Variation in assessment of oxidatively damaged DNA in mononuclear blood cells by the comet assay with visual scoring

Abstract

The comet assay is popular for assessments of genotoxicity, but the comparison of results between studies is challenging because of differences in experimental procedures and reports of DNA damage in different units. We investigated the variation of DNA damage in mononuclear blood cells (MNBCs) measured by the comet assay with focus on the variation related to alkaline unwinding and electrophoresis time, number of cells scored, as well as the putative benefits of transforming the primary end points to common units by the use of reference standards and calibration curves. Eight experienced investigators scored pre-made slides of nuclei differently, but each investigator scored constantly over time. Scoring of 200 nuclei per treatment was associated with the lowest residual variation. Alkaline unwinding for 20 or 40 min and electrophoresis for 20 or 30 min yielded different dose-response relationships of cells exposed to gamma-radiation and it was possible to reduce the variation in oxidized purines in MNBCs from humans by adjusting the level of lesions with protocol-specific calibration curves. However, there was a difference in the level of DNA damage measured by different investigators and this variation could not be reduced by use of investigator-specific calibration curves. The mean numbers of lesions per 10(6) bp in MNBCs from seven humans were 0.23 [95% confidence interval (CI): 0.14-0.33] and 0.31 (95% CI: 0.20-0.55) for strand breaks (SBs) and oxidized guanines, respectively. In conclusion, our results indicate that inter-investigator difference in scoring is a strong determinant of DNA damage levels measured by the comet assay.

Comet assay: a reliable tool for the assessment of DNA damage in different models

“…Prospective cohort studies have not been conducted to find the predictive value of the Comet assay in human biomonitoring, further limiting its application (Moller 2006a). Cell to cell, gel to gel, culture to culture, animal to animal variability as well as use of various image analysis systems or visual scoring (Forchhammer et al 2008) and use of different Comet parameters, e.g., Olive tail moment and tail (%) DNA, are the other factors contributing to interlaboratory differences in the results.…”

E ect of mometasone furoate nasal spray on the DNA of nasal mucosal cells

“…Certain differences (agarose concentration, number of applied cells, electrophoresis time) at technical implementation can affect results, and there can be differences among results from different laboratories even if the same protocol is applied (43). These differences occur primarily because of the subjective nature of comet classification.…”

Relationship between Genotoxic Effects of Breast Cancer Treatments and Patient Basal DNA Integrity

“…However, the visual method can be influenced by the observer experience; thus, it is more subjective. 40,41 Although image analysis provides more objective information, Duez et al reported 4% variability among operators. 42 Thus, and as in most analytical techniques, it is important to maintain the same operator throughout the study to avoid these variations.…”