Cryopreserving whole blood for functional assays using viable lymphocytes in molecular epidemiology studies

There is an increasing need for viable lymphocytes in performing phenotypic assays for biomarker studies. Both fresh and cryopreserved lymphocytes have been used for cell culture-based functional assays. However, fresh lymphocytes do not allow assays to be done in batches and cryopreservation of isolated lymphocytes results in a considerable loss of viable cells. To investigate the feasibility of using cryopreserved whole blood as a source of viable lymphocytes in molecular epidemiology studies, two well-established biomarkers, the host-cell reactivation (HCR) and mutagen sensitivity assays, were used to compare the method of cryopreserving whole blood with the traditional methods. In 25 paired blood samples assayed for DNA repair capacity (DRC) by the HCR assay, the DRC values of frozen whole blood (mean +/- SD, 11.59 +/- 3.07) were similar to those of frozen isolated lymphocytes (11.08 +/- 3.50). The correlation between the paired DRC values was 0.77 (P < 0.001). In 31 paired blood samples assayed for the gamma-radiation-induced chromatid breaks by the mutagen sensitivity assay, there was no significant difference between the baseline level of chromatid breaks in lymphocytes from frozen blood (0.05 +/- 0.03) and fresh blood (0.06 +/- 0.03). The blastogenic rate and mitotic index of the cells used for the two assays were compared between the different processing methods. The lymphocytes from frozen whole blood were more sensitive to gamma-radiation, with a higher mean level of chromatid breaks (0.68 +/- 0.21) than that in fresh blood (0.42 +/- 0.12, P < 0.01), and the correlation between the numbers of chromatid breaks in the paired samples was statistically significant (r = 0.61, P < 0.001). These data suggest that within the limits of the parameters investigated here, cryopreserved whole blood is a good source of viable lymphocytes for biomarker assays in molecular epidemiological studies.