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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.

Total calcium content, determined by atomic absorption spectroscopy, Ca2+ influx, and cytosolic free Ca2+ concentration ([Ca]1), estimated by a method involving the incorporation of a Ca2+ chelator (Quin 2), were measured in erythrocytes from β-thalassemic (β-thal) and hemoglobin C (CC) patients. Elevation of the total calcium content was observed in the cells from all patients, particularly in CC and splenectomized β-thal. However, [Ca]1was within the normal range (-25 nmol/L) in all the pathologic cells. Ca2+ influx in CC cells and in cells from nonsplenectomized β-thal patients was also within the same range as that observed in control erythrocytes. In cells from splenectomized β-thal patients, the kinetic of 45Ca influx was biphasic, indicating the existence of two pools of exchangeable Ca2+. Density fractionation of the cells from one splenec- tomized β-thal patient showed that the rapid pool corresponded to the lightest cell fraction, which was also found to have the highest calcium content. The dense cells exhibited a normal Ca2+ influx as well as a smaller increase in total calcium content. It is suggested that, as in sickle cell anemia, the excess of Ca2+ in β-thal cells is not free in the cytoplasm but trapped within endocytic vacuoles, especially in a population of abnormal cells that are normally removed by the spleen. In CC patients, who have a functional spleen, a different mechanism could be responsible for the calcium retention. In conclusion, the present results demonstrate that in these two cases of hemolytic anemia associated with high calcium content, Ca2+ permeability and the level of cytosolic Ca2+ are normal.© 1987 by Grune & Stratton, Inc.0006-4971/87/7003-0031$3.00/0

A human leukemic cell line KU 812 was recently established and described as a basophilic cell line. In the present study we show that KU 812 and two of its clones are at least bipotent: in addition to a minor component of basophils, the majority of KU 812 cells belongs to the erythroid cell lineage with a significant percentage (about 15%) of mature hemoglobinized erythroblasts. This terminal differentiation is associated with the synchronized synthesis of the main erythroid proteins, including glycophorins, spectrin beta chain, band 3, and hemoglobin. The predominant hemoglobins are adult, fetal, and Bart's hemoglobin. Adult hemoglobin represented up to 75% of all hemoglobins in the KU 812 F clone in passages containing a high number of mature erythroblasts. Transcripts of all human globin chains were present with ten times less embryonic chain messenger RNA (mRNA) than alpha-, beta- or gamma-chain mRNA. Hemin slightly increased the total hemoglobin production of the cell line, especially gamma-globin chain synthesis, but did not modify the percentage of hemoglobinized cells. Phorbol myristate acetate (PMA) had a complex effect, inducing a proportion of KU 812 cells to adhere to the plastic culture flask. The adherent cell fraction expressed a very low level of specific erythroid proteins, but their ultrastructure was consistent with immature erythroid cells. In contrast, approximately 40% of the nonadherent cells were mature erythroid cells. Cell-sorting experiments showed that this paradoxic effect of PMA is mostly due to cell selection, the more mature cells being unable to adhere. In addition, KU 812 F was found to be sensitive to erythropoietin, which slightly increased its plating efficiency range (from 0% to 50%) in semisolid medium and enhanced hemoglobin accumulation twofold. In binding experiments using 125I erythropoietin, a single class of high-affinity Epo receptors (Kd: 250 pM) was detected by binding with a density of 205 receptors per cell. The KU 812 cell line is therefore a unique model for studying cell commitment toward different hematopoietic lineages and erythroid differentiation.

