Approaches of Whole Blood Thawing for Genotoxicity Analysis in Rats

The comet assay is a sensitive method for detecting DNA damage and is widely used in isolated human lymphocytes. This test involves time-consuming experiments and the samples should immediately be processed after collection. Hininger et al. froze human whole blood samples to use for future analyzes and thawed them after four months. Herein, the objective of this study was to validate a protocol in rat whole blood samples following modified Hininger et al. protocol for future analyses of DNA damage. After rat blood collection, samples were randomly processed using four different tests: Test 1: Following Lima et al. protocol with freshly isolated rat lymphocytes, which are used as gold standard for comet assay. Test 2: This test as performed according to modified Hininger et al. protocol. Test 3: similar test to Hininger et al. freezing process, but with changes in thawing technique. Test 4: The fresh whole blood was used and considered as the control experiment. All the samples were prepared for DNA damage levels by comet assay. The results showed that the tests 1, 2, 3 and 4 showed no statistically significant differences (p> 0.05) among themselves, when analyzed using comet assay by software (tail intensity) or by visual score. Therefore, the tests with frozen whole blood were viable showing greater efficiency and reducing cost and time in laboratory practice. Thus, the present study also offers a new approach in experimental investigation, since most articles that address this issue is held in humans.