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).

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.

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 dose-response 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.

Recruitment of medical graduates from Prague to foundation programme jobs in a UK hospital: results of a 6-year project.

Introduction: International medical graduates (IMGs) are required to fill foundation-level posts in the NHS. Recruiting for these posts is difficult and time consuming. In 2016, we created a link between the Great Western Hospital, Swindon and Charles University, Prague to recruit to these posts. Here, we describe in detail the recruitment process, level of support and outcomes after 6 years, including the success rate of completion of foundation training and their career progression. Methods: Formal structured interviews occurred in Prague or online. Support included tailored educational/clinical supervision and additional simulation sessions. Data collected included exception reports and sick leave records. In 2022, a survey established satisfaction levels with the project and subsequent career progression. Results: All 51 doctors (100%) recruited as IMGs completed foundation training. Exception reporting and sick leave were below the Trust average. Of those doctors, 39 (76%) replied to the survey. All reported that the appointments helped them progress in their careers and 23 (45%) have subsequently been appointed successfully to a national Specialty Training Scheme. Conclusions: This project improved recruitment of non-deanery foundation doctors, prevented rota gaps and provided adequate staffing of junior doctors in our hospital. Concerns around employment of IMGs can be offset by providing a supportive and developmental environment.

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.