Outcomes of comet assay analysis using freshly prepared and cryopreserved leukocytes

Abstract

Maneli Mozaffarieh, Katarzyna Konieczka, Andreas Schoetzau, Josef Flammer

Comet assay analysis is a useful method which can be used to quantify systemic oxidative stress. Different laboratories use slightly different approaches when performing comet assay analysis. While some use cryopreserved leukocytes, others perform comet assays on freshly prepared cells. The purpose of this investigation was to compare comet assay results for single-stranded (ss)- DNA breaks in freshly prepared and cryopreserved leukocytes. A comparative quantification of ss-DNA breaks was performed on circulating leukocytes from two groups of ten healthy subjects (age- and sex-matched). Comet assay analysis of ss-DNA breaks was performed on all individuals on the same day. In the first group of ten subjects (group 1), leukocytes were isolated from the whole blood and comet assay was directly performed on the freshly isolated leukocytes. In the second group of ten subjects (group 2), blood samples were taken the day before comet assay analysis and the leukocytes were isolated and cryopreserved for 24 h. DNA damage (ss-DNA breaks) was quantified in the two groups, and the values of DNA breaks were calculated as comet tail moment. As result, group 2, where cryopreserved cells were used for comet assay analysis, had a significantly higher amount of ss-DNA damage in the circulating leukocytes than group1, in which freshly prepared leukocytes were used for analysis (tail moment: 3.28 AU in comparison to 0.38 AU, respectively). In conclusion, cryopreservation of leukocytes increases the number of ssDNA breaks, and therefore, affects the results of comet assay analysis. Performing comet assay analysis on freshly prepared leukocytes is an approach that is likely to yield more accurate results.