| Source: |
Forchhammer, L, Johansson, C, Loft, S, Moller, L, Godschalk, R W, Langie, S A, Jones, G D, Kwok, R W, Collins, A R, Azqueta, A, 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 2010, 'Variation in the measurement of DNA damage by comet assay measured by the ECVAG inter-laboratory validation trial', Mutagenesis, vol. 25, no. 2, pp. 113-123. https://doi.org/10.1093/mutage/gep048 |
| Description: |
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. |