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Homozygous β thalassemic mice received 50 U (1,660 U/kg) of recombinant human erythropoietin (rhEpo) 5 days a week for 2 weeks. Hemoglobin increased from 9.2 ± 0.6 g/dL to 10.5 ± 0.4 g/dL (P = .002) and hematocrit increased from 29.2% ± 0.9% to 34.1% ± 1.9% (P = .0014). The β minor/α globin chain synthesis ratio increased slightly but significantly between day -4 (0.75 ± 0.07) and day 4 (0.81 ± 0.04) (P = .01) and reached a minimum ratio (0.67 ± 0.03) on day 15 (P = .001), being parallel to reticulocyte counts and to the incorporated trichloracetic acid (TCA)-insoluble radioactivity, therefore parallel to the erythropoietic output in thalassemic mice, as in normal mice. Erythrocyte defects were improved in β thalassemic mice treated by rhEpo: membrane-associated α globin was significantly decreased (P < .01), thiol group reactivity of ankyrin was significantly improved (P < .05), spectrin alterations were reduced, and deformabil-ity of mouse thalassemic red blood cells was normalized. These results provide experimental criteria for modulating globin chain imbalance necessary for the therapy of human β thalassemia intermedia, and suggest that rhEpo might be of interest to improve the red blood cell mass and reduce erythrocyte alterations in this disease. © 1991 by The American Society of Hematology.

Hemoglobin variants can be successfully identified in cord blood samples. The methods most commonly used include cellulose acetate (CAC) and citrate agar (CAG) electrophoresis. Recently thin layer isoelectric focusing (TLIF) has been shown to be an excellent method for identifying hemoglobin variants. To determine the applicability of TLIF for cord blood screening, we compared the results of 835 samples obtained by TLIF with that obtained by CAC, CAG, and the combination of both CAC and CAG. In 100 of these samples we detected an abnormal hemoglobin pattern using TLIF. In contrast, we detected only 80 abnormal samples by CAC, 70 by CAG, and 80 by using the combination of CAC and CAG. Due to the increased resolution provided by TLIF, we correctly diagnosed two sickle cell trait samples by TLIF that were incorrectly suspected to be homozygous for sickle cell disease by CAC and CAG. We identified 41 samples containing Bart's hemoglobin by TLIF in contrast to only 21 using CAC and 14 using CAG. The time and cost of TLIF was comparable to that using the combination of both methods. We, therefore, conclude that TLIF is the method of choice for cord blood screening.

Isoelectric focusing (IEF) of hemoglobin was compared to the classical chromatography of labeled globin chains for 22 antenatal diagnoses of hemoglobinopathies: 11 for beta thalassemia, and 11 for sickle cell disease. In all cases, the two methods gave identical results. The diagnosis was confirmed after birth or abortion. Three fetuses homozygous for beta thalassemia and one homozygous for sickle cell disease exhibited no Hb A by IEF, in contrast to normal fetuses or those heterozygous for one of the two hemoglobinopathies. In addition, blood samples obtained in other centers after abortion of 22 fetuses homozygous for beta + or beta 0 thalassemia exhibited no Hb A when analyzed by IEF. When Hb A was present, the respective proportions of Hb A and acetylated Hb F were determined by densitometry of the IEF gel. The Hb A/acetylated Hb F ratio obtained by IEF correlated well with the beta A/gamma ratio of globin chain synthesis, IEF requires 0.1 mg of unlabeled hemoglobin. It is performed in 90 min and several samples can be analyzed simultaneously. If present, maternal contamination of fetal blood must be eliminated by selective lysis of maternal (RBC) using the Orskov reaction. Improvements in this method to obtain suitable samples for IEF analysis are described.

Isoelectric focusing on slabs of acrylamide gel was adapted for the screening of abnormal hemoglobins, the characterization of 70 human variants, and the study of minor fractions of normal hemoglobin. The screening method was as fast and inexpensive as conventional techniques, allowed the simultaneous analysis of some 50 samples of whole blood, and yielded resolution superior to that obtained by other methods with hemolysates. Among the 70 variants, 31 mutants could not be separated from HbS by cellulose acetate electrophoresis. The characterization technique of electrofocusing allowed us to distinguish between most variants. Only one mutant, Hb Galveston, could be confused with HbS. Hb Koln, the most frequent unstable mutant, exhibited a special pattern. HbA1C was separated from HbA. Preliminary results indicate that quantitation of HbA1C by gel scanning is feasible.

Deoxygenation of sickle cells is known to increase cation permeabilities (Na+, K+, and Ca2+). The possible mechanisms involved in the increased uptake of Ca2+were investigated: activation of Ca2+channels, involvement of the anion channel, and the formation of endocytic vacuoles. The Ca2+channel blocker nifedipine reduced the deoxy-stimulated Ca2+uptake by about 30% to 40%. The anion channel inhibitor DIDS (4,4‘ diisothiocyanate stilbene 2,2‘ disulfonate) inhibited the deoxy-stimulated Ca2+uptake by approximately 50%. Maximal possible endocytic uptake, measured by using an impermeant marker ([3H] inuline), accounted for 6% to 9% of the total Ca2+uptake. These data indicate that the deoxygenation-induced increase in Ca2+permeability could result from both the activation of a Ca2+channel and of a transport system for cations involving interactions between polymerized hemoglobin S, band 3 and other membrane components. Endocytosis appears to play only a minor role in the Ca2+uptake of deoxygenated sickle cells.