Development of a novel flow cytometry-based approach for reticulocytes micronucleus test in rat peripheral blood.

The micronucleus test (MNT) is the most widely applied short-term assay to detect clastogens or spindle disruptors. The use of flow cytometry (FCM) has been reported for micronucleated erythrocytes scoring in peripheral blood. The aim of this study was to develop a novel and practical protocol for MNT in rat peripheral blood by FCM, with the method validation. CD71-fluorescein isothiocyanate and DRAQ5 were adopted for the fluorescent staining of proteins and DNA, respectively, to detect micronuclei. To validate the method, groups of male Sprague-Dawley rats (five per group) received two oral gavage doses at 0 and 24 h of six chemicals (four positive mutagens: ethyl methanesulphonate [EMS], cyclophosphamide [CP], colchicine [COL], and ethyl nitrosourea [ENU]; two nongenotoxic chemicals: sodium saccharin and eugenol). Blood samples were collected from the tail vein before and on the five continuous days after treatments; all of which were analyzed for micronuclei presence by both the manual (Giemsa staining) and FCM methods. The FCM-based method consistently demonstrated highly sensitive responses for micronucleus detection at all concentrations and all time points for EMS, CP, COL, and ENU. Sodium saccharin and eugenol could be identified as negative in this protocol. Results obtained with the FCM-based method correlated well with the micronucleus frequencies (r = 0.659-0.952), and the proportion of immature erythrocytes (r = 0.915-0.981) tested by Giemsa staining. The method reported here, with easy operation, low background, and requirement for a regular FCM, could be an efficient system for micronucleus scoring.