Anderson D, Yu T W, Dobrzyńska M M, Ribas G, Marcos R
Department of Genetic and Reproductive Toxicology, BIBRA International, Carshalton, Surrey, United Kingdom.
Teratog Carcinog Mutagen. 1997;17(3):115-25.
The Comet assay is a rapid and sensitive method for analyzing single cells for DNA damage. Using human lymphocytes, the assay is particularly useful for human monitoring studies, as well as for in vitro genotoxicity testing of chemicals. In such studies, it is not always possible to collect and process matched samples on the same day as the blood is taken. It would be useful if some samples could be stored and examined at a different time, without loss of viability or other factors affecting responses. It is thus important to understand the effects of storage conditions on blood to be used in such studies and how exposure or treatment might modify such responses. In a joint study in two laboratories, blood was taken from various donors and stored under different conditions. It was examined on day 1 (day on which sample was taken) and days 2, 3, 4, 5, or 8 at room temperature, 4 degrees C, or -20 degrees C. Cells were treated after storage (from day 2 onward) with bleomycin (BLM) and ethylnitrosourea (ENU). The data were analyzed either by eye (classifying cells with different categories of damage) or by using a computerized image analysis system (Kinetic Imaging Ltd., Liverpool UK. Software Package Comet 3.0) where the tail moment, which is considered to be a sensitive measurement, has been analyzed. There was no loss of cell viability at 4 degrees C or room temperature up to 8 days when measured by trypan blue dye exclusion. Findings suggest that on days 1-4 for the untreated samples at room temperature or 4 degrees C there were no biologically meaningful changes in both the different categories of cell damage and tail moment data. In treated cultures up to day 4, either at room temperature or at 4 degrees C, responses were only minimally affected and changes were considered not to be of biological significance. However, there was slightly less variability between samples at 4 degrees C than at room temperature in one laboratory. The reverse was true in the other. This would suggest that samples can probably be stored up to day 4 at 4 degrees C or room temperature without any untoward effects. Provided samples can be processed within this 4-day time frame, it would not seem necessary to cryopreserve samples at -196 degrees C.
彗星试验是一种用于分析单细胞DNA损伤的快速且灵敏的方法。使用人类淋巴细胞，该试验对于人体监测研究以及化学品的体外遗传毒性测试尤为有用。在这类研究中，并非总是能够在采血当天收集并处理配对样本。如果一些样本能够在不同时间进行储存和检测，而不丧失活力或不受其他影响反应的因素干扰，那将是很有用的。因此，了解储存条件对用于此类研究的血液的影响以及暴露或处理如何改变此类反应非常重要。在两个实验室的一项联合研究中，从不同捐赠者采集血液并在不同条件下储存。在第1天（采样日）以及第2、3、4、5或8天在室温、4℃或-20℃下对其进行检测。储存后（从第2天起）用博来霉素（BLM）和乙基亚硝基脲（ENU）处理细胞。通过肉眼（将具有不同损伤类别的细胞分类）或使用计算机图像分析系统（英国利物浦的动力学成像有限公司。软件包Comet 3.0）分析数据，其中已对被认为是灵敏测量指标的尾矩进行了分析。通过台盼蓝染料排除法测量，在4℃或室温下长达8天细胞活力没有丧失。研究结果表明，对于在室温或4℃下未处理的样本，在第1 - 4天，不同类别的细胞损伤和尾矩数据均未出现生物学上有意义的变化。在室温或4℃下直至第4天的处理培养物中，反应仅受到极小影响，且变化被认为不具有生物学意义。然而，在一个实验室中，4℃下样本之间的变异性略低于室温。在另一个实验室中则相反。这表明样本在4℃或室温下可能可以储存至第4天而不会产生任何不良影响。只要样本能够在这4天时间内进行处理，似乎没有必要在-196℃下冷冻保存样本。
