Variation in the measurement of DNA damage by comet assay measured by the ECVAG inter-laboratory validation trial.
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.
Forchhammer
Lykke
L
Department of Public Health, Section of Environmental Health, University of Copenhagen, Copenhagen, Denmark.
Johansson
Clara
C
Loft
Steffen
S
Möller
Lennart
L
Godschalk
Roger W L
RW
Langie
Sabine A S
SA
Jones
George D D
GD
Kwok
Rachel W L
RW
Collins
Andrew R
AR
Azqueta
Amaya
A
Phillips
David H
DH
Sozeri
Osman
O
Stepnik
Maciej
M
Palus
Jadwiga
J
Vogel
Ulla
U
Wallin
Håkan
H
Routledge
Michael N
MN
Handforth
Catherine
C
Allione
Alessandra
A
Matullo
Giuseppe
G
Teixeira
João Paulo
JP
Costa
Solange
S
Riso
Patrizia
P
Porrini
Marisa
M
Møller
Peter
P
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
D043382:DNA-Formamidopyrimidine Glycosylase