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Alkaline comet assay results on fresh and one-year frozen whole blood in small volume without cryo-protection in a group of people with different health status.

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Title: Alkaline comet assay results on fresh and one-year frozen whole blood in small volume without cryo-protection in a group of people with different health status.
Authors: Milić, Mirta1 (AUTHOR) mmilic@imi.hr, Ožvald, Ivan2 (AUTHOR) iozvald@gmail.com, Vinković Vrček, Ivana3 (AUTHOR) ivinkovic@imi.hr, Vučić Lovrenčić, Marijana4 (AUTHOR) vucic@idb.hr, Oreščanin, Višnja5 (AUTHOR) vorescan@gmail.com, Bonassi, Stefano6,7 (AUTHOR) stefano.bonassi@sanraffaele.it, Del Castillo, Emilio Rojas8 (AUTHOR) emilior@biomedicas.unam.mx
Source: Mutation Research - Genetic Toxicology & Environmental Mutagenesis. Jul2019, Vol. 843, p3-10. 8p.
Subject Terms: Type 2 diabetes, DNA damage, Blood volume, Comets
Abstract: • To evaluate the impact of small volume blood storage without cryopreservative on the basal level of DNA damage using the comet assay. • Tail intensity values of frozen samples after 1 year at −80 °C were within 11% of allowed values for background controls. • There was significant correlation between TL and TI parameters between fresh and frozen samples in nondiabetic group. • Different health status of individuals can influence on the level of correlation between fresh and frozen samples. • Frozen samples did not have higher percentage of heavily damaged cells and demonstrated that can be used in future studies. Using alkaline comet assay, DNA damage tail length (TL) and tail intensity (TI) parameters were compared between fresh whole blood and 1-year frozen small volume whole blood in EDTA at -80 °C without cryo-preservation. The studied group consisted of 25 volunteers with different health conditions who served as their own controls for frozen blood results. Without the purification step after thawing, the results and the usefulness of this protocol for future/retrospective (including re-analysations of putative outliers) studies were analysed. Medical surveillance and blood sampling were done at Merkur University Hospital Zagreb. No significant differences between fresh and frozen blood samples in terms of the mean TL values (mean ± SD: 29.03 ± 12.26 vs. 25.36 ± 6.97, respectively) and the mean TI values (9.19 ± 10.37 vs. 10.17 ± 8.55, respectively), and highly damaged cell percentage were determined among 25 volunteers. Median TI frozen samples values of entire group were within the allowed 10–11% (8.24). At the individual levels, no correlation between fresh and frozen whole blood samples was observed in 11 volunteers who suffered from diabetes mellitus type 2. Strong correlation between fresh/frozen samples was seen for TL (r = 0.64, p < 0.015) and TI (r = 0.71, p < 0.005) in nondiabetic subgroup. Overall, the results demonstrated the usefulness of the 1-year frozen blood without induction of heavily damaged DNA. Due to the different DNA damage behaviour connected with different health conditions, future studies should involve more volunteers, oxidative DNA damage comet assay measurements, the inclusion of a washing step after thawing and inclusion of disease/antioxidant biomarkers. [ABSTRACT FROM AUTHOR]
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Abstract:• To evaluate the impact of small volume blood storage without cryopreservative on the basal level of DNA damage using the comet assay. • Tail intensity values of frozen samples after 1 year at −80 °C were within 11% of allowed values for background controls. • There was significant correlation between TL and TI parameters between fresh and frozen samples in nondiabetic group. • Different health status of individuals can influence on the level of correlation between fresh and frozen samples. • Frozen samples did not have higher percentage of heavily damaged cells and demonstrated that can be used in future studies. Using alkaline comet assay, DNA damage tail length (TL) and tail intensity (TI) parameters were compared between fresh whole blood and 1-year frozen small volume whole blood in EDTA at -80 °C without cryo-preservation. The studied group consisted of 25 volunteers with different health conditions who served as their own controls for frozen blood results. Without the purification step after thawing, the results and the usefulness of this protocol for future/retrospective (including re-analysations of putative outliers) studies were analysed. Medical surveillance and blood sampling were done at Merkur University Hospital Zagreb. No significant differences between fresh and frozen blood samples in terms of the mean TL values (mean ± SD: 29.03 ± 12.26 vs. 25.36 ± 6.97, respectively) and the mean TI values (9.19 ± 10.37 vs. 10.17 ± 8.55, respectively), and highly damaged cell percentage were determined among 25 volunteers. Median TI frozen samples values of entire group were within the allowed 10–11% (8.24). At the individual levels, no correlation between fresh and frozen whole blood samples was observed in 11 volunteers who suffered from diabetes mellitus type 2. Strong correlation between fresh/frozen samples was seen for TL (r = 0.64, p < 0.015) and TI (r = 0.71, p < 0.005) in nondiabetic subgroup. Overall, the results demonstrated the usefulness of the 1-year frozen blood without induction of heavily damaged DNA. Due to the different DNA damage behaviour connected with different health conditions, future studies should involve more volunteers, oxidative DNA damage comet assay measurements, the inclusion of a washing step after thawing and inclusion of disease/antioxidant biomarkers. [ABSTRACT FROM AUTHOR]
ISSN:13835718
DOI:10.1016/j.mrgentox.2019.03.009