Flow cytometric chemosensitivity analysis of blasts from patients with acute myeloblastic leukemia and myelodysplastic syndromes: The use of 7AAD with antibodies to CD45 or CD34

Background: Flow cytometry is potentially suited to the chemosensitivity analysis of peripheral blood or bone marrow subpopulations in patients with leukaemia and myelodysplastic syndromes. Methods: The use of the fluorescent dye 7‐amino‐actinomycin (7AAD) on unfixed cells to measure loss of viability at a range of cytosine arabinoside (ara‐C) doses was evaluated. A six‐tube flow cytometric assay for measuring the sensitivity to ara‐C of CD45/side‐scatter‐gated or of CD34‐positive leukemic blasts with 7AAD was established, using fixed stained normal mononuclear cells as an internal standard for quantitation of viable cells following culture. Results: 7AAD dose response curves for 10 patients with acute myeloblastic leukemia (AML) showed a wide range of sensitivities at 2.5–5 μM araC (3.7–97%, mean 54% of control cell viability at 2.5 μM and 4.1–94.6 %, mean 27% at 5 μM). Parallel assays for ATP bioluminescence agreed reasonably well with the 7AAD method, r s = 0.78. The chemosensitivity of CD45/SSC‐gated blast cells at 2.5 μM araC showed no consistent relationship with the ungated cell populations, such that CD45/SSC‐gated blast sensitivity of seven samples ranged from 86% more to 38% less than that of the total population. Similarly, the chemosensitivities of the CD34‐gated subpopulations ranged from 51% more to 78% less than those of the total populations. Conclusions: These results emphasize the necessity of measuring the chemosensitivity of the population of interest rather than of the sample as a whole in heterogeneous clinical material. Cytometry 37:308–313, 1999. © 1999 Wiley‐Liss, Inc.