[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.