title: Variation in the measurement of DNA damage by comet assay measured by the ECVAG 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.
authors.affiliation: Department of Public Health, Section of Environmental Health, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Lykke
authors.initials: L
authors.lastname: Forchhammer
authors.forename: Clara
authors.initials: C
authors.lastname: Johansson
authors.forename: Steffen
authors.initials: S
authors.lastname: Loft
authors.forename: Lennart
authors.initials: L
authors.lastname: Möller
authors.forename: Roger W L
authors.initials: RW
authors.lastname: Godschalk
authors.forename: Sabine A S
authors.initials: SA
authors.lastname: Langie
authors.forename: George D D
authors.initials: GD
authors.lastname: Jones
authors.forename: Rachel W L
authors.initials: RW
authors.lastname: Kwok
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Collins
authors.forename: Amaya
authors.initials: A
authors.lastname: Azqueta
authors.forename: David H
authors.initials: DH
authors.lastname: Phillips
authors.forename: Osman
authors.initials: O
authors.lastname: Sozeri
authors.forename: Maciej
authors.initials: M
authors.lastname: Stepnik
authors.forename: Jadwiga
authors.initials: J
authors.lastname: Palus
authors.forename: Ulla
authors.initials: U
authors.lastname: Vogel
authors.forename: Håkan
authors.initials: H
authors.lastname: Wallin
authors.forename: Michael N
authors.initials: MN
authors.lastname: Routledge
authors.forename: Catherine
authors.initials: C
authors.lastname: Handforth
authors.forename: Alessandra
authors.initials: A
authors.lastname: Allione
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Matullo
authors.forename: João Paulo
authors.initials: JP
authors.lastname: Teixeira
authors.forename: Solange
authors.initials: S
authors.lastname: Costa
authors.forename: Patrizia
authors.initials: P
authors.lastname: Riso
authors.forename: Marisa
authors.initials: M
authors.lastname: Porrini
authors.forename: Peter
authors.initials: P
authors.lastname: Møller
mesh_terms: D002138:Calibration; D002478:Cells, Cultured; D020552:Comet Assay; D015925:Cryopreservation; D004249:DNA Damage / Q000528:radiation effects*; D043382:DNA-Formamidopyrimidine Glycosylase / Q000378:metabolism*; D006801:Humans; D007753:Laboratories / Q000592:standards*; D009000:Monocytes / Q000166:cytology / Q000378:metabolism* / Q000528:radiation effects; D015588:Observer Variation; D011839:Radiation, Ionizing; D012015:Reference Standards
chemical_list: D043382:DNA-Formamidopyrimidine Glycosylase