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

Lykke Forchhammer; Clara Johansson; Steffen Loft; Lennart Möller; Roger W L Godschalk; Sabine A S Langie; George D D Jones; Rachel W L Kwok; Andrew R Collins; Amaya Azqueta; David H Phillips; Osman Sozeri; Maciej Stepnik; Jadwiga Palus; Ulla Vogel; Håkan Wallin; Michael N Routledge; Catherine Handforth; Alessandra Allione; Giuseppe Matullo; João Paulo Teixeira; Solange Costa; Patrizia Riso; Marisa Porrini; Peter Møller

doi:10.1093/mutage/gep048

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

Lykke Forchhammer, Clara Johansson, Steffen Loft, Lennart Möller, Roger W L Godschalk, Sabine A S Langie, George D D Jones, Rachel W L Kwok, Andrew R Collins, Amaya Azqueta, David H Phillips, Osman Sozeri, Maciej Stepnik, Jadwiga Palus, Ulla Vogel, Håkan Wallin, Michael N Routledge, Catherine Handforth, Alessandra Allione, Giuseppe MatulloJoão Paulo Teixeira, Solange Costa, Patrizia Riso, Marisa Porrini, Peter Møller

111 Citations (Scopus)

Abstract

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.

Original language	English
Journal	Mutagenesis
Volume	25
Issue number	2
Pages (from-to)	113-23
Number of pages	10
ISSN	0267-8357
DOIs	https://doi.org/10.1093/mutage/gep048
Publication status	Published - Mar 2010

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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, A., Phillips, D. H., Sozeri, O., Stepnik, M., Palus, J., Vogel, U., Wallin, H., Routledge, M. N., Handforth, C., Allione, A., ... Møller, P. (2010). Variation in the measurement of DNA damage by comet assay measured by the ECVAG{dagger} inter-laboratory validation trial. Mutagenesis, 25(2), 113-23. https://doi.org/10.1093/mutage/gep048

Forchhammer, L, Johansson, C, Loft, S, Möller, L, Godschalk, RWL, Langie, SAS, Jones, GDD, Kwok, RWL, Collins, AR, Azqueta, A, Phillips, DH, Sozeri, O, Stepnik, M, Palus, J, Vogel, U, Wallin, H, Routledge, MN, Handforth, C, Allione, A, Matullo, G, Teixeira, JP, Costa, S, Riso, P, Porrini, M & Møller, P 2010, 'Variation in the measurement of DNA damage by comet assay measured by the ECVAG{dagger} inter-laboratory validation trial', Mutagenesis, vol. 25, no. 2, pp. 113-23. https://doi.org/10.1093/mutage/gep048

@article{afd06650269411df8ed1000ea68e967b,

title = "Variation in the measurement of DNA damage by comet assay measured by the ECVAG{dagger} inter-laboratory validation trial",

abstract = "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.",

author = "Lykke Forchhammer and Clara Johansson and Steffen Loft and Lennart M{\"o}ller and Godschalk, {Roger W L} and Langie, {Sabine A S} and Jones, {George D D} and Kwok, {Rachel W L} and Collins, {Andrew R} and Amaya Azqueta and Phillips, {David H} and Osman Sozeri and Maciej Stepnik and Jadwiga Palus and Ulla Vogel and H{\aa}kan Wallin and Routledge, {Michael N} and Catherine Handforth and Alessandra Allione and Giuseppe Matullo and Teixeira, {Jo{\~a}o Paulo} and Solange Costa and Patrizia Riso and Marisa Porrini and Peter M{\o}ller",

year = "2010",

month = mar,

doi = "10.1093/mutage/gep048",

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journal = "Mutagenesis",

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T1 - Variation in the measurement of DNA damage by comet assay measured by the ECVAG{dagger} inter-laboratory validation trial

AU - Forchhammer, Lykke

AU - Johansson, Clara

AU - Loft, Steffen

AU - Möller, Lennart

AU - Godschalk, Roger W L

AU - Langie, Sabine A S

AU - Jones, George D D

AU - Kwok, Rachel W L

AU - Collins, Andrew R

AU - Azqueta, Amaya

AU - Phillips, David H

AU - Sozeri, Osman

AU - Stepnik, Maciej

AU - Palus, Jadwiga

AU - Vogel, Ulla

AU - Wallin, Håkan

AU - Routledge, Michael N

AU - Handforth, Catherine

AU - Allione, Alessandra

AU - Matullo, Giuseppe

AU - Teixeira, João Paulo

AU - Costa, Solange

AU - Riso, Patrizia

AU - Porrini, Marisa

AU - Møller, Peter

PY - 2010/3

Y1 - 2010/3

N2 - 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.

AB - 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.

U2 - 10.1093/mutage/gep048

DO - 10.1093/mutage/gep048

M3 - Journal article

C2 - 19910383

SN - 0267-8357

VL - 25

SP - 113

EP - 123

JO - Mutagenesis

JF - Mutagenesis

IS - 2

ER -

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

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