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ToxSci Advance Access originally published online on August 3, 2006
Toxicological Sciences 2006 94(1):92-107; doi:10.1093/toxsci/kfl076
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Published by Oxford University Press 2006.

Flow Cytometric Analysis of Micronuclei in Peripheral Blood Reticulocytes: II. An Efficient Method of Monitoring Chromosomal Damage in the Rat

James T. MacGregor*,1, Michelle E. Bishop{dagger}, James P. McNamee{ddagger}, Makoto Hayashi§, Norhide Asano, Akihiro Wakata||, Madoka Nakajima|||, Junichiro Saito||||, Anane Aidoo{dagger}, Martha M. Moore{dagger} and Stephen D. Dertinger#

* FDA National Center for Toxicological Research, Rockville, Maryland 21012 {dagger} FDA National Center for Toxicological Research, Jefferson, Arkansas 72079 {ddagger} Health Canada, Ottawa, Ontario, Canada K1A 0L2 § National Institute of Health Sciences, Tokyo 158-8501, Japan Nitto Denko Corporation, Osaka 567-8680, Japan || Astellas Pharma, Inc., Osaka 532-8514, Japan ||| An-Pyo Center, Shizuoka 437-1213, Japan |||| Astellas Pharma Inc., Tokyo 174-8511, Japan # Litron Laboratories, Rochester, New York 14623

Received February 2, 2006; accepted July 31, 2006

We have evaluated a flow cytometric method that allows assessment of micronucleated reticulocytes (MN-RETs) in microliter quantities of peripheral blood and compared results using this assay with those of established microscopic methods of scoring bone marrow and peripheral blood from rats treated with well-characterized genotoxic agents. Young reticulocytes (RETs) are labeled with FITC–anti-CD71 (transferrin receptor) and micronuclei with propidium iodide (with RNase treatment). Red blood cells parasitized with Plasmodia serve as a calibration standard for DNA content. Microscopic scoring used acridine orange (AO) staining of methanol-fixed slides or supravital AO staining. The effect of the rat spleen on the parameters evaluated was determined by comparing age- and sex-matched normal and splenectomized rats treated with cyclophosphamide, cis-platin, or vinblastine under treatment conditions that established a steady-state frequency of MN-RETs in the bone marrow and peripheral blood compartments. The data demonstrate the sensitivity and reproducibility of the flow cytometric assay in the Sprague-Dawley rat, and comparative studies using identical blinded samples at multiple laboratories show that inter- and intra-laboratory reproducibility is much higher with the flow method than with the microscopic methods currently employed for regulatory studies. A significant effect of splenic selection against genotoxicant-induced MN-RETs was observed with each of the three scoring methodologies, despite the fact that the flow and supravital AO techniques restrict analysis to the youngest fraction of RETs. The high precision of flow-based measurements also demonstrated a slight but statistically significant level of selection against spontaneously arising MN-RET. Despite these spleen effects, assay sensitivity for blood-based analyses was maintained by the flow method as it was shown to have superior counting statistics, lower variability, and higher sensitivity than manual scoring. The data suggest that flow cytometric assessment of micronucleus induction can be integrated into routine toxicity testing, eliminating the need for a separate bioassay.

Key Words: flow cytometric analysis; micronucleated reticulocytes; erythrocytes; cyclophosphamide, cis-platin; vinblastine.


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