Collection and storage of human white blood cells for analysis of DNA damage and repair activity using the comet assay in molecular epidemiology studies

Abstract

DNA damage and repair activity are often assessed in blood s#38les from humans in different types of molecular epidemiology studies. However, it is not always feasible to analyse the s#38les on the day of collection without any type of storage. For instance, certain studies use repeated s#38ling of cells from the same subject or s#38les from different subjects collected at different time-points, and it is desirable to analyse all these s#38les in the same comet assay experiment. In addition, flawless comet assay analyses on frozen s#38les opens up for the possibility of using this technique on biobank material. In this article we discuss the use of cryopreserved peripheral blood mononuclear cells (PBMCs), buffy coat (BC) and whole blood (WB) for analysis of DNA damage and repair using the comet assay. The published literature and the authors’ experiences indicate that various types of blood s#38les can be cryopreserved with only minor effect on the basal level of DNA damage. There is evidence to suggest that WB and PBMCs can be cryopreserved for several years without much effect on the level of DNA damage. However, care should be taken when cryopreserving WB and BCs. It is possible to use either fresh or frozen s#38les of blood cells, but results from fresh and frozen cells should not be used in the same dataset. The article outlines detailed protocols for the cryopreservation of PBMCs, BCs and WB s#38les.

Biomarkers of DNA Oxidation Products: Links to Exposure and Disease in Public Health Studies

“…This trial was superseded by the European Comet Assay Validation Group (ECVAG) . ECVAG was superseded by the comet assay in human biomonitoring (hCOMET) project that has disseminated recommendations on technical procedures, , molecular epidemiological studies, and reporting of results . The hCOMET project also included a ring trial on the Fpg-modified comet assay, aiming at testing KBrO 3 as positive assay control …”

“…It is possible to cryopreserve tissue samples for later analysis of oxidatively damaged DNA using chromatographic techniques. PBMCs are cryopreserved in a medium containing an agent that prohibits the formation of ice crystals (e.g., dimethyl sulfoxide) and serum . It has been a limiting factor in public health research that biobank-based cohort studies may only have stored whole blood or buffy coat .…”

“…Still, it should be mentioned that there is uncertainty about the stability of DNA damage levels in whole blood and buffy coat samples after long-term storage, as the level of Fpg-sensitive sites might increase . The effect of cryopreservation on background levels of oxidatively damaged DNA in isolated PBMCs is not resolved; however, evidence suggests that it is stable, and careful freezing and thawing procedures reduce the risk of artifactual generation of DNA damage …”

“…PBMCs are cryopreserved in a medium containing an agent that prohibits the formation of ice crystals (e.g., dimethyl sulfoxide) and serum. 67 based cohort studies may only have stored whole blood or buffy coat. 62 However, the enzyme-modified comet assay uses relatively few cells, and it is possible to use small volumes of whole blood (i.e., 200−400 μL) that have been cryopreserved.…”

“…69 The effect of cryopreservation on background levels of oxidatively damaged DNA in isolated PBMCs is not resolved; however, evidence suggests that it is stable, and careful freezing and thawing procedures reduce the risk of artifactual generation of DNA damage. 67 ■ BACKGROUND LEVELS OF OXIDATIVELY…”