An ECVAG inter-laboratory validation study of the comet assay: inter-laboratory and intra-laboratory variations of DNA strand breaks and FPG-sensitive sites in human mononuclear cells
An ECVAG inter-laboratory validation study of the comet assay: inter-laboratory and intra-laboratory variations of DNA strand breaks and FPG-sensitive sites in human mononuclear cells
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DOI：
10.1093/mutage/get001
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发表时间：
2013-05-01
期刊：
影响因子：
2.7
通讯作者：
Moller, Lennart
中科院分区：
文献类型：
--
作者：
Ersson, Clara;Moller, Peter;Moller, Lennart
碱性彗星测定是一种广泛用于生物监测研究的成熟、灵敏的方法。可以修改该方法来测量一系列不同类型的 DNA 损伤。然而，不同研究小组使用的方案存在很大差异，影响了实验室间结果的比较。本研究的目的是评估实验室间、实验室内、样品和残留（无法解释的）DNA 链断裂和甲酰胺嘧啶 DNA 糖基化酶 (FPG) 敏感位点的变异，这些变异是通过彗星试验使用平衡拉丁方设计测量的。 14 个参与实验室使用自己的彗星检测方案，测量了包含外周血单核细胞 (PBMC) 的编码样本中 DNA 链断裂和 FPG 敏感位点的水平，以及在不同分析日的编码校准曲线样本（暴露于不同剂量电离辐射的细胞）中 DNA 链断裂的水平。十一个实验室在两到三天的分析中发现编码校准曲线样本中存在剂量反应关系，而三个实验室在检测中存在技术问题。在编码校准曲线样本中，电离辐射剂量、实验室间变异、实验室内变异和残余变异分别占总变异的60.9%、19.4%、0.1%和19.5%。在编码的 PBMC 样本中，实验室间变异解释了 DNA 链断裂总体变异的最大部分 (79.2%)，残余变异 (19.9%) 远大于实验室内 (0.3%) 和受试者间 (0.5%) 变异。 PBMC 样本中 FPG 敏感位点总体变异的相同划分表明，实验室间变异是最强的贡献者 (56.7%)，而残留变异 (42.9%)、实验室内变异 (0.2%) 和受试者间变异 (0.3%) 对整体变异的贡献较小。结果表明，通过彗星试验测量的健康受试者 PBMC 中 DNA 损伤的变化是检测变化，而不是受试者之间的变化。
The alkaline comet assay is an established, sensitive method extensively used in biomonitoring studies. This method can be modified to measure a range of different types of DNA damage. However, considerable differences in the protocols used by different research groups affect the inter-laboratory comparisons of results. The aim of this study was to assess the inter-laboratory, intra-laboratory, sample and residual (unexplained) variations in DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites measured by the comet assay by using a balanced Latin square design. Fourteen participating laboratories used their own comet assay protocols to measure the level of DNA strand breaks and FPG-sensitive sites in coded samples containing peripheral blood mononuclear cells (PBMC) and the level of DNA strand breaks in coded calibration curve samples (cells exposed to different doses of ionising radiation) on three different days of analysis. Eleven laboratories found doseresponse relationships in the coded calibration curve samples on two or three days of analysis, whereas three laboratories had technical problems in their assay. In the coded calibration curve samples, the dose of ionising radiation, inter-laboratory variation, intra-laboratory variation and residual variation contributed to 60.9, 19.4, 0.1 and 19.5%, respectively, of the total variation. In the coded PBMC samples, the inter-laboratory variation explained the largest fraction of the overall variation of DNA strand breaks (79.2%) and the residual variation (19.9%) was much larger than the intra-laboratory (0.3%) and inter-subject (0.5%) variation. The same partitioning of the overall variation of FPG-sensitive sites in the PBMC samples indicated that the inter-laboratory variation was the strongest contributor (56.7%), whereas the residual (42.9%), intra-laboratory (0.2%) and inter-subject (0.3%) variations again contributed less to the overall variation. The results suggest that the variation in DNA damage, measured by comet assay, in PBMC from healthy subjects is assay variation rather than variation between subjects.