Browsing by Author "Higgins, J.A."
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- An ECVAG inter-laboratory validation study of the comet assay: inter-laboratory and intra-laboratory variation of DNA strand breaks and FPG-sensitive sites in human mononuclear cellsPublication . Ersson, C.; Møller, P.; Forchhammer, L.; Loft, S.; Azqueta, A.; Godschalk, R.W.; van Schooten, F.J.; Jones, G.D.; Higgins, J.A.; Cooke, M.S.; Mistry, V.; Karbaschi, M.; Phillips, D.H.; Sozeri, O.; Routledge, M.N.; Nelson-Smith, K.; Riso, P.; Porrini, M.; Matullo, G.; Allione, A.; Stepnik, M.; Ferlińska, M.; Teixeira, João Paulo; Costa, S.; Corcuera, L.A.; López de Cerain, A.; Laffon, B.; Valdiglesias, V.; Collins, A.R.; Möller, L.The alkaline comet assay is an established, sensitive method extensively used in biomonitoring studies. This method can be modified to measure a range of different types of DNA damage. However, considerable differences in the protocols used by different research groups affect the inter-laboratory comparisons of results. The aim of this study was to assess the inter-laboratory, intra-laboratory, sample and residual (unexplained) variations in DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites measured by the comet assay by using a balanced Latin square design. Fourteen participating laboratories used their own comet assay protocols to measure the level of DNA strand breaks and FPG-sensitive sites in coded samples containing peripheral blood mononuclear cells (PBMC) and the level of DNA strand breaks in coded calibration curve samples (cells exposed to different doses of ionising radiation) on three different days of analysis. Eleven laboratories found dose-response relationships in the coded calibration curve samples on two or three days of analysis, whereas three laboratories had technical problems in their assay. In the coded calibration curve samples, the dose of ionising radiation, inter-laboratory variation, intra-laboratory variation and residual variation contributed to 60.9, 19.4, 0.1 and 19.5%, respectively, of the total variation. In the coded PBMC samples, the inter-laboratory variation explained the largest fraction of the overall variation of DNA strand breaks (79.2%) and the residual variation (19.9%) was much larger than the intra-laboratory (0.3%) 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%), whereas the residual (42.9%), intra-laboratory (0.2%) and inter-subject (0.3%) variations again contributed less to the overall variation. The results suggest that the variation in DNA damage, measured by comet assay, in PBMC from healthy subjects is assay variation rather than variation between subjects.
- Variation of DNA damage levels in peripheral blood mononuclear cells isolated in different laboratoriesPublication . Godschalk, R.W.; Ersson, C.; Stępnik, M.; Ferlińska, M.; Palus, J.; Teixeira, João Paulo; Costa, S.; Jones, G.D.; Higgins, J.A.; Kain, J.; Möller, L.; Forchhammer, L.; Loft, S.; Lorenzo, Y.; Collins, A.R.; van Schooten, F.J.; Laffon, B.; Valdiglesias, V.; Cooke, M.; Mistry, V.; Karbaschi, M.; Phillips, D.H.; Sozeri, O.; Routledge, M.N.; Nelson-Smith, K.; Riso, P.; Porrini, M.; López de Cerain, A.; Azqueta, A.; Matullo, G.; Allione, A.; Møller, P.This study investigated the levels of DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG) sensitive sites, as assessed by the comet assay, in peripheral blood mononuclear cells (PBMC) from healthy women from five different countries in Europe. The laboratory in each country (referred to as 'centre') collected and cryopreserved PBMC samples from three donors, using a standardised cell isolation protocol. The samples were analysed in 13 different laboratories for DNA damage, which is measured by the comet assay. The study aim was to assess variation in DNA damage in PBMC samples that were collected in the same way and processed using the same blood isolation procedure. The inter-laboratory variation was the prominent contributor to the overall variation. The inter-laboratory coefficient of variation decreased for both DNA strand breaks (from 68 to 26%) and FPG sensitive sites (from 57 to 12%) by standardisation of the primary comet assay endpoint with calibration curve samples. The level of DNA strand breaks in the samples from two of the centres (0.56-0.61 lesions/10(6) bp) was significantly higher compared with the other three centres (0.41-0.45 lesions/10(6) bp). In contrast, there was no difference between the levels of FPG sensitive sites in PBMC samples from healthy donors in the different centres (0.41-0.52 lesion/10(6) bp).