it to analyze the gamma chain composition of Hb F in 47 adults with Hb F levels between 0.1-3.4%. The method involves the isolation of Hb F from as little as 50 mul of whole blood on an analytical size cation -exchange HPLC column, followed by concentration in a Centricon micro concentrator unit and by reversed-phase HPLC analysis. The entire proc edure can be completed in one day and 3-4 analyses can be made simulta neously. We reanalyzed the blood samples from 22 subjects with known b eta-globin gene duster haplotypes, and confirmed the association of hi gh, low, and very low G(gamma) levels with haplotypes A, B, and C, res pectively. Also included are the results of DNA sequence analyses of t he G(gamma) and beta promoters, and of the locus-control-region hypers ensitive site-2 (LCR-HS-2) of the beta-globin gene cluster in five sub jects homozygous for haplotypes A, B or C; the data obtained failed to provide a satisfactory explanation for all the variations in the G(ga mma) levels that have been observed.