An ECVAG inter-laboratory validation study of the comet assay: inter-laboratory and intra-laboratory variations of DNA strand breaks and FPG-sensitive sites in human mononuclear cells.
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Godschalk, Roger W. L.
van Schooten, Frederik-Jan
Jones, George D. D.
Higgins, Jennifer A.
Cooke, Marcus S.
Phillips, David H.
Routledge, Michael N.
Teixeira, João Paulo
López de Cerain, Adela
Collins, Andrew R.
MetadataShow full item record
AbstractThe alkaline comet assay is an established, sensitive method extensively used in biomonitoring studies. This method can be modified to measure a range of different types of DNA damage. However, considerable differences in the protocols used by different research groups affect the inter-laboratory comparisons of results. The aim of this study was to assess the inter-laboratory, intra-laboratory, sample and residual (unexplained) variations in DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites measured by the comet assay by using a balanced Latin square design. Fourteen participating laboratories used their own comet assay protocols to measure the level of DNA strand breaks and FPG-sensitive sites in coded samples containing peripheral blood mononuclear cells (PBMC) and the level of DNA strand breaks in coded calibration curve samples (cells exposed to different doses of ionising radiation) on three different days of analysis. Eleven laboratories found dose-response relationships in the coded calibration curve samples on two or three days of analysis, whereas three laboratories had technical problems in their assay. In the coded calibration curve samples, the dose of ionising radiation, inter-laboratory variation, intra-laboratory variation and residual variation contributed to 60.9, 19.4, 0.1 and 19.5%, respectively, of the total variation. In the coded PBMC samples, the inter-laboratory variation explained the largest fraction of the overall variation of DNA strand breaks (79.2%) and the residual variation (19.9%) was much larger than the intra-laboratory (0.3%) and inter-subject (0.5%) variation. The same partitioning of the overall variation of FPG-sensitive sites in the PBMC samples indicated that the inter-laboratory variation was the strongest contributor (56.7%), whereas the residual (42.9%), intra-laboratory (0.2%) and inter-subject (0.3%) variations again contributed less to the overall variation. The results suggest that the variation in DNA damage, measured by comet assay, in PBMC from healthy subjects is assay variation rather than variation between subjects.
CitationMutagenesis 2013, 28 (3):279-286
SponsorsThis work was supported by Environmental Cancer Risk, Nutrition and Individual Susceptibility (ECNIS), a network of excellence operating within the European Union 6th Framework Program, Priority 5: ‘Food Quality and Safety’ (Contract No 513943), the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS) and the Swedish Research Council (Vetenskapsrådet). ECVAG (the European Comet assay Validation Group) was created within the ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility) network of excellence, in order to validate the comet assay.
- Inter-laboratory variation in DNA damage using a standard comet assay protocol.
- Authors: Forchhammer L, Ersson C, Loft S, Möller L, Godschalk RW, van Schooten FJ, Jones GD, 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, Stępnik M, Komorowska M, Teixeira JP, Costa S, Corcuera LA, López de Cerain A, Laffon B, Valdiglesias V, Møller P
- Issue date: 2012 Nov
- Variation of DNA damage levels in peripheral blood mononuclear cells isolated in different laboratories.
- Authors: Godschalk RW, Ersson C, Stępnik M, Ferlińska M, Palus J, Teixeira JP, Costa S, Jones GD, Higgins JA, Kain J, Möller L, Forchhammer L, Loft S, Lorenzo Y, Collins AR, van Schooten FJ, Laffon B, Valdiglesias V, Cooke M, Mistry V, Karbaschi M, Phillips DH, Sozeri O, Routledge MN, Nelson-Smith K, Riso P, Porrini M, López de Cerain A, Azqueta A, Matullo G, Allione A, Møller P
- Issue date: 2014 Jul
- An ECVAG trial on assessment of oxidative damage to DNA measured by the comet assay.
- Authors: Johansson C, Møller P, Forchhammer L, Loft S, Godschalk RW, Langie SA, Lumeij S, Jones GD, Kwok RW, Azqueta A, Phillips DH, Sozeri O, Routledge MN, Charlton AJ, Riso P, Porrini M, Allione A, Matullo G, Palus J, Stepnik M, Collins AR, Möller L
- Issue date: 2010 Mar
- Intra-laboratory comet assay sample scoring exercise for determination of formamidopyrimidine DNA glycosylase sites in human mononuclear blood cell DNA.
- Authors: Møller P, Friis G, Christensen PH, Risom L, Plesner G, Kjaersgaard J, Vinzents P, Loft S, Jensen A, Tved M
- Issue date: 2004 Nov
- Variation in the measurement of DNA damage by comet assay measured by the ECVAG inter-laboratory validation trial.
