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

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We have designed an assay for the simultaneous measurement of cell surface phenotype, S-phase fraction, and DNA content by single laser instrumentation for the purpose of determining the labeling index (LI), duration of S-phase (Ts), and the potential doubling time (Tpot) of leukocyte subpopulations. The procedure was optimized with regard to: mode of bromodeoxyuridine (BrdUrd) incorporation, selection of suitable leukocyte differentiation antigens (LDAs) as well as PE-conjugated monoclonal antibodies (MoAbs) against myeloid cells, overnight permeabilization and fixation (paraformaldehyde 1% and 0.05% Nonidet P40), DNase I treatment (250 Kunitz units), concentration of FITC-conjugated anti-BrdUrd MoAb (dilution 1:5), and DNA staining with 7-amino-actinomycin (7-AAD) (10 microg/ml). We validated this assay by measuring LI, Ts, and Tpot repeatedly in four leukemic cell lines and found these to be stable (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 valuable data regarding the cell cycle kinetics of subsets of leukocytes in heterogeneous mixtures of hematopoietic cells.