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 H*1 and the Sysmex R-1000 flow cytometers, instruments which produce a full blood count and a differential leucocyte count, and an automated reticulocyte count, respectively. In Experiment 1, a single dose of busulphan was administered 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 given at 0, 35 or 45 mg/kg and sequential blood, marrow and spleen samples examined up to day 10. In the first experiment there was a dose-related depression in the numbers of all leucocyte types. Values for Hb, RBC and HCT were not affected, whereas MCV and percentage macrocytic erythrocytes were increased, and MCHC was decreased, at high dose levels. Platelet numbers showed marked dose-related decreases. There were dose-related decreases in the numbers of all leucocyte types in Experiment 2 at days 14 and 42. Large unstained cell (LUC) numbers were reduced, and the mean neutrophil peroxidase index (MPXI) was increased, at high busulphan levels. Hb, RBC and HCT were reduced, whereas MCV, MCH and percentage macrocytic and percentage hypochromic erythrocytes were increased, in a dose-related fashion. Reticulocyte numbers showed a dose-related upward trend, but platelet counts illustrated a dose-related decrease, at days 14 and 42. In Experiment 3, busulphan caused a depression with a 'U'-shaped curve, in the numbers of monocytes, eosinophils, lymphocytes and neutrophils. Decreased values and 'U'-shaped curves were also seen for Hb, RBC, HCT and reticulocyte counts. Reticulocyte fluorescence ratio analysis showed that the high fluorescence ratio (HFR) was affected first and most profoundly. Calculation of the reticulocyte maturation index also demonstrated a dose-related effect on the earliest reticulocytes, and a 'rebound' effect. Total nucleated cell counts of the spleen and femur showed decreasing cell numbers and 'U'-shaped responses with 45 mg/kg busulphan. This series of three experiments has established the use of a 6 week dosing regimen, with busulphan administered at fortnightly intervals, to induce myelotoxicity in a range of haematological parameters in female B6C3F, mice. We consider the use of the newly-developed flow cytometers and associated software, 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.