Mutagenesis Advance Access published online on September 27, 2005
Mutagenesis, doi:10.1093/mutage/gei053
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1 Litron Laboratories, 200 Canal View Boulevard, Rochester, NY 14623, USA
* To whom correspondence should be addressed. To confirm the performance and statistical power of a flow cytometric method for scoring micronucleated erythrocytes, reconstruction experiments were performed. For these investigations, peripheral blood erythrocytes from untreated mice, with a micronucleated erythrocyte frequency of
Received May 23, 2005
Revised July 21, 2005
Accepted July 22, 2005
Original article
Performance of flow cytometric analysis for the micronucleus assay--a reconstruction model using serial dilutions of malaria-infected cells with normal mouse peripheral blood
2 Toxicological Research Center, Nitto Denko Corporation, 1-1-2 Shimohozumi, Ibaraki, Osaka 567-8680, Japan
3 Division of Safety Information on Drug, Food and Chemicals, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
4 Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
Dorothea Torous, E-mail: dtorous{at}litronlabs.com
Abstract 
0.1% were combined with known quantities of Plasmodium berghei (malaria) infected mouse erythrocytes. These cells had an infected erythrocyte frequency of 0.7%, and mimic the DNA content of micronuclei (MN). For an initial experiment, samples with a range of MN/malaria (Mal) content were constructed and analysed in triplicate by flow cytometry until 2000, 20 000 and 200 000 total erythrocytes were acquired. In a second experiment, each specimen was analysed in triplicate until 2000, 20 000, 200 000 and 1 000 000 erythrocytes were acquired. As expected, the sensitivity of the assay to detect small changes in rare erythrocyte sub-population frequencies was directly related to the number of cells analysed. For example, when 2000 cells were scored, increases in MN/Mal frequencies of 3.9- or 2.7-fold were detected as statistically significant. When 200 000 cells were analysed, a 1.2-fold increase was detected. These data have implications for the experimental design and interpretation of micronucleus assays that are based on automated scoring procedures, since previously unattainable numbers of cells can now be readily scored.
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