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

Abstract

The 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.

Hepatic Oxidative Stress, Genotoxicity and Vascular Dysfunction in Lean or Obese Zucker Rats

“…The level of FPG or hOGG1 sensitive sites was calculated as the difference in DNA damage between slides that had been treated with the FPG or hOGG1 enzyme and buffer. We used Ro19-8022 and light exposed monocytic THP-1 cells as reference controls, which has been used as control in comet assay validation trials [21–23]. Ro19-8022 was a gift from F. Hoffmann-La Roche (Basel, Switzerland).The levels of SB, FPG- and hOGG1-sensitive sites were 0.51 ± 0.31, 0.77 ± 0.19 and 0.48 ± 0.12 lesions/10 6 base pairs, respectively.…”

Look back in anger – what clinical studies tell us about preclinical work

“…There is promise of new methods, most obviously of the micronucleus test, which was formally validated and led to an OECD test guideline. There is some validation for the COMET assay (Ersson et al, 2013), which compared 27 samples in 14 laboratories using their own protocols; the variance observed was mainly between laboratories/protocols, i.e., 79%. Thus standardization of the COMET assay is essential, and we are desperately awaiting the results of the Japanese validation study for the COMET assay in vivo and in vitro .…”

On the search for an intelligible comet assay descriptor

“…A subsequent ECVAG trial looked into a standard comet assay protocol, but was only partly successful because some laboratories observed no difference in calibration curve samples and obtained negative values of FPG sensitive sites in human PBMCs (Forchhammer et al, 2012). ECVAG also showed that the overall variation of FPG-sensitive sites in the PBMCs could be partitioned into inter-laboratory (56.7%), residual (42.9%), intra-laboratory (0.2%) and inter-subject (0.3%) variation (Ersson et al, 2013). The most important finding in this trial was that the variation within each laboratory was relatively low.…”