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  • Title: Inter-laboratory validation of the in-vivo flow cytometric micronucleus analysis method (MicroFlow®) in China.
    Author: Chang Y, Zhou C, Huang F, Torous DK, Luan Y, Shi C, Wang H, Wang X, Wei N, Xia Z, Zhong Z, Zhang M, An F, Cao Y, Geng X, Jiang Y, Ju Q, Yu Y, Zhu J, Dertinger SD, Li B, Liao M, Yuan B, Zhang T, Yu J, Zhang Z, Wang Q, Ma J.
    Journal: Mutat Res Genet Toxicol Environ Mutagen; 2014 Sep 15; 772():6-13. PubMed ID: 25308541.
    Abstract:
    Although inter-laboratory validation efforts of the in-vivo micronucleus (MN) assay based on flow cytometry (FCM) have taken place in the EU and US, none have been organized in China. Therefore, an inter-laboratory study that included eight laboratories in China and one experienced reference laboratory in the US was coordinated to validate the in-vivo FCM MicroFlow(®) method to determine the frequency of micro-nucleated reticulocytes (MN-RETs) in rat blood. Assay reliability and reproducibility were evaluated with four known genotoxicants, and the results obtained with the FCM method were compared with the outcome of the traditional evaluation of bone-marrow micronuclei by use of microscopy. Each of the four chemicals was tested at three sites (two in China and the one US reference laboratory). After three consecutive daily exposures to a genotoxicant, blood and bone-marrow samples were obtained from rats 24h after the third dose. MN-RET frequencies were measured in 20,000 RET in blood by FCM, and micro-nucleated polychromatic erythrocyte (MN-PCE) frequencies were measured in 2,000 PCEs in bone marrow by microscopy. For both methods, each genotoxicant was shown to induce a statistically significant increase in the frequency of MN after treatment with at least one dose. Where more doses than one caused an increase, responses occurred in a dose-dependent manner. Spearman's correlation coefficient (rs) for FCM-based MN-RET vs microscopy-based MN-PCE measurements (eight experiments, 200 paired measurements) was 0.723, indicating a high degree of correspondence between methods and compartments. The rs value for replicate FCM MN-RET measurements performed at the eight collaborative laboratories was 0.940 (n=200), and between the eight FCM laboratories with the reference laboratory was 0.933 (n=200), suggesting that the automated method is very well transferable between laboratories. The FCM micronucleus analysis method is currently used in many countries worldwide, and these data support its use for evaluating the in-vivo genotoxic potential of test chemicals in China.
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