Flow Cytometric Determination of Micronucleus Frequency

During the last two decades the micronucleus (MN) test has been extensively used as a genotoxicity screening tool of chemicals and in a variety of exploratory and mechanistic investigations. The MN is a biomarker for chromosomal damage or mitotic abnormalities, since it can originate from chromosome fragments or whole chromosomes that fail to be incorporated into daughter nuclei during mitosis (Fenech et al., Mutagenesis 26:125–132, 2011; Kirsch-Volders et al., Arch Toxicol 85:873–899, 2011). The simplicity of scoring, accuracy, amenability to automation by image analysis or flow cytometry, and readiness to be applied to a variety of cell types either in vitro or in vivo have made it a versatile tool that has contributed to a large extent in our understanding of key toxicological issues related to genotoxins and their effects at the cellular and organism levels. Recently, the final acceptance of the in vitro MN test guideline 487 (OECD Guideline for Testing of Chemicals, In vitro mammalian cell micronucleus test 487. In vitro mammalian cell micronucleus test (MNVIT). Organization for Economic Cooperation and Development, Paris, 2010) together with the standard in vivo MN test OECD guideline 474 (OECD Guideline for The Testing of Chemicals, Mammalian erythrocyte micronucleus test no. 474. Organization for Economic Cooperation and Development, Paris, 1997) will further position the assay as a key driver in the determination of the genotoxicity potential in exploratory research as well as in the regulatory environment. This chapter covers to some extent the protocol designs and experimental steps necessary for a successful performance of the MN test and an accurate analysis of the MN by the flow cytometry technique.