RENEB Inter-Laboratory Comparison 2021: Inter-Assay Comparison of Eight Dosimetry Assays.

Tools for radiation exposure reconstruction are required to support the medical management of radiation victims in radiological or nuclear incidents. Different biological and physical dosimetry assays can be used for various exposure scenarios to estimate the dose of ionizing radiation a person has absorbed. Regular validation of the techniques through inter-laboratory comparisons (ILC) is essential to guarantee high quality results. In the current RENEB inter-laboratory comparison, the performance quality of established cytogenetic assays [dicentric chromosome assay (DCA), cytokinesis-block micronucleus assay (CBMN), stable chromosomal translocation assay (FISH) and premature chromosome condensation assay (PCC)] was tested in comparison to molecular biological assays [gamma-H2AX foci (gH2AX), gene expression (GE)] and physical dosimetry-based assays [electron paramagnetic resonance (EPR), optically or thermally stimulated luminescence (LUM)]. Three blinded coded samples (e.g., blood, enamel or mobiles) were exposed to 0, 1.2 or 3.5 Gy X-ray reference doses (240 kVp, 1 Gy/min). These doses roughly correspond to clinically relevant groups of unexposed to low exposed (0-1 Gy), moderately exposed (1-2 Gy, no severe acute health effects expected) and highly exposed individuals (>2 Gy, requiring early intensive medical care). In the frame of the current RENEB inter-laboratory comparison, samples were sent to 86 specialized teams in 46 organizations from 27 nations for dose estimation and identification of three clinically relevant groups. The time for sending early crude reports and more precise reports was documented for each laboratory and assay where possible. The quality of dose estimates was analyzed with three different levels of granularity, 1. by calculating the frequency of correctly reported clinically relevant dose categories, 2. by determining the number of dose estimates within the uncertainty intervals recommended for triage dosimetry (±0.5 Gy or ±1.0 Gy for doses <2.5 Gy or >2.5 Gy), and 3. by calculating the absolute difference (AD) of estimated doses relative to the reference doses. In total, 554 dose estimates were submitted within the 6-week period given before the exercise was closed. For samples processed with the highest priority, earliest dose estimates/categories were reported within 5-10 h of receipt for GE, gH2AX, LUM, EPR, 2-3 days for DCA, CBMN and within 6-7 days for the FISH assay. For the unirradiated control sample, the categorization in the correct clinically relevant group (0-1 Gy) as well as the allocation to the triage uncertainty interval was, with the exception of a few outliers, successfully performed for all assays. For the 3.5 Gy sample the percentage of correct classifications to the clinically relevant group (≥2 Gy) was between 89-100% for all assays, with the exception of gH2AX. For the 1.2 Gy sample, an exact allocation to the clinically relevant group was more difficult and 0-50% or 0-48% of the estimates were wrongly classified into the lowest or highest dose categories, respectively. For the irradiated samples, the correct allocation to the triage uncertainty intervals varied considerably between assays for the 1.2 Gy (29-76%) and 3.5 Gy (17-100%) samples. While a systematic shift towards higher doses was observed for the cytogenetic-based assays, extreme outliers exceeding the reference doses 2-6 fold were observed for EPR, FISH and GE assays. These outliers were related to a particular material examined (tooth enamel for EPR assay, reported as kerma in enamel, but when converted into the proper quantity, i.e. to kerma in air, expected dose estimates could be recalculated in most cases), the level of experience of the teams (FISH) and methodological uncertainties (GE). This was the first RENEB ILC where everything, from blood sampling to irradiation and shipment of the samples, was organized and realized at the same institution, for several biological and physical retrospective dosimetry assays. Almost all assays appeared comparably applicable for the identification of unexposed and highly exposed individuals and the allocation of medical relevant groups, with the latter requiring medical support for the acute radiation scenario simulated in this exercise. However, extreme outliers or a systematic shift of dose estimates have been observed for some assays. Possible reasons will be discussed in the assay specific papers of this special issue. In summary, this ILC clearly demonstrates the need to conduct regular exercises to identify research needs, but also to identify technical problems and to optimize the design of future ILCs.

