Variation in the measurement of DNA damage by comet assay measured by the ECVAG inter-laboratory validation trial
Variation in the measurement of DNA damage by comet assay measured by the ECVAG inter-laboratory validation trial
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DOI：
10.1093/mutage/gep048
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发表时间：
2010-03-01
期刊：
影响因子：
2.7
通讯作者：
Moller, Peter
中科院分区：
文献类型：
--
作者：
Forchhammer, Lykke;Johansson, Clara;Moller, Peter
彗星试验已经成为生物监测研究和遗传毒理学中评估DNA损伤的一种流行方法。然而，很少有研究涉及通过彗星试验测量的DNA损伤的实验室间变异性的问题。在这项研究中，12个实验室分析了单核细胞来源的THP-1细胞的DNA损伤水平，方法是对预先制作的玻片、编码的冷冻细胞样本和参考标准细胞(校准曲线样本)进行视觉分类或计算机辅助图像分析。参考标准样品用电离辐射(0-10GY)照射，并用来构建校准曲线，以计算每10(6)个碱基对的病变数量。所有实验室都在电离辐射(1.5-7GY)照射的编码样本中检测到剂量-反应关系，但不同实验室报告的DNA损伤水平存在明显差异，实验室间变异系数(CV)为47%。通过每个实验室准备的校准曲线调整初级彗星试验的终点，将变异系数降低到28%，这在统计学上具有显著意义(P<；0.05，Levene‘s检验)。很大一部分实验室间的差异源于图像分析的差异，而实验室内的差异比实验室间的差异小得多。总而言之，根据参考标准调整初级彗星分析结果，减少了碱性版本彗星分析所测量的DNA损伤水平的实验室间差异。
The comet assay has become a popular method for the assessment of DNA damage in biomonitoring studies and genetic toxicology. However, few studies have addressed the issue of the noted inter-laboratory variability of DNA damage measured by the comet assay. In this study, 12 laboratories analysed the level of DNA damage in monocyte-derived THP-1 cells by either visual classification or computer-aided image analysis of pre-made slides, coded cryopreserved samples of cells and reference standard cells (calibration curve samples). The reference standard samples were irradiated with ionizing radiation (0-10 Gy) and used to construct a calibration curve to calculate the number of lesions per 10(6) base pair. All laboratories detected dose-response relationships in the coded samples irradiated with ionizing radiation (1.5-7 Gy), but there were overt differences in the level of DNA damage reported by the different laboratories as evidenced by an inter-laboratory coefficient of variation (CV) of 47%. Adjustment of the primary comet assay end points by a calibration curve prepared in each laboratory reduced the CV to 28%, a statistically significant reduction (P < 0.05, Levene's test). A large fraction of the inter-laboratory variation originated from differences in image analysis, whereas the intra-laboratory variation was considerably smaller than the variation between laboratories. In summary, adjustment of primary comet assay results by reference standards reduces inter-laboratory variation in the level of DNA damage measured by the alkaline version of the comet assay.