ToxSci Advance Access originally published online on August 3, 2006
Toxicological Sciences 2006 94(1):83-91; doi:10.1093/toxsci/kfl075
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Published by Oxford University Press 2006.
Flow Cytometric Analysis of Micronuclei in Peripheral Blood Reticulocytes: I. Intra- and Interlaboratory Comparison with Microscopic Scoring
* Litron Laboratories, Rochester, New York 14623
U.S. Food and Drug Administration, National Center for Toxicological Research, Jefferson, Arkansas 72079
Health Canada, Ottawa, Ontario, Canada K1A 0L2
National Institute of Health Sciences, Tokyo 158-8501, Japan
¶ Nitto Denko Corporation, Osaka 567-8680, Japan
|| An-Pyo Center, Shizuoka 437-1213, Japan
||| Astellas Pharma Inc., Tokyo 174-8511, Japan
|||| U.S. Food and Drug Administration, National Center for Toxicological Research, Rockville, Maryland 21012
Received February 2, 2006; accepted July 31, 2006
Accumulating evidence suggests that reticulocytes (RETs) 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., 2003, Toxicol. Sci. 74, 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 6 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 intralaboratory variability were evaluated for each method. Scoring reproducibility over time was also evaluated. These data support the premise that rat RETs harvested from peripheral blood are a suitable cell population to assess genotoxicant-induced micronucleus formation. The interlaboratory 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 intralaboratory variation and higher statistical sensitivity.
Key Words: flow cytometric analysis; reticulocytes; micronucleus test; CD71.
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