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Variation in the measurement of DNA damage by comet assay measured by the ECVAG inter-laboratory validation trial

Autores: Forchhammer, L.; Johansson, C.; Loft, S.; Möller, L.; Godschalk, R. W. L.; Langie, S. A. S.; Jones, G. D. D.; Kwok, R. W. L.; Collins, A. R.; Azqueta Oscoz, Amaya; Phillips, D. H.; Sozeri, O.; Stepnik, M.; Palus, J.; Vogel, U.; Wallin, H.; Routledge, M. N.; Handforth, C.; Allione, A.; Matullo, G.; Teixeira, J. P.; Costa, S.; Riso, P.; Porrini, M.; Moller, P.
Título de la revista: MUTAGENESIS
ISSN: 0267-8357
Volumen: 25
Número: 2
Páginas: 113 - 123
Fecha de publicación: 2010
The comet assay has become a popular method for the assessment of DNA damage in biomonitoring studies and genetic toxicology. However, few studies have addressed the issue of the noted inter-laboratory variability of DNA damage measured by the comet assay. In this study, 12 laboratories analysed the level of DNA damage in monocyte-derived THP-1 cells by either visual classification or computer-aided image analysis of pre-made slides, coded cryopreserved samples of cells and reference standard cells (calibration curve samples). The reference standard samples were irradiated with ionizing radiation (0-10 Gy) and used to construct a calibration curve to calculate the number of lesions per 10(6) base pair. All laboratories detected dose-response relationships in the coded samples irradiated with ionizing radiation (1.5-7 Gy), but there were overt differences in the level of DNA damage reported by the different laboratories as evidenced by an inter-laboratory coefficient of variation (CV) of 47%. Adjustment of the primary comet assay end points by a calibration curve prepared in each laboratory reduced the CV to 28%, a statistically significant reduction (P < 0.05, Levene's test). A large fraction of the inter-laboratory variation originated from differences in image analysis, whereas the intra-laboratory variation was considerably smaller than the variation between laboratories. In summary, adjustment of primary comet assay results by reference standards reduces inter-laboratory variation in the level of DNA damage measured by the alkaline version of the comet assay.