Application of high-performance liquid chromatographic methodology to the analysis of hemoglobins synthesized in...

High-performance liquid chromatography (HPLC) has been successfully used in the quantitation of the relatively minute amounts of hemoglobin types recovered from in vitro cultures of hemoglobin-synthesizing erythroid progenitor (BFU-E) cells. This reversed-phase HPLC method uses the Vydac C4 column and water-acetonitrile-trifluoroacetic acid as mobile phases; it has been applied to the study of fetal hemoglobin synthesis patterns in ten homozygous sickle cell anemia patients and a similar number of their heterozygous relatives along with a few normal control subjects. A significant increase in the total gamma chain level was observed in the BFU-E lysate samples corresponding to the whole blood lysates of all the patients and their heterozygous relatives, except in one patient with the beta S haplotype Mor. On the other hand, the relative level of the G gamma chains appeared to be decreased in the BFU-E lysate samples of all except the individuals carrying the Mor haplotype, where it is reversed. The method has considerable advantages over other chromatographic and electrophoretic procedures; it is extremely sensitive and allows quantitation of all different globin chains in one single chromatogram.