Suitability of cryopreserved isolated lymphocytes for the analysis of micronuclei with the cytokinesis-block method

Abstract

In order to assess the applicability of the cytokinesis-block micronucleus assay to frozen cells in human biomonitoring and in vitro radiosensitivity studies, basal and radiation-induced micronuclei and nucleoplasmic bridges (NPBs) were analysed in 28 lymphocyte samples stored frozen from 18 to 123 weeks. All samples successfully proliferated and produced a sufficient number of binucleated cells to be analysed. The length of the cryopreservation period did not influence cell proliferation, nor the incidence of micronuclei and NPBs, both in untreated and irradiated cells. Spontaneous levels of micronuclei were modulated by age (P=0.007) and by gender (P=0.024), as previously shown for cultures set up using fresh cell samples. Irradiation with 2 Gy gamma-rays significantly increased both micronuclei and NPBs, which were significantly correlated to each other (P=0.004). Radiation-induced micronuclei significantly increased with the age of donors (P=0.035), confirming previous findings obtained with fresh cell samples. The spontaneous incidences of micronuclei observed in cultures set up with frozen lymphocytes were compared with data recorded from the same subjects 5 years before using fresh blood samples. A high correlation was observed between the two data sets (P=0.004 after removing age and gender effects), highlighting the stability during the time of micronuclei as a biomarker of genomic stability, and supporting the suitability of frozen cells for cytogenetic analyses in biomonitoring and susceptibility studies.