USE OF ACRIDINE-ORANGE IN - FLOW CYTOMETRIC EVALUATION OF ERYTHROPOIETIC CYTOTOXICITY

Cytotoxic insult to bone marrow frequently impairs the proliferating a nd maturational abilities of erythroid cells. Typically, a ratio of en ucleated, immature polychromatic erythrocytes (PCE) to mature normochr omatic erythrocytes (NCE) is used to assess cytotoxicity in the micron ucleus (MN) assay. The effects of cyclophosphamide (CP) on PCE/NCE rat io in rat bone marrow and spleen were assessed by a newly developed fl ow cytometric procedure using glutaraldehyde-fixed, acridine orange (A O)-stained cells, and compared to manual scoring of PCE/NCE in Wright stained slides. Comparison of methods showed that manual and flow cyto metric determination of PCE were not statistically different. Several other parameters of cytotoxicity could be simultaneously assessed beca use the method allowed use of unfractionated whole bone marrow/spleen cell samples. Absolute numbers of total nucleated cells (TNC), a ratio of TNC to total erythrocytes (TE), and determination of RNA content w ithin the PCE population demonstrated dose-and time-dependent effects with CP treatment. Shifts in RNA content were particularly sensitive, correctly identifying all CP-treated from control specimens, even in t hose samples where PCE/NCE ratio was similar. The AO methodology provi ded a more rapid, statistically-superior, and thorough approach in the assessment of bone marrow and spleen cytotoxicity than the convention al manual method of scoring PCE/NCE ratio alone. (C) 1998 Elsevier Sci ence B.V. All rights reserved.