An ECVAG inter-laboratory validation study of the comet assay: inter-laboratory and intra-laboratory variations of DNA strand breaks and FPG-sensitive sites in human mononuclear cells.

The alkaline comet assay is an established, sensitive method extensively biomonitoring studies. This method can be modified to measure a range of different types of DNA damage. However, considerable differences in the used by different research groups affect the inter-laboratory results. The aim of this study was to assess the inter-laboratory, intra-laboratory, sample and residual (unexplained) variations in DNA breaks and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites the comet assay by using a balanced Latin square design. Fourteen laboratories used their own comet assay protocols to measure the level strand breaks and FPG-sensitive sites in coded samples containing blood mononuclear cells (PBMC) and the level of DNA strand breaks in calibration curve samples (cells exposed to different doses of ionising radiation) on three different days of analysis. Eleven laboratories dose-response relationships in the coded calibration curve samples on three days of analysis, whereas three laboratories had technical their assay. In the coded calibration curve samples, the dose of radiation, inter-laboratory variation, intra-laboratory variation and variation contributed to 60.9, 19.4, 0.1 and 19.5%, respectively, of the variation. In the coded PBMC samples, the inter-laboratory variation the largest fraction of the overall variation of DNA strand breaks the residual variation (19.9%) was much larger than the intra-laboratory and inter-subject (0.5%) variation. The same partitioning of the overall variation of FPG-sensitive sites in the PBMC samples indicated that the inter-laboratory variation was the strongest contributor (56.7%), residual (42.9%), intra-laboratory (0.2%) and inter-subject (0.3%) again contributed less to the overall variation. The results suggest variation in DNA damage, measured by comet assay, in PBMC from healthy is assay variation rather than variation between subjects.