Impact of 12-month cryopreservation on endogenous DNA damage in whole blood and isolated mononuclear cells evaluated by the comet assay

Mirko Marino,Letizia Gigliotti,Marisa Porrini,Cristian Del Bo,Peter Møller,Patrizia Riso

doi:10.1038/s41598-020-79670-8

Mirko Marino, Letizia Gigliotti + Show 4 more

Open Access

https://doi.org/10.1038/s41598-020-79670-8

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Journal: Scientific Reports	Publication Date: Jan 11, 2021
Citations: 10	License type: open-access

Affiliation: University of Milan, University of Copenhagen

Abstract

The comet assay is an electrophoretic technique used to assess DNA damage, as a marker of genotoxicity and oxidative stress, in tissues and biological samples including peripheral blood mononuclear cells (PBMCs) and whole blood (WB). Although numerous studies are performed on stored samples, the impact of cryopreservation on artifactual formation of DNA damage is not widely considered. The present study aims to evaluate the impact of storage at different time-points on the levels of strand breaks (SBs) and formamidopyrimidine DNA glycosylase (Fpg)-sensitive sites in isolated PBMCs and WB. Samples were collected, aliquoted and stored at − 80 °C. DNA damage was analyzed on fresh samples, and subsequently on frozen samples every 2 months up to a year. Results have shown no changes in DNA damage in samples of PBMCs and WB stored for up to 4 months, while a significant increase in SBs and Fpg-sensitive sites was documented starting from 6-month up to 12-month storage of both the samples. In addition, fresh and frozen WB showed higher basal levels of DNA damage compared to PBMCs. In conclusion, WB samples show high levels of DNA damage compared to PBMCs. One-year of storage increased the levels of SBs and Fpg-sensitive sites especially in the WB samples. Based on these findings, the use of short storage times and PBMCs should be preferred because of low background level of DNA damage in the comet assay.

Highlights

The comet assay is an electrophoretic technique used to assess DNA damage, as a marker of genotoxicity and oxidative stress, in tissues and biological samples including peripheral blood mononuclear cells (PBMCs) and whole blood (WB)
The present study aims to evaluate the impact of storage at different time-points on the levels of strand breaks (SBs) and formamidopyrimidine DNA glycosylase (Fpg)-sensitive sites in isolated PBMCs and WB
Taken together our results documented that WB and isolated PBMCs can be safely cryopreserved for only several months at − 80 °C because of the DNA damage progresses after longer storage

Summary

Introduction

The comet assay is an electrophoretic technique used to assess DNA damage, as a marker of genotoxicity and oxidative stress, in tissues and biological samples including peripheral blood mononuclear cells (PBMCs) and whole blood (WB). There are advantages to use cryopreserved samples; they can be analyzed in whole batches, as opposed to blood samples that are procured over a period of time in a biomonitoring study and entails some kind of storage before they are processed in the comet assay at even inconvenient times of the day. For large volumes of WB it has been suggested to dilute the sample with an equal volume of cell culture medium containing 20% dimethyl sulfoxide (DMSO, a cryoprotectant) or alternatively, to freeze directly small volumes of blood (max 250 μL) without cryopreservation solutions These two approaches allow a rapid freezing process and reduce the risk of ice crystals formation and damages[3]. The possibility to use fresh or cryopreserved WB and/or PBMCs is under debate due the lack of standardized e procedures that may represent an additional source of variability in the evaluation of DNA damage as already widely observed in several studies[14,15,16,17,18]

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Conclusion