IMPROVED METHOD FOR DIAGNOSIS OF POLYCYTHEMIA-VERA BASED ON FLOW CYTOMETRIC ANALYSIS OF AUTONOMOUS GROWTH OF ERYTHROID PRECURSORS IN LIQUIDCULTURE

''Autonomous'' development of erythroid colonies in erythropoietin (EP O)-free semi-solid culture has been used as an in vitro assay for diag nosis of polycythemia vera (PV). These colonies, however, are small an d poorly hemoglobinized, rendering the assay in many cases unreliable. We report here on the use of a novel assay; it combines a modified cu lture procedure that maximizes the growth of EPO-independent erythroid cells, and immunofluorescence flow cytometry for their detection and quantitation. Peripheral blood mononuclear cells are cultured for 2-5 days in the presence of a combination of growth factors. During this p hase, early erythroid committed progenitors, burst forming units (BFUe ), proliferate and differentiate into colony forming units (CFUe)-like progenitors. In the second phase, the latter cells, in the presence o f stem cell factor, hemin, and iron-saturated transferrin, continue to proliferate and mature into hemoglobin (Hb)-containing orthochromatic normoblasts. Neither phases contained EPO. The culture produced large , pure, and synchronized erythroid cell populations. The cells were th en dually labeled with fluorescent probes, nuclear DNA with thiazole o range and intracellular hemoglobin (Hb) with phycoerythrin-conjugated monoclonal antibodies against human Hb. Cells positive for both labels were assigned as Hb-containing nucleated precursors. The presence of such cells in EPO-free cultures indicated ''autonomous growth.'' None of the EPO-free cultures derived from normal donors or patients with s econdary polycythemia contained such cells. Cultures derived from PV p atients contained from 5 to 92% ''autonomously-grown'' cells. These cu lture and analysis methods should minimize false negative results with PV patients and provide objective and quantitative data. (C) 1997 Wil ey-Liss, Inc.