Use of whole blood directly for single-cell gel electrophoresis (comet) assay in vivo and white blood cells for in vitro assay.
The present study investigated the use of whole blood from humans and rats directly for single-cell gel electrophoresis (comet) assay. As little as 20 microl of whole blood was sufficient for comet assay, and the comet images obtained from whole blood were not different from those obtained from isolated lymphocytes. The DNA remained intact up to 4 h at 4 degrees C after isolation and had no observable strand breakage, when whole blood was cryopreserved (at -80 degrees C) in 10% pre-cooled DMSO up to 60 days. To demonstrate that the whole-blood technique could be applied to in vivo studies, we injected rats with a known carcinogen Fe/NTA and measured DNA strand breaks in whole blood in comparison with isolated lymphocytes. We showed that Fe/NTA injection resulted in similar extent of DNA strand breakage in both whole blood and lymphocytes, indicating that whole-blood method can be used for in vivo genotoxic studies. One disadvantage of the whole-blood technique is that whole blood cannot be used for in vitro studies because of the interferences from red blood cell (RBC) components. However, this problem can be overcome by prior hemolysis of RBCs and a brief centrifugation to obtain white blood cells (WBCs), which can then be used for in vitro incubation with genotoxic compounds before comet assay. Overall, this whole-blood technique for comet assay is expected to provide a simple, rapid, and cost-effective alternative for the existing comet assay using isolated lymphocytes in situations such as when time and cost are limiting factors.
Department of Food Science, National Chung-Hsing University, 250 Kuo-Kuang Raod, Taichung 402, Taiwan, ROC.
Cheng-Hung
CH
Chuang
Miao-Lin
ML
Hu
D000818:Animals; D001769:Blood; D001793:Blood Preservation; D020552:Comet Assay / Q000379:methods*; D015925:Cryopreservation; D004247:DNA / Q000187:drug effects; D004249:DNA Damage; D005290:Ferric Compounds / Q000633:toxicity; D006801:Humans; D008214:Lymphocytes / Q000166:cytology / Q000502:physiology*; D009153:Mutagens / Q000633:toxicity; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives* / Q000633:toxicity; D051381:Rats
D005290:Ferric Compounds; D009153:Mutagens; D004247:DNA; D009571:Nitrilotriacetic Acid; C020326:ferric nitrilotriacetate