Isolation of large and pure samples of human erythroid precursors at different stages of maturation using immunomagnetic separation of cells from liquid cultures.

The two-phase liquid culture, which supports the development of human erythroid progenitors into hemoglobin-containing orthochromatic normoblasts, provides high yield of erythroblasts with synchronized development. However, other cell types are also present in the culture. In the present report, we used immunomagnetic separation for the purification of the developing erythroid precursors. At different stages of the culture, aliquots of the cells were incubated with anti-glycophorin A, B monoclonal antibodies, followed by anti-mouse IgG-coated magnetic beads, and separated by a magnet. In mature cultures, all isolated glycophorin-positive cells contained hemoglobin, as indicated by their staining with benzidine. All glycophorin-negative cells were benzidine negative. In earlier cultures, morphological examination and measurements of hemoglobin content showed that the erythroid precursors could be isolated at different stages of maturation. Thus, the combination of the liquid culture procedure with the immunomagnetic separation technique permitted to obtain large samples of pure erythroid cells, which were synchronized at subsequent stages of maturation. The method enables comprehensive studies of developing erythroid cells from normal donors as well as from patients with various disorders of erythropoiesis.