CHARACTERIZATION OF BUSULFAN-INDUCED MYELOTOXICITY IN B6C3F(1) MICE USING FLOW-CYTOMETRY

Three experiments were carried out to investigate the myelotoxicity of busulphan in female B6C3F1 mice using the Technicon H1 and the Sysme x R-1000 flow cytometers, instruments which produce a full blood count and a differential leucocyte count, and an automated reticulocyte cou nt, respectively. In Experiment 1, a single dose of busulphan was admi nistered at levels from 0 to 60 mg/kg and blood parameters measured at day 14. In Experiment 2, four doses of busulphan, from 0 to 40 mg/kg, were given at fortnightly intervals, and blood samples taken at days 14 and 42. In the third experiment, a single dose of busulphan was giv en at 0, 35 or 45 mg/kg and sequential blood, marrow and spleen sample s examined up to day 10. In the first experiment there was a dose-rela ted depression in the numbers of all leucocyte types. Values for Hb, R BC and HCT were not affected, whereas MCV and percentage macrocytic er ythrocytes were increased, and MCHC was decreased, at high dose levels . Platelet numbers showed marked dose-related decreases. There were do se-related decreases in the numbers of all leucocyte types in Experime nt 2 at days 14 and 42. Large unstained cell (LUC) numbers were reduce d, and the mean neutrophil peroxidase index (MPXI) was increased, at h igh busulphan levels. Hb, RBC and HCT were reduced, whereas MCV, MCH a nd percentage macrocytic and percentage hypochromic erythrocytes were increased, in a dose-related fashion. Reticulocyte numbers showed a do se-related upward trend, but platelet counts illustrated a dose-relate d decrease, at days 14 and 42. In Experiment 3, busulphan caused a dep ression with a 'U'-shaped curve, in the numbers of monocytes, eosinoph ils, lymphocytes and neutrophils. Decreased values and 'U'-shaped curv es were also seen for Hb, RBC, HCT and reticulocyte counts. Reticulocy te fluorescence ratio analysis showed that the high fluorescence ratio (HFR) was affected first and most profoundly. Calculation of the reti culocyte maturation index also demonstrated a dose-related effect on t he earliest reticulocytes, and a 'rebound' effect. Total nucleated cel l counts of the spleen and femur showed decreasing cell numbers and 'U '-shaped responses with 45 mg/kg busulphan. This series of three exper iments has established the use of a 6 week dosing regimen, with busulp han administered at fortnightly intervals, to induce myelotoxicity in a range of haematological parameters in female B6C3F, mice. We conside r the use of the newly-developed flow cytometers and associated softwa re, and the measurement of 'non-standard parameters' such as LUC, HFR and MPXI, to be particularly effective in the charcterisation of these busulphan-induced haematological changes.