COMPARATIVE MICRONUCLEUS QUANTITATION IN PRECOLUMN AND POSTCOLUMN FRACTIONATED MOUSE BONE-MARROW BY MANUAL AND FLOW METHODS

In the present study, cellulose-column fractionation methodology which has been used to eliminate nucleated cells in bone marrow was verified for its usefulness in micronucleus analysis and compared to standard smear methodology using cyclophosphamide as the test compound. Also, the possibility of using column-fractionated cells in the evaluation of micronucleus frequency by flow cytometry has been explored and comparative results are reported. The results indicated that column fractionation was effective in removing nucleated cells from mouse bone marrow and provided clean preparations of polychromatic and normochromatic erythrocytes (PCEs and NCEs). An initial comparison of manual scoring of cyclophosphamide-induced (10, 20 or 40 mg/kg) micronucleus frequency between standard whole bone-marrow smear and column-fractionated cytospun smears from the same animals showed comparable results. In a definitive study, manual scoring of micronuclei in whole bone marrow was compared with the column-fractionated cell preparations quantified manually and using flow cytometry. Statistically significant positive dose-related trends were detected with all 3 methods, with each treatment group having significantly elevated micronucleated PCEs (MNPCEs) compared to the control group. The 3 methods provided comparable MNPCE values for the lower dose groups but diverged somewhat for the high dose group. The flow method yielded similar individual animal variability in the data when compared to the other two methods. These results support the use of column fractionation in the enumeration of MNPCEs and indicate that coupling this technique with flow cytometry may provide a rapid and sensitive method for the conduct of mouse bone-marrow micronucleus studies.