TY - JOUR

T1 - Variation in assessment of oxidatively damaged DNA in mononuclear blood cells by the comet assay with visual scoring

AU - Forchhammer, Lykke

AU - Bräuner, Elvira Vaclavik

AU - Folkmann, Janne Kjaersgaard

AU - Danielsen, Pernille Høgh

AU - Nielsen, Claus

AU - Jensen, Annie

AU - Loft, Steffen

AU - Friis, Gitte

AU - Møller, Peter

N1 - Keywords: Cell Nucleus; Comet Assay; DNA; DNA Damage; Data Interpretation, Statistical; Humans; Leukocytes, Mononuclear; Observer Variation; Oxidative Stress

PY - 2008

Y1 - 2008

N2 - 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.

AB - 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.

U2 - 10.1093/mutage/gen006

DO - 10.1093/mutage/gen006

M3 - Journal article

C2 - 18326868

VL - 23

SP - 223

EP - 231

JO - Mutagenesis

JF - Mutagenesis

SN - 0267-8357

IS - 3

ER -