OPTIMIZATION OF A FLOW CYTOMETRIC METHOD FOR THE SIMULTANEOUS MEASUREMENT OF CELL-SURFACE ANTIGEN, DNA CONTENT, AND IN-VITRO BRDURD INCORPORATION INTO NORMAL AND MALIGNANT HEMATOPOIETIC-CELLS

We hare designed an assay for the simultaneous measurement of cell sur face phenotype, S-phase fraction, and DNA content by single laser inst rumentation for the purpose of determining the labeling index (II), du ration of S-phase (Ts), and the potential doubling time (Tpot) of leuk ocyte subpopulations, The procedure was optimized with regard to: mode of bromodeoxyuridine (BrdUrd) incorporation, selection of suitable le ukocyte differentiation antigens (LDAs) as well as PE-conjugated monoc lonal antibodies (MoAbs) against myeloid cells, overnight permeabiliza tion and fixation (paraformaldehyde 1% and 0.05% Nonidet P40), DNase I treatment (250 Kunitz units), concentration of FITC-conjugated anti-B rdUrd MoAb (dilution 1:5), and DNA staining with 7-amino-actinomycin ( 7-AAD) (10 mu g/ml). We validated this assay by measuring LI, Ts, and Tpot repeatedly in four leukemic cell Lines and found these to be stab le (coefficients of variation (CV): 0.06, 0.13, and 0.08, respectively ). Finally, we employed the assay on different leukocyte preparations from normal donors (including purified CD34+ cells) and patients with malignant myeloid disorders, and we concluded that it will-yield valua ble data regarding the cell cycle kinetics of subsets of leukocytes in heterogeneous mixtures of hematopoietic cells. (C) 1998 Wiley-Liss, I nc.