Mutagenesis, Vol. 16, No. 3, 189-195,
May 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
Evaluation of inter-scorer and inter-laboratory reliability of the mouse epididymal sperm aneuploidy (m-ESA) assay
Institut für Säugetiergenetik, GSF-Forschungszentrum für Umwelt und Gesundheit GmbH, Neuherberg, Ingolstädter Landstraße 1, D-85764, Neuherberg, Germany, 1 Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, PO Box 808, 7000 East Avenue, Livermore, CA 94550, USA, 2 Laboratory of Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA and 3 BioStatistics Branch, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA
The mouse epididymal sperm aneuploidy (mESA) assay using 3-chromosome fluorescence in situ hybridization (FISH) was recently developed for assessing the aneugenic potential of chemicals on male germ cells. This study was designed to identify the major technical factors that affect inter-scorer and inter-laboratory variability of the mESA assay. Two laboratories participated in this study (GSF and Lawrence Livermore National Laboratory, LLNL). Mice (102/ElxC3H/El) F1 were exposed in one laboratory (GSF) to vinblastine (VBL; single intraperitoneal injection of 0, 0.5, 1.0 or 2.0 mg/kg), one of the 10 priority compounds of the Commission of the European Communities (CEC) Aneuploidy Program. Twenty-two days later the mESA assay was applied to analyze sperm aneuploidy. In the initial evaluation, small but statistically significant differences were found between the two laboratories in baseline frequencies and there was also disagreement in the determination of a VBL aneuploid effect. Therefore, experiments were conducted to identify the sources of the inter-laboratory differences and technical factors that affected assay reliability and the VBL study was repeated. A harmonization experiment was conducted by bringing the microscope scorers from both laboratories to the same site (LLNL) for a cross-training exercise. Following this exercise, a second group of VBL-treated and control mice were evaluated, and we concluded that VBL is not a sperm aneugen. Our research has identified scoring criteria as the major source of inter-laboratory variation and emphasizes the importance of strict technical controls for the mESA assay, including controlling slide preparations for treatment-induced reductions in sperm count, coding of slides and selection of statistical tests. These considerations are particularly important for the interpretation of small effects (
2-fold) on sperm aneuploidy. Our findings suggest that 2-fold differences in frequencies can result from differences among scorers, samples and treatment groups, and are readily within the normal variation for the mESA assay. Such small differences should be viewed with caution until independently confirmed.
4 These authors contributed equally to this work
5 Present address: Department of Psychiatry, California Pacific Medical Center, San Francisco, CA 94120, USA
6 To whom correspondence should be addressed. Tel: +1 925 422 6296; Fax: +1 925 424 3130; Email: wyrobek1{at}llnl.gov
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