We cultured marrow and peripheral blood erythropoietic precrusors in methylcellulose clonal assay and measured the synthetic rates of HbA, A2, F, and S in patients with and without sickle cell anemia. Hb was labeled with 14C-amino acid in culture, separated by slab gel isoelectric focusing techniques, and quantitated by autoradiographic methods. Comparison of marrow late (CFU-E) and early (BFU-E) precursors from patients without hemoglobinopathies showed that preferential synthesis of HbF is limited to early precursors. Simultaneous examinations of Hb synthesis by blood and marrow early erythropoietic precursors confirmed the similarity of the biosynthetic capabilities of the precursors from the two sources. Increasing concentrations of erythropoietin (Ep) in culture corresponded with increases in the percentages of HbF synthesized by blood BFU-E of normal individuals. HbF biosynthesis by blood BFU-E from sickle cell anemia patients was significantly higher than that synthesized by nonanemic individuals and showed significant individual variations. HbF synthesis in patients with sickle cell anemia was partially dependent on Ep concentrations in culture. Cell culture of circulating erythropoietic precursors in man appears to provide a unique tool for studying the control mechanisms of Hb synthesis in man.
We cultured human umbilical cord blood erythropoietic precursors in methyl cellulose clonal assay and analyzed the synthetic rates of Hb A and Hb F in individual erythropoietic bursts. Hemoglobin was labeled with 14C-amino acids in culture, separated by slab gel isoelectric focusing techniques, and quantitated by fluorographic methods. Almost all bursts exhibited both Hb A and Hb F in varying ratios. Frequencies of the individual bursts differing in percentage Hb F biosynthesis had normal distributions. Natal erythropoietic precursors appeared to be randomly committed to Hb F synthesis.
By using a methylcellulose clonal assay, we cultured peripheral blood erythropoietic precursors (BFU-E) from an adult couple whose child had HbF Malta-I(gamma 117 His leads to Arg), a G gamma variant, and measured the synthetic rates of HbA, HbF, and HbF Malta-I. Hemoglobin was labeled with 14C-amino acid in culture, separated by slab gel isoelectric focusing technique, and quantitated by autoradiographic or fluorographic method. Culture of BFU-E from both parents revealed significant HbF biosynthesis. HbF Malta-I was present in culture of the father's cells and comprised about 24% of total HbF. When we analyzed Hb biosynthesis in individual bursts, all bursts contained HbA and HbF in varying ratios. The frequency distribution of the individual bursts differing in percentages of HbF biosynthesis approached normal distribution. While the relative ratio of HbF Malta-I to total HbF biosynthesis in individual bursts also revealed significant variation, its frequency distribution did not show a normal distribution. There was a positive correlation between the ratios of HbF/Hb and HbF Malta- I/HbF in individual bursts.
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