DNA strand break levels in cryopreserved mononuclear blood cell lines measured by the alkaline comet assay: results from the hCOMET ring trial

Research output: Contribution to journalJournal articleResearchpeer-review

  • Amaya Azqueta
  • Adriana Rodriguez-Garraus
  • Tamara Bakuradze
  • Elke Richling
  • Ezgi Eyluel Bankoglu
  • Helga Stopper
  • Victoria Claudino Bastos
  • Sabine A S Langie
  • Sara Ristori
  • Francesca Scavone
  • Lisa Giovannelli
  • Maria Wojewódzka
  • Marcin Kruszewski
  • Vanessa Valdiglesias
  • Blanca Laffon
  • Carla Costa
  • Solange Costa
  • João Paulo Teixeira
  • Mirko Marino
  • Cristian Del Bo'
  • Patrizia Riso
  • Congying Zhang
  • Sergey Shaposhnikov
  • Andrew Collins

The comet assay is widely used in biomonitoring studies for the analysis of DNA damage in leukocytes and peripheral blood mononuclear cells. Rather than processing blood samples directly, it can be desirable to cryopreserve whole blood or isolated cells for later analysis by the comet assay. However, this creates concern about artificial accumulation of DNA damage during cryopreservation. In this study, ten laboratories used standardized cryopreservation and thawing procedures of monocytic (THP-1) or lymphocytic (TK6) cells. Samples were cryopreserved in small aliquots in 50% foetal bovine serum, 40% cell culture medium and 10% dimethyl sulphoxide. Subsequently, cryopreserved samples were analysed by the standard comet assay on three occasions over a three-year period. Levels of DNA strand breaks in THP-1 cells were increased (4 laboratories), unaltered (4 laboratories) or decreased (2 laboratories) by long-term storage. Pooled analysis indicates only a modest positive association between storage time and levels of DNA strand breaks in THP-1 cells (0.37% Tail DNA per year, 95% confidence interval: -0.05, 0.78). In contrast, DNA strand break levels were not increased by cryopreservation in TK6 cells. There was inter-laboratory variation in levels of DNA strand breaks in THP-1 cells (SD = 3.7% Tail DNA) and TK6 reference sample cells (SD = 9.4% Tail DNA), whereas the intra-laboratory residual variation was substantially smaller (i.e. SD = 0.4% to 2.2% Tail DNA in laboratories with the smallest and largest variation). In conclusion, the study shows that accumulation of DNA strand breaks in cryopreserved mononuclear blood cell lines is not a matter of concern.

Original languageEnglish
Issue number5
Pages (from-to)273–282
Number of pages10
Publication statusPublished - 2023

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© The Author(s) 2023. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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