What We Have Learned from RENEB Inter-Laboratory Comparisons Since 2012 With Focus on ILC 2021.

Inter-laboratory exercises are important tools within the European network for biological dosimetry and physical retrospective dosimetry (RENEB) to validate and improve the performance of member laboratories and to ensure an operational network with high quality standards for dose estimations in case of a large-scale radiological or nuclear event. In addition to the RENEB inter-laboratory comparison 2021, several inter-laboratory comparisons have been performed in the frame of RENEB for a number of assays in recent years. This publication gives an overview of RENEB inter-laboratory comparisons for biological dosimetry assays in the past and a final summary of the challenges and lessons learnt from the RENEB inter-laboratory comparison 2021. In addition, the dose estimates of all RENEB inter-laboratory comparisons since 2013 that have been conducted for the dicentric chromosome assay, the most established and applied assay, are compared and discussed.

RENEB Inter-Laboratory Comparison 2021: The Cytokinesis-Block Micronucleus Assay.

The goal of the RENEB inter-laboratory comparison 2021 exercise was to simulate a large-scale radiation accident involving a network of biodosimetry labs. Labs were required to perform their analyses using different biodosimetric assays in triage mode scoring and to rapidly report estimated radiation doses to the organizing institution. This article reports the results obtained with the cytokinesis-block micronucleus assay. Three test samples were exposed to blinded doses of 0, 1.2 and 3.5 Gy X-ray doses (240 kVp, 13 mA, ∼75 keV, 1 Gy/min). These doses belong to 3 triage categories of clinical relevance: a low dose category, for no exposure or exposures inferior to 1 Gy, requiring no direct treatment of subjects; a medium dose category, with doses ranging from 1 to 2 Gy, and a high dose category, after exposure to doses higher than 2 Gy, with the two latter requiring increasing medical attention. After irradiation the test samples (no. 1, no. 2 and no. 3) were sent by the organizing laboratory to 14 centers participating in the micronucleus assay exercise. Laboratories were asked to setup micronucleus cultures and to perform the micronucleus assay in triage mode, scoring 500 binucleated cells manually, or 1,000 binucleated cells in automated/semi-automated mode. One laboratory received no blood samples, but scored pictures from another lab. Based on their calibration curves, laboratories had to provide estimates of the administered doses. The accuracy of the reported dose estimates was further analyzed by the micronucleus assay lead. The micronucleus assay allowed classification of samples in the corresponding clinical triage categories (low, medium, high dose category) in 88% of cases (manual scoring, 88%; semi-automated scoring, 100%; automated scoring, 73%). Agreement between scoring laboratories, assessed by calculating the Fleiss' kappa, was excellent (100%) for semi-automated scoring, good (83%) for manual scoring and poor (53%) for fully automated scoring. Correct classification into triage scoring dose intervals (reference dose ±0.5 Gy for doses ≤2.5 Gy, or reference dose ±1 Gy for doses >2.5 Gy), recommended for triage biodosimetry, was obtained in 79% of cases (manual scoring, 73%; semi-automated scoring, 100%; automated scoring, 67%). The percentage of dose estimates whose 95% confidence intervals included the reference dose was 58% (manual scoring, 48%; semiautomated scoring, 72%; automated scoring, 60%). For the irradiated samples no. 2 and no. 3, a systematic shift towards higher dose estimations was observed. This was also noticed with the other cytogenetic assays in this intercomparison exercise. Accuracy of the rapid triage modality could be maintained when the number of manually scored cells was scaled down to 200 binucleated cells. In conclusion, the micronucleus assay, preferably performed in a semi-automated or manual scoring mode, is a reliable technique to perform rapid biodosimetry analysis in large-scale radiation emergencies.

RENEB Inter-Laboratory Comparison 2021: The Dicentric Chromosome Assay.

