Inter-laboratory variation in DNA damage using a standard comet assay protocol

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Lykke Forchhammer, Clara Ersson, Steffen Loft, Lennart Möller, Roger W L Godschalk, Frederik J van Schooten, George D D Jones, Jennifer A Higgins, Marcus Cooke, Vilas Mistry, Mahsa Karbaschi, Andrew R Collins, Amaya Azqueta, David H Phillips, Osman Sozeri, Michael N Routledge, Kirsty Nelson-Smith, Patrizia Riso, Marisa Porrini, Giuseppe Matullo & 10 more Alessandra Allione, Maciej Steepnik, Magdalena Komorowska, João Paulo Teixeira, Solange Costa, Laura-Ana Corcuera, Adela López de Cerain, Blanca Laffon, Vanessa Valdiglesias, Peter Møller

There are substantial inter-laboratory variations in the levels of DNA damage measured by the comet assay. The aim of this study was to investigate whether adherence to a standard comet assay protocol would reduce inter-laboratory variation in reported values of DNA damage. Fourteen laboratories determined the baseline level of DNA strand breaks (SBs)/alkaline labile sites and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites in coded samples of mononuclear blood cells (MNBCs) from healthy volunteers. There were technical problems in seven laboratories in adopting the standard protocol, which were not related to the level of experience. Therefore, the inter-laboratory variation in DNA damage was only analysed using the results from laboratories that had obtained complete data with the standard comet assay protocol. This analysis showed that the differences between reported levels of DNA SBs/alkaline labile sites in MNBCs were not reduced by applying the standard assay protocol as compared with the laboratory's own protocol. There was large inter-laboratory variation in FPG-sensitive sites by the laboratory-specific protocol and the variation was reduced when the samples were analysed by the standard protocol. The SBs and FPG-sensitive sites were measured in the same experiment, indicating that the large spread in the latter lesions was the main reason for the reduced inter-laboratory variation. However, it remains worrying that half of the participating laboratories obtained poor results using the standard procedure. This study indicates that future comet assay validation trials should take steps to evaluate the implementation of standard procedures in participating laboratories.
Original languageEnglish
JournalMutagenesis
Volume27
Issue number6
Pages (from-to)665-72
Number of pages8
ISSN0267-8357
DOIs
Publication statusPublished - Nov 2012

    Research areas

  • Calibration, Comet Assay, DNA Damage, DNA-Formamidopyrimidine Glycosylase, Endpoint Determination, Humans, Laboratories, Leukocytes, Mononuclear, Linear Models

ID: 45158153