On the search for an intelligible comet assay descriptor

The comet assay has developed over the past 30 years and today, a variety of different DNA lesions and DNA repair can be measured by different versions of the assay (Collins, 2004). In the final step of the method, an image resembling a comet with a head (the nuclear core) and a tail (consisting of mainly single stranded DNA that has migrated out from the cell nuclei) is analyzed. The magnitude of the comet's DNA-tail provides information about the level of DNA lesions in the cell. The results from comet assay analyses are reported using different descriptors, the most frequently used being percentage of DNA in the tail (%T), tail length and tail moment (the product of %T and tail length). These descriptors can be reported in different ways, i.e., as means, medians or as distribution patterns. To compile the information on the migration of thousands of comets into a single value that is meaningful to convey to other researchers, is difficult. The solution has been practical and controlled by those researchers with the longest experience with the comet assay. In this opinion paper, we revisit the search for a commonly accepted descriptor for DNA damage measured by the comet assay. We define the “best” comet assay descriptor as a measurement that best describes the migration of DNA in each comet in the agarose, fits the distribution of comets in the gel, and conveys the technical measurement of comets as a descriptor that other researchers can understand. It should be emphasized that we do not embark on a mission to promote only one comet assay descriptor

Variation of DNA damage levels in peripheral blood mononuclear cells isolated in different laboratories

This study investigated the levels of DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG) sensitive sites, as assessed by the comet assay, in peripheral blood mononuclear cells (PBMC) from healthy women from five different countries in Europe. The laboratory in each country (referred to as 'centre') collected and cryopreserved PBMC samples from three donors, using a standardised cell isolation protocol. The samples were analysed in 13 different laboratories for DNA damage, which is measured by the comet assay. The study aim was to assess variation in DNA damage in PBMC samples that were collected in the same way and processed using the same blood isolation procedure. The inter-laboratory variation was the prominent contributor to the overall variation. The inter-laboratory coefficient of variation decreased for both DNA strand breaks (from 68 to 26%) and FPG sensitive sites (from 57 to 12%) by standardisation of the primary comet assay endpoint with calibration curve samples. The level of DNA strand breaks in the samples from two of the centres (0.56-0.61 lesions/10(6) bp) was significantly higher compared with the other three centres (0.41-0.45 lesions/10(6) bp). In contrast, there was no difference between the levels of FPG sensitive sites in PBMC samples from healthy donors in the different centres (0.41-0.52 lesion/10(6) bp)

Inter-laboratory variation in DNA damage using a standard comet assay protocol

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