ToxSci Advance Access published online on August 3, 2006
Toxicological Sciences, doi:10.1093/toxsci/kfl075
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1 Litron Laboratories, Rochester, NY
* To whom correspondence should be addressed. Accumulating evidence suggests that reticulocytes in the peripheral blood of rats may represent a suitable cell population for use in the micronucleus assay, despite the ability of the rat spleen to selectively remove micronucleated erythrocytes from the peripheral circulation. To evaluate the analytical performance of a previously described flow cytometric method (Torous et al., Tox. Sci., 74 (2003) 309-314) that may allow this assay to be conducted using peripheral blood in lieu of bone marrow sampling, we compared the sensitivity and performance characteristics of the flow cytometric technique with two established microscopy-based scoring methods. Peripheral blood samples from single Sprague-Dawley rats treated for six days with either vehicle or cyclophosphamide were prepared in replicate for scoring by the three methods at different laboratories. These blood-based measurements were compared to those derived from bone marrow specimens from the same animals, stained with acridine orange and scored by microscopy. Through the analysis of replicate specimens, inter- and intra-laboratory variability were evaluated for each method. Scoring reproducibility over time was also evaluated. These data support the premise that rat reticulocytes harvested from peripheral blood are a suitable cell population to assess genotoxicant-induced micronucleus formation. The inter-laboratory comparison provides evidence of the general robustness of the micronucleus endpoint using different analytical approaches. Furthermore, data presented herein demonstrate a clear advantage of flow cytometry-based scoring over microscopy--significantly lower inter- and intra-laboratory variation and higher statistical sensitivity.
Received February 2, 2006
Accepted July 31, 2006
Genetic Toxicology
Flow Cytometric Analysis of Micronuclei in Peripheral Blood Reticulocytes: I. Intra- and Inter-Laboratory Comparison with Microscopic Scoring
Stephen D. Dertinger 1, Michelle E. Bishop 2, James P. McNamee 3, Makoto Hayashi 4, Takayoshi Zuzuki 4, Norihide Asano 5, Madoka Nakajima 6, Junichiro Saito 7, Martha Moore 2, Dorothea K. Torous 1, and James T. MacGregor 8 *
2 U.S. Food and Drug Administration, National Center for Toxicological Research, Jefferson, AR
3 Health Canada, Ottawa, Ontario, Canada
4 National Institute of Health Sciences, Tokyo, Japan
5 Nitto Denko Corporation, Osaka, Japan
6 An-Pyo Center, Shizuoka, Japan
7 Astellas Pharma Inc., Tokyo, Japan
8 U.S. Food and Drug Administration, National Center for Toxicological Research, Rockville, MD; Present address: Toxicology Consulting Services, Arnold, MD
James T. MacGregor, E-mail: jtmacgregor{at}earthlink.net
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