An in vitro study on thalassemic erythroid precursors in liquid culture.

Thalassemia is an inherited hematological disorder which can generally be classified according to the affected globin imbalance (alpha- or beta-globin) into two main types, i.e. alpha-thalassemia and beta-thalassemia, respectively. There is a wide range of cellular abnormalities associated with thalassemic erythrocytes such as hypochromia, microcytosis, reduced cellular deformability and membrane oxidative damage. The red cell abnormalities lead to premature destruction with marrow erythroid hyperplasia and ineffective erythropoiesis. The abnormalities in thalassemic red blood cells have been found along the erythroid differentiation pathway other than the mature stage as previously shown in bone marrow erythroid precursors and in reticulocytes, the penultimate stage of erythroid differentiation. However, there is a lag in our understanding of the more primitive erythroid stages due to the difficult and hazardous marrow aspiration and heterogeneity of cells derived. We have utilized a novel method of Two-Phase Liquid Culture (TPLC) of beta-thalassemia/HbE erythroid precursors instead of conventional semisolid culture. This type of liquid culture can given higher cell yield with quite synchronous cell differentiation stages and easily be applied for other cellular analytical techniques. The peripheral blood mononuclear cells (PBMC) obtained from non-splenectomized and splenectomized beta-thalassemia/HbE patients were first cultured in medium supplemented with 5637 conditioned medium for a 6-day period (phase I) and then transferred to medium supplemented with recombinant human erythropoietin to allow the terminal differentiation of erythroid precursors (phase II). During the phase I or II, the cultured cells were periodically sampled to determine the cell number, cytocentrifuged on glass slides and stained with Wright stain for morphological assessment of their differentiation stages and analyzed flow cytometrically by staining with fluoresceinated anti-transferrin receptor (anti-CD71) and R-phycoerythrin-conjugated anti-glycophorin A. After assessment by flow cytometry, the remaining stained cells were cytocentrifuged on glass slides and photographed by a fluorescent microscope and a laser scanning confocal microscope. The results of morphological assessment, flow cytometric analysis and microscopic pictures will be presented.

Ultrastructural aspects of the final stage of hemoglobin biosynthesis

Electron microscopic observations on rabbit embryo, adult rabbit, guinea pig, and human immature erythroid cells showed characteristic hemoglobinized organelles distinguishable from mitochondria by their highly dense matrix, two or three longitudinally arranged double lamellae, and smaller diameters. The presence of hemoglobin (Hb) within these organelles was also demonstrated by electrophoresis of the concentrated supernatant from the isolated, washed and osmotically lysec organelle fraction. The term hemosome has been suggested for these organelles because of their Hb content. We propose that they are the sites of heme integration into the four polypeptide globin chains. The frequency of hemosomes is higher in the peripheral blood erythorid cells of embryos than in the liver erythroid cells, coinciding with the higher Hb synthesis rate in peripheral blood than in the liver. Peripheral blood reticulocytes of rabbits with anemia induced by bleeding presented a lower hemosome frequency than normal reticulocytes. The decrease paralleled the decay of Hb biosynthesis activity. Moreover, Hb biosynthesis induced in HeLa cell and epithelial cell tissue cultures was always associated with the formation of hemosomes. Hemosomegenesis was studied in epithelial tissue culture cells experimentally induced to synthesize Hb, allowing the identification of several stages of hemosome formation in erythroid cells. The morphological data suggest that mitochondria are successively modified to lamellated...