After large-scale radiation accidents where many individuals are suspected to be exposed to ionizing radiation, biological and physical retrospective dosimetry assays are important tools to aid clinical decision making by categorizing individuals into unexposed/minimally, moderately or highly exposed groups. Quality-controlled inter-laboratory comparisons of simulated accident scenarios are regularly performed in the frame of the European legal association RENEB (Running the European Network of Biological and Physical retrospective Dosimetry) to optimize international networking and emergency readiness in case of large-scale radiation events. In total 33 laboratories from 22 countries around the world participated in the current RENEB inter-laboratory comparison 2021 for the dicentric chromosome assay. Blood was irradiated in vitro with X rays (240 kVp, 13 mA, ∼75 keV, 1 Gy/min) to simulate an acute, homogeneous whole-body exposure. Three blood samples (no. 1: 0 Gy, no. 2: 1.2 Gy, no. 3: 3.5 Gy) were sent to each participant and the task was to culture samples, to prepare slides and to assess radiation doses based on the observed dicentric yields from 50 manually or 150 semi-automatically scored metaphases (triage mode scoring). Approximately two-thirds of the participants applied calibration curves from irradiations with γ rays and about 1/3 from irradiations with X rays with varying energies. The categorization of the samples in clinically relevant groups corresponding to individuals that were unexposed/minimally (0-1 Gy), moderately (1-2 Gy) or highly exposed (>2 Gy) was successfully performed by all participants for sample no. 1 and no. 3 and by ≥74% for sample no. 2. However, while most participants estimated a dose of exactly 0 Gy for the sham-irradiated sample, the precise dose estimates of the samples irradiated with doses >0 Gy were systematically higher than the corresponding reference doses and showed a median deviation of 0.5 Gy (sample no. 2) and 0.95 Gy (sample no. 3) for manual scoring. By converting doses estimated based on γ-ray calibration curves to X-ray doses of a comparable mean photon energy as used in this exercise, the median deviation decreased to 0.27 Gy (sample no. 2) and 0.6 Gy (sample no. 3). The main aim of biological dosimetry in the case of a large-scale event is the categorization of individuals into clinically relevant groups, to aid clinical decision making. This task was successfully performed by all participants for the 0 Gy and 3.5 Gy samples and by 74% (manual scoring) and 80% (semiautomatic scoring) for the 1.2 Gy sample. Due to the accuracy of the dicentric chromosome assay and the high number of participating laboratories, a systematic shift of the dose estimates could be revealed. Differences in radiation quality (X ray vs. γ ray) between the test samples and the applied dose effect curves can partly explain the systematic shift. There might be several additional reasons for the observed bias (e.g., donor effects, transport, experimental conditions or the irradiation setup) and the analysis of these reasons provides great opportunities for future research. The participation of laboratories from countries around the world gave the opportunity to compare the results on an international level.

Absence of genotoxic impact assessed by micronucleus frequency in circulating lymphocytes of workers exposed to cadmium.

PURPOSE: Cd is considered as a genotoxic carcinogen for which a threshold can be identified. This threshold has, however, not been established and the shape of the relationship between Cd exposure and genotoxic effects is unknown. The aim of the present study was to analyse the shape of the dose-response relationship for the genotoxic effects of Cd in occupational settings. METHODS: The study has a cross-sectional design and includes 60 healthy male and female workers with known Cd exposure selected from two plants manufacturing or recycling nickel-Cd batteries. The frequency of MN was measured in circulating lymphocytes, and related to internal Cd doses (Cd-B, Cd-U). Determinants of MN frequency were traced by multivariate regression analysis. RESULTS: Cd exposure covered a wide range as measured by Cd-B (0.02-1.26 µg/dL), Cd-U (0.26-15.80 µg/g creat) and seniority in the plant (1-42 years). Gender was the only parameter significantly associated with MN frequency, women having on average 8.5 additional MN/1000 BN cells compared to men. Cd-B, Cd-U or Ni-U did not influence MN frequency when adjusted for gender and other potential confounders. CONCLUSION: This finding is consistent with the existing knowledge on the mechanisms governing the genotoxic activity of Cd, which are all non-stochastic and thresholded. The threshold for systemic genotoxic effects of Cd is thus beyond the range of internal exposure considered in the present investigation.

Radiation Sensitivity of Human CD34(+) Cells Versus Peripheral Blood T Lymphocytes of Newborns and Adults: DNA Repair and Mutagenic Effects.