- Authors: Forchhammer L, Johansson C, Loft S, Möller L, Godschalk RW, Langie SA, Jones GD, Kwok RW, 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
- Issue date: 2010 Mar
Showing items related by title, author, creator and subject.
An ECVAG trial on assessment of oxidative damage to DNA measured by the comet assay.Johansson, Clara; Moller, Peter; Forchhammer, Lykke; Loft, Steffen; Godschalk, Roger W. L.; Langie, Sabine A. S.; Lumeij, Stijn; Jones, George D. D.; Kwok, Rachel W. L.; Azqueta, Amaya; Phillips, David H.; Sozeri, Osman; Routledge, Michael N.; Charlton, Alexander J.; Riso, Patrizia; Porrini, Marisa; Allione, Alessandra; Matullo, Giuseppe; Palus, Jadwiga; Stepnik, Maciej; Collins, Andrew R.; Moller, Lennart (2010-03)The increasing use of single cell gel electrophoresis (the comet assay) highlights its popularity as a method for detecting DNA damage, including the use of enzymes for assessment of oxidatively damaged DNA. However, comparison of DNA damage levels between laboratories can be difficult due to differences in assay protocols (e.g. lysis conditions, enzyme treatment, the duration of the alkaline treatment and electrophoresis) and in the end points used for reporting results (e.g. %DNA in tail, arbitrary units, tail moment and tail length). One way to facilitate comparisons is to convert primary comet assay end points to number of lesions/10(6) bp by calibration with ionizing radiation. The aim of this study was to investigate the inter-laboratory variation in assessment of oxidatively damaged DNA by the comet assay in terms of oxidized purines converted to strand breaks with formamidopyrimidine DNA glycosylase (FPG). Coded samples with DNA oxidation damage induced by treatment with different concentrations of photosensitizer (Ro 19-8022) plus light and calibration samples irradiated with ionizing radiation were distributed to the 10 participating laboratories to measure DNA damage using their own comet assay protocols. Nine of 10 laboratories reported the same ranking of the level of damage in the coded samples. The variation in assessment of oxidatively damaged DNA was largely due to differences in protocols. After conversion of the data to lesions/10(6) bp using laboratory-specific calibration curves, the variation between the laboratories was reduced. The contribution of the concentration of photosensitizer to the variation in net FPG-sensitive sites increased from 49 to 73%, whereas the inter-laboratory variation decreased. The participating laboratories were successful in finding a dose-response of oxidatively damaged DNA in coded samples, but there remains a need to standardize the protocols to enable direct comparisons between laboratories.
Assessment and reduction of comet assay variation in relation to DNA damage: studies from the European Comet Assay Validation Group.Moller, Peter; Moller, Lennart; Godschalk, Roger W. L.; Jones, George D. D. (2010-03)The alkaline single cell gel electrophoresis (comet) assay has become a widely used method for the detection of DNA damage and repair in cells and tissues. Still, it has been difficult to compare results from different investigators because of differences in assay conditions and because the data are reported in different units. The European Comet Assay Validation Group (ECVAG) was established for the purpose of validation of the comet assay with respect to measures of DNA damage formation and its repair. The results from this inter-laboratory validation trail showed a large variation in measured level of DNA damage and formamidopyrimidine DNA glycosylase-sensitive sites but the laboratories could detect concentration-dependent relationships in coded samples. Standardization of the results with reference standards decreased the inter-laboratory variation. The ECVAG trail indicates substantial reliability for the measurement of DNA damage by the comet assay but there is still a need for further validation to reduce both assay and inter-laboratory variation.
Interlaboratory comparison of methodologies for the measurement of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine.Cooke, Marcus S.; Barregard, Lars; Mistry, Vilas; Potdar, Neelam; Rozalski, Rafal; Gackowski, Daniel; Siomek, Agnieszka; Foksinski, Marek; Svoboda, Peter; Kasai, Hiroshi; Konje, Justin C.; Sallsten, Gerd; Evans, Mark D.; Olinski, Ryszard (2009-03)Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) is widely used as a marker of oxidative stress. Here we report the comparison of two, distinct chromatographic assays with an enzyme-linked immunosorbent assay (ELISA). The chromatographic assays displayed good agreement (r =:0.89, p < 0.0001), whereas there was markedly worse, albeit still significant, agreement with the ELISA (high-pressure liquid chromatography followed by gas chromatography (HPLC-GC/MS), r = 0.43; HPLC with electrochemical detection (HPLC-EC), r = 0.56; p < 0.0001). Mean values differed significantly between the chromatographic assays and the ELISA (HPLC-GC/MS 3.86, HPLC-EC 4.20, ELISA 18.70 ng mg(-1) creatinine; p < 0.0001). While it is reassuring to note good agreement between chromatographic assays, this study reveals significant short-comings in the ELISA, which brings into question its continued use in its present form.