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Summary A population-based screening of newborns for the structural variants of fetal hemoglobin was carried out in Osaka Prefecture, Japan, by isoelectric focusing of globin chains using dried blood on filter paper. Of 80,000 newborns, 18 had α globin variants and 55 had γ globin variants. The incidence of γ globin variants (1/1,455) was much higher than that of α globin variants (1/4,444). Structural studies were then carried out on the abnormal γ globins in 36 samples, and revealed that 25 of them were Hb F Yamaguchi (AγT 80 Asp→Asn). The prevalence of this variant in Japanese was estimated to be as high as one per 2,100.

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.

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.

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.

We cultured human umbilical cord blood and adult peripheral blood erythropoietic precursors in methylcellulose clonal assay and measured the synthetic rates of HbA, A2, and F. Hb was labeled with 14C-amino acid in culture and separated by slab-gel isoelectric focusing and quantitated by autoradiography. While the mean percentage of HbF synthesized by adult cells was only 20.1%, that of umbilical cord blood cells was 53.9%, which corresponds closely to the biosynthetic capabilities of umbilical cord blood reticulocytes. Variations in the erythropoietin concentrations did not influence the percentage of HbF. Erythropoietic cell cultures of human umbilical cord blood may provide an important means for studying the molecular mechanisms controlling physiological Hb switching in the perinatal period.

The automatic oxygenation technique of Imai et al (Biochimica Biophysica Acta, 200: 189-196, 1970) was slightly modified and applied to the study of oxygen equilibrium curves of dilute, red-cell suspensions from normal subjects and individuals with hemoglobinopathies, enzymopathies, and other hematologic disorders. the p50 values of non-smoking, normal adults were 25.9 + 0.6 mm Hg at pH 7.4 and 37+C, and corresponded to the values for whole blood reported in the literature. the oxygen equilibrium curves of suspensions from subjects with enzymopathies revealed shifts in position which are thought to be due to alterations in the concentration of 2,3-DPG of the red cells. Abnormalities in shape of the equilibrium curves were observed only for the hemoglobinopathic red cells, and could best be illustrated by the abnormally low Hill's exponent (n*). Analyses of the n* values of 34 patients with various red-cell disorders of unknown causes led to the identification of ten cases showing low values. in five of the ten patients, the presence of an abnormal hemoglobin was confirmed using column chromatography on Amberlite CG-50. These results point to the usefulness of the Hill Plot analysis of red-cell oxygen dissociation curves in functional screening for clinically important hemoglobinopathies.