As hematopoietic stem and progenitor cells (HSPCs) self-renew throughout life, accumulation of genomic alterations can potentially give rise to radiation carcinogenesis. In this study we examined DNA double-strand break (DSB) induction and repair as well as mutagenic effects of ionizing radiation in CD34(+) cells and T lymphocytes from the umbilical cord of newborns. The age dependence of DNA damage repair end points was investigated by comparing newborn T lymphocytes with adult peripheral blood T lymphocytes. As umbilical cord blood (UCB) contains T lymphocytes that are practically all phenotypically immature, we examined the radiation response of separated naive (CD45RA(+)) and memory (CD45RO(+)) T lymphocytes. The number of DNA DSBs was assessed by microscopic scoring of γ-H2AX/53BP1 foci 0.5 h after low-dose radiation exposure, while DNA repair was studied by scoring the number of residual γ-H2AX/53BP1 foci 24 h after exposure. Mutagenic effects were studied by the cytokinesis block micronucleus (CBMN) assay. No significant differences in the number of DNA DSBs induced by low-dose (100-200 mGy) radiation were observed among the three different cell types. However, residual γ-H2AX/53BP1 foci levels 24 h postirradiation were significantly lower in CD34(+) cells compared to newborn T lymphocytes, while newborn T lymphocytes showed significantly higher foci yields than adult T lymphocytes. No significant differences in the level of radiation-induced micronuclei at 2 Gy were observed between CD34(+) cells and newborn T lymphocytes. However, newborn T lymphocytes showed a significantly higher number of micronuclei compared to adult T lymphocytes. These results confirm that CD34(+) cell quiescence promotes mutagenesis after exposure. Furthermore, we can conclude that newborn peripheral T lymphocytes are significantly more radiosensitive than adult peripheral T lymphocytes. Using the results from the comparative study of radiation-induced DNA damage repair end points in naive (CD45RA(+)) and memory (CD45RO(+)) T lymphocytes, we could demonstrate that the observed differences between newborn and adult T lymphocytes can be explained by the immunophenotypic change of T lymphocytes with age, which is presumably linked with the remodeling of the closed chromatin structure of naive T lymphocytes.

BRCA1, BRCA2 and PALB2 mutations and CHEK2 c.1100delC in different South African ethnic groups diagnosed with premenopausal and/or triple negative breast cancer.

BACKGROUND: Current knowledge of the aetiology of hereditary breast cancer in the four main South African population groups (black, coloured, Indian and white) is limited. Risk assessments in the black, coloured and Indian population groups are challenging because of restricted information regarding the underlying genetic contributions to inherited breast cancer in these populations. We focused this study on premenopausal patients (diagnosed with breast cancer before the age of 50; n = 78) and triple negative breast cancer (TNBC) patients (n = 30) from the four South African ethnic groups. The aim of this study was to determine the frequency and spectrum of germline mutations in BRCA1, BRCA2 and PALB2 and to evaluate the presence of the CHEK2 c.1100delC allele in these patients. METHODS: In total, 108 South African breast cancer patients underwent mutation screening using a Next-Generation Sequencing (NGS) approach in combination with Multiplex Ligation-dependent Probe Amplification (MLPA) to detect large rearrangements in BRCA1 and BRCA2. RESULTS: In 13 (12 %) patients a deleterious mutation in BRCA1/2 was detected, three of which were novel mutations in black patients. None of the study participants was found to have an unequivocal pathogenic mutation in PALB2. Two (white) patients tested positive for the CHEK2 c.1100delC mutation, however, one of these also carried a deleterious BRCA2 mutation. Additionally, six variants of unknown clinical significance were identified (4 in BRCA2, 2 in PALB2), all in black patients. Within the group of TNBC patients, a higher mutation frequency was obtained (23.3 %; 7/30) than in the group of patients diagnosed before the age of 50 (7.7 %; 6/78). CONCLUSION: This study highlights the importance of evaluating germline mutations in major breast cancer genes in all of the South African population groups. This NGS study shows that mutation analysis is warranted in South African patients with triple negative and/or in premenopausal breast cancer.

