ERYTHROPOIETIN RECEPTOR EXPRESSION ON HUMAN BONE-MARROW ERYTHROID PRECURSOR CELLS BY A NEWLY-DEVISED QUANTITATIVE FLOW-CYTOMETRIC ASSAY

In order to develop a non-isotopic quantitative assay of erythropoieti n (Epo) receptor (EpoR) on human cells, we devised a flow-cytometric a ssay using cells stained with biotin-labelled and a streptavidine-RED6 70 conjugate. For quantification, we applied the Kolmogorov-Smirnov te st and calculated the D value. The D value was evaluated from the degr ee of shift in two profiles according to the increase of fluorescence intensity due to the specific binding of biotin-labelled Epo to EpoR. A good correlation was observed between the number of EpoR calculated by I-125-Epo binding assay and the D value. Then, EpoR expression on b one marrow cells from normal individuals was studied by three-colour f low cytometry. In normal bone marrow, the number of EpoR on cells was highest in CD34(+)CD38(-) cells (approximately 1600 sites/cell), and d ecreased in the following order: CD34(+)CD38(-) cells > CD34(+)CD38(+) cells > CD34(-)CD38(+) cells. Glycophorin A (GpA) positive erythroid cells also expressed EpoR, and their CD34(+) fraction expressed more E poR than their CD34(-) fraction. However, the expression levels of Epo R of these fractions were lower than CD34(+)CD38(-) cells. These resul ts indicated that EpoR was highly expressed on CD34(+) haemopoietic pr ogenitors from very early stages of differentiation without expression of CD38 antigen, and that the level of expression decreased with eryt hroid differentiation as well as with various lineage commitment in hu man bone marrow cells.