Influence of counting methodology on erythrocyte ratios in the mouse micronucleus test
Article first published online: 6 DEC 2012
Copyright © 2012 Wiley Periodicals, Inc.
Environmental and Molecular Mutagenesis
Volume 54, Issue 3, pages 222–228, April 2013
How to Cite
LeBaron, M. J., Schisler, M. R., Torous, D. K., Dertinger, S. D. and Gollapudi, B. B. (2013), Influence of counting methodology on erythrocyte ratios in the mouse micronucleus test. Environ. Mol. Mutagen., 54: 222–228. doi: 10.1002/em.21754
- Issue published online: 12 MAR 2013
- Article first published online: 6 DEC 2012
- Manuscript Accepted: 29 OCT 2012
- Manuscript Revised: 24 SEP 2012
- Manuscript Received: 12 MAY 2012
The mammalian erythrocyte micronucleus test is widely used to investigate the potential interaction of a test substance with chromosomes or mitotic apparatus of replicating erythroblasts. In addition to the primary endpoint, micronucleated erythrocyte frequency, the proportion of immature erythrocytes is measured to assess the influence of treatment on erythropoiesis. The guideline recommendation for an acceptable limit of the immature erythrocyte fraction of not < 20% of the controls was based on traditional scoring methods that consider RNA content. Flow-based sample analysis (e.g., MicroFlow®) characterizes a subpopulation of RNA-containing reticulocytes (RETs) based on CD71 (transferrin receptor) expression. As CD71+ cells represent a younger cohort of RETs, we hypothesized that this subpopulation may be more responsive than the RNA+ fraction for acute exposures. This study evaluated RET population in the peripheral blood of two strains of mice treated by oral gavage with three clastogens (cyclophosphamide, N-ethyl-N-nitrosourea, and methyl methanesulfonate). Although CD71+ frequencies correlated with RNA-based counts, the relative treatment-related reductions were substantially greater. Accordingly, when using the flow cytometry-based CD71+ values for scoring RETs in an acute treatment design, it is suggested that a target value ≥ 5% CD71+ reticulocytes (i.e., 95% depression in reticulocytes proportion) be considered as acceptable for a valid assay. Environ. Mol. Mutagen. 54:222–228, 2013. © 2013 Wiley Periodicals, Inc.