Rapid detection of mutagen induced micronucleated erythrocytes by flow cytometry
The micronucleus test is a cytogenetic method for the screening of mutagens and carcinogens which exhibit clastogenic mechanisms of action. After application of clastogenic agents chromosomal fragments or even whole chromsomes can remain as conspicuous structures (micronuclei) in a small fraction of anucleated polychromatic erythrocytes which can be visually scored using a microscope following staining with May-Grünwald-Giemsa solutions. These time-consuming, painstaking, and tedious manual evaluations are often sources of unreliability and uncertainty. Here, a fluorescence technique is presented which applies DNA and protein fluorochromes to discriminate normal anucleated erythrocytes from micronucleated erythrocytes using a fluorescence microscope. This method is particularly tailored to be applied to flow cytometric instrumentation. Data obtained manually and automatically in flow show a strong linear correlation with high significance (r=0.96) as far as the percentage of micronucleated erythrocytes as an indicator for the mutagenicity of a given drug is concerned. These results have been obtained by means of the established clastogens cyclophosphamide and mitomycin C.