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

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; Alessandra Allione; Maciej Steepnik; Magdalena Komorowska; João Paulo Teixeira; Solange Costa; Laura-Ana Corcuera; Adela López de Cerain; Blanca Laffon; Vanessa Valdiglesias; Peter Møller

doi:10.1093/mutage/ges032

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

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 MatulloAlessandra Allione, Maciej Steepnik, Magdalena Komorowska, João Paulo Teixeira, Solange Costa, Laura-Ana Corcuera, Adela López de Cerain, Blanca Laffon, Vanessa Valdiglesias, Peter Møller

50 Citations (Scopus)

Abstract

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 language	English
Journal	Mutagenesis
Volume	27
Issue number	6
Pages (from-to)	665-72
Number of pages	8
ISSN	0267-8357
DOIs	https://doi.org/10.1093/mutage/ges032
Publication status	Published - Nov 2012

Keywords

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

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10.1093/mutage/ges032

https://academic.oup.com/mutage/article-pdf/27/6/665/17087447/ges032.pdf

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Forchhammer, L., Ersson, C., Loft, S., Möller, L., Godschalk, R. W. L., van Schooten, F. J., Jones, G. D. D., Higgins, J. A., Cooke, M., Mistry, V., Karbaschi, M., Collins, A. R., Azqueta, A., Phillips, D. H., Sozeri, O., Routledge, M. N., Nelson-Smith, K., Riso, P., Porrini, M., ... Møller, P. (2012). Inter-laboratory variation in DNA damage using a standard comet assay protocol. Mutagenesis, 27(6), 665-72. https://doi.org/10.1093/mutage/ges032

Forchhammer, L, Ersson, C, Loft, S, Möller, L, Godschalk, RWL, van Schooten, FJ, Jones, GDD, Higgins, JA, Cooke, M, Mistry, V, Karbaschi, M, Collins, AR, Azqueta, A, Phillips, DH, Sozeri, O, Routledge, MN, Nelson-Smith, K, Riso, P, Porrini, M, Matullo, G, Allione, A, Steepnik, M, Komorowska, M, Teixeira, JP, Costa, S, Corcuera, L-A, López de Cerain, A, Laffon, B, Valdiglesias, V & Møller, P 2012, 'Inter-laboratory variation in DNA damage using a standard comet assay protocol', Mutagenesis, vol. 27, no. 6, pp. 665-72. https://doi.org/10.1093/mutage/ges032

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title = "Inter-laboratory variation in DNA damage using a standard comet assay protocol",

abstract = "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.",

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

author = "Lykke Forchhammer and Clara Ersson and Steffen Loft and Lennart M{\"o}ller and Godschalk, {Roger W L} and {van Schooten}, {Frederik J} and Jones, {George D D} and Higgins, {Jennifer A} and Marcus Cooke and Vilas Mistry and Mahsa Karbaschi and Collins, {Andrew R} and Amaya Azqueta and Phillips, {David H} and Osman Sozeri and Routledge, {Michael N} and Kirsty Nelson-Smith and Patrizia Riso and Marisa Porrini and Giuseppe Matullo and Alessandra Allione and Maciej Steepnik and Magdalena Komorowska and Teixeira, {Jo{\~a}o Paulo} and Solange Costa and Laura-Ana Corcuera and {L{\'o}pez de Cerain}, Adela and Blanca Laffon and Vanessa Valdiglesias and Peter M{\o}ller",

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T1 - Inter-laboratory variation in DNA damage using a standard comet assay protocol

AU - Forchhammer, Lykke

AU - Ersson, Clara

AU - Loft, Steffen

AU - Möller, Lennart

AU - Godschalk, Roger W L

AU - van Schooten, Frederik J

AU - Jones, George D D

AU - Higgins, Jennifer A

AU - Cooke, Marcus

AU - Mistry, Vilas

AU - Karbaschi, Mahsa

AU - Collins, Andrew R

AU - Azqueta, Amaya

AU - Phillips, David H

AU - Sozeri, Osman

AU - Routledge, Michael N

AU - Nelson-Smith, Kirsty

AU - Riso, Patrizia

AU - Porrini, Marisa

AU - Matullo, Giuseppe

AU - Allione, Alessandra

AU - Steepnik, Maciej

AU - Komorowska, Magdalena

AU - Teixeira, João Paulo

AU - Costa, Solange

AU - Corcuera, Laura-Ana

AU - López de Cerain, Adela

AU - Laffon, Blanca

AU - Valdiglesias, Vanessa

AU - Møller, Peter

PY - 2012/11

Y1 - 2012/11

N2 - 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.

AB - 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.

KW - Calibration

KW - Comet Assay

KW - DNA Damage

KW - DNA-Formamidopyrimidine Glycosylase

KW - Endpoint Determination

KW - Humans

KW - Laboratories

KW - Leukocytes, Mononuclear

KW - Linear Models

U2 - 10.1093/mutage/ges032

DO - 10.1093/mutage/ges032

M3 - Journal article

C2 - 22844078

SN - 0267-8357

VL - 27

SP - 665

EP - 672

JO - Mutagenesis

JF - Mutagenesis

IS - 6

ER -

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

Abstract

Keywords

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