Total calcium content, determined by atomic absorption spectroscopy, Ca2+ influx, and cytosolic free Ca2+ concentration ( [Ca]i), estimated by a method involving the incorporation of a Ca2+ chelator (Quin 2), were measured in erythrocytes from beta-thalassemic (beta-thal) and hemoglobin C (CC) patients. Elevation of the total calcium content was observed in the cells from all patients, particularly in CC and splenectomized beta-thal. However, [Ca]i was within the normal range (approximately 25 nmol/L) in all the pathologic cells. Ca2+ influx in CC cells and in cells from nonsplenectomized beta-thal patients was also within the same range as that observed in control erythrocytes. In cells from splenectomized beta-thal patients, the kinetic of 45Ca influx was biphasic, indicating the existence of two pools of exchangeable Ca2+. Density fractionation of the cells from one splenectomized beta-thal patient showed that the rapid pool corresponded to the lightest cell fraction, which was also found to have the highest calcium content. The dense cells exhibited a normal Ca2+ influx as well as a smaller increase in total calcium content. It is suggested that, as in sickle cell anemia, the excess of Ca2+ in beta- thal cells is not free in the cytoplasm but trapped within endocytic vacuoles, especially in a population of abnormal cells that are normally removed by the spleen. In CC patients, who have a functional spleen, a different mechanism could be responsible for the calcium retention. In conclusion, the present results demonstrate that in these two cases of hemolytic anemia associated with high calcium content, Ca2+ permeability and the the level of cytosolic Ca2+ are normal.

Nine cases of early erythroblastic leukemia, unidentified by usual criteria, have been diagnosed using a panel of antibodies. Three cases arose in patients with Down's syndrome, one in a patient with therapy- related leukemia, and four patients were in blast crisis of chronic myeloid leukemia; only one case arose de novo. Blast cells could be assigned to two main stages of erythroid differentiation: presence of all erythroid-specific proteins in two patients, a phenotype corresponding to an immature erythroblast; absence of the erythroid markers such as glycophorin A and spectrin in the presence of carbonic anhydrase isoenzyme I, ABH group antigens, and the antigen defined by FA6 152 monoclonal antibody in six patients, a phenotype related to a late erythroid progenitor (CFU-E). One patient had an intermediate phenotype. All patients except one demonstrated a megakaryocytic component. In three patients, chromosomal abnormalities were present, detected both in blasts and in erythroid colonies. In conclusion, these findings indicate that most “cryptic erythroleukemias” are blocked at a “CFU-E-like” stage of differentiation, it may be a frequent event in Down's syndrome and chronic myeloid leukemia, and these erythroleukemias are phenotypically heterogeneous.

Erythroid colonies from cord blood in which both late (CFU-E) and early (BFU-E) erythroid precursors are present, were grown by the plasma clot technique. Hemoglobin (Hb) synthesis was studied and compared in fresh reticulocytes, 7-day-old colonies, and 14-day-old colonies. In the 8 cases studied, the proportion of HbA synthesis progresssively increased from circulating reticulocytes to 7-day-old colonies and finally in 14-day-old colonies. This result brings evidence that Hb switch is programmed at least at the level of early erythroid precursors. In order to modify the cellular environment of the culture and to examine their influence on globin genes expression, neonate and adult irradiated light density blood cells were added. Irradiated cells from adults, in contrast to those from neonates, were able to increase HbA synthesis in colonies derived from early erythroid progenitors. Under optimal conditions of culture (i.e., a high concentration of neonate plated cells), adult cells elicited a constant increase (22%) in the proportion of HbA synthesis. A linear relationship between this increase of HbA synthesis and the number of added cells was observed. In contrast, the plating efficiency was not significantly modified; however, the size of the erythroid bursts was increased upon the addition of adult irradiated cells. Under suboptimal conditions of culture (i.e., a low concentration of plated cells), adult irradiated cells markedly increased the plating efficiency, the size of the colonies, and HbA synthesis (85%). In contrast to the dramatic effects of irradiated adult cells, the cord blood irradiated cells had a very slight effect on HbA synthesis, the plating efficiency, and the size of the colonies derived from cord blood precursors. All these results suggest that only adult cells are able to amplify the Hb switch in the newborn at the level of BFU-E. Two possible mechanisms can be hypothesized: the adult irradiated cells may either modify the differentiation of the BFU-E or recruit precursors that have a higher capacity to express HbA in their progeny.