Abstract: To overcome time-consuming, laborious, subjective and other shortcomings of the traditional microscopic examination method of micronuclei (MN), we established flow cytometry (FCM) method for automatic analysis of micronuclei in mouse peripheral blood stained with anti-CD71-FITC, anti-CD61-PE and PI. METHOD: Mice received a single oral dose of 0, 20, 40, 80 mg/kg cyclophosphamide and blood was sampled at different times before and after treatment. Additional mice were orally treated with 40 mg/kg cyclophosphamide for 5 consecutive days and blood was collected before and after treatment at 24 h interval. Blood cells were fixed in -80 ℃ methanol and stained with anti-CD71-FITC, anti-CD61-PE, RNase and PI. After instrument was calibrated with malaria-infected erythrocytes as a biological standard, the frequencies of micronucleated CD71-positive reticulocytes were measured by FCM. RESULTS: The highest frequency of MN-RET was observed at 48 h after single treatment, and MN-RET showed dose-dependent accumulation (r=0.9849, P<0.01). These data acquired by FCM demonstrated that the steady-state of MN-RET was attained approximately 24 h after the second administration with 5 consecutive days treatment. Parallel analysis of micronucleus induction in peripheral blood by FCM and bone marrow using traditional microscopy-based method showed concordant results (r=0.9212, P<0.01). CONCLUSION: The three-color flow cytometric assessment of MN-RET in mouse peripheral blood can be considered as an acceptable substitute to microscopy-based analysis in mouse bone marrow micronucleus test in vivo.

Key words: CD71, flow cytometry, reticulocyte, micronucleus, cyclophosphamide