Section of Environmental Health, Department of Public Health, University of Copenhagen, Copenhagen K, Denmark.
Mutagenesis. 2012 Nov;27(6):665-72. doi: 10.1093/mutage/ges032. Epub 2012 Jul 27.
There are substantial inter-laboratory variations in the levels of DNA damage measured by the comet assay. The aim of this study was to investigate whether adherence to a standard comet assay protocol would reduce inter-laboratory variation in reported values of DNA damage. Fourteen laboratories determined the baseline level of DNA strand breaks (SBs)/alkaline labile sites and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites in coded samples of mononuclear blood cells (MNBCs) from healthy volunteers. There were technical problems in seven laboratories in adopting the standard protocol, which were not related to the level of experience. Therefore, the inter-laboratory variation in DNA damage was only analysed using the results from laboratories that had obtained complete data with the standard comet assay protocol. This analysis showed that the differences between reported levels of DNA SBs/alkaline labile sites in MNBCs were not reduced by applying the standard assay protocol as compared with the laboratory's own protocol. There was large inter-laboratory variation in FPG-sensitive sites by the laboratory-specific protocol and the variation was reduced when the samples were analysed by the standard protocol. The SBs and FPG-sensitive sites were measured in the same experiment, indicating that the large spread in the latter lesions was the main reason for the reduced inter-laboratory variation. However, it remains worrying that half of the participating laboratories obtained poor results using the standard procedure. This study indicates that future comet assay validation trials should take steps to evaluate the implementation of standard procedures in participating laboratories.
彗星试验检测到的 DNA 损伤水平存在很大的实验室间差异。本研究旨在探讨遵循标准彗星试验方案是否会降低报告的 DNA 损伤值的实验室间差异。14 个实验室测定了来自健康志愿者单核细胞（MNBC）的编码样本中的 DNA 链断裂（SBs）/碱性不稳定位点和经型嘧啶 DNA 糖基化酶（FPG）敏感位点的基线水平。七个实验室在采用标准方案时存在技术问题，这些问题与经验水平无关。因此，仅使用具有标准彗星试验方案获得完整数据的实验室的结果分析了 DNA 损伤的实验室间差异。该分析表明，与实验室自身方案相比，应用标准检测方案并未降低报告的 MNBC 中 DNA SBs/碱性不稳定位点水平的差异。通过实验室特异性方案测量的 FPG 敏感位点存在很大的实验室间差异，当通过标准方案分析时，该差异会降低。在相同的实验中测量了 SBs 和 FPG 敏感位点，表明后者损伤的广泛分布是实验室间差异降低的主要原因。然而，仍令人担忧的是，一半的参与实验室使用标准程序获得了较差的结果。本研究表明，未来的彗星试验验证试验应采取措施评估参与实验室标准程序的实施情况。
