Isolation of leukocytes from frozen buffy coat for comet assay analysis of DNA damage

Frozen buffy coat fractions are often stored in human biomonitoring trials but their use for biomarker purposes has been limited. The purpose of the current study was to study whether frozen buffy coats can be used to monitor DNA damage levels. EDTA blood samples were provided from 9 healthy, non-smoking female volunteers, aged 26-48. Pre-existing DNA damage (strand breaks and oxidised purines) was measured with the comet assay in thawed resuspended buffy coat samples and washed leukocytes from these buffy coats, as well as resistance to DNA damage induced exogenously by H₂O₂ in the latter, and compared with damage measured in peripheral blood mononuclear cells isolated from fresh blood using percoll gradient centrifugation. Basal DNA damage levels (strand breaks) were significantly higher in the leukocytes isolated from frozen buffy coats in the untreated samples compared with peripheral blood mononuclear cells. However, the levels of strand breaks were still low (<4% tail DNA), indicating that little damage is caused by freezing or processing. Base oxidation was significantly higher in isolated buffy coat leukocytes than in peripheral blood mononuclear cells from fresh blood, but showed a good correlation (r = 0.67) between the two cell types. The correlation for strand breaks was stronger (r = 0.85). H₂O₂ induced DNA breaks in the cells both from fresh blood and buffy coats. The results indicate that buffy coat samples stored from cohort studies might be usefully analysed for DNA damage in retrospective epidemiological investigations. However, caution should be exercised when comparing the absolute levels of DNA damage in buffy coat leukocytes and peripheral blood mononuclear cells.