5-bromodeoxyuridine (BUdR) quenching of acridine orange fluorescence distinguishes cycling and non-cycling normal and malignant bone marrow cells in vitro.

Suppression of the green fluorescence of acridine orange by 5-bromodeoxyuridine incorporation into cellular DNA was measured by flow cytometry. Bone marrow cells from normal volunteers and patients with chronic myelogenous leukemia acute lymphocytic leukemia acute myelogenous leukemia and multiple myeloma were incubated with BUdR in vitro. By 24 h acridine orange stained cycling cells that had synthesized DNA in the presence of BUdR were differentiated from quiescent cells as a second peak with quenched green fluorescence (DNA). After 72 h in culture 11-65% of the G0/G1 cells from normal bone marrows and bone marrows with myeloid leukemia were identified as cycling in culture by the presence of a second peak with quenched green fluorescence. A greater percentage of cells with BUdR quenched AO fluorescence was associated of acridine orange was higher in the cycling cells that had synthesized DNA in the presence of BUdR than in the non-cycling G0/G1 cells. In one patient with AML there was quenching of the DNA fluorescence of the aneuploid population but not the diploid population indicating that the aneuploid leukemia cells were proliferating. In contrast in patients with multiple myeloma, the quenched fluorescence of the diploid cell population and not the aneuploid cells, indicated that the diploid cells were proliferating. The cells from patients with untreated ALL failed to proliferation prohibiting an in vivo assessment of growth. Although measurements of proliferation obtained by this method are clearly influenced by the cell's adaptation to culture, measurement of BUdR quenching of acridine orange fluorescence is technically feasible and can identify and allow characterization of the cycling population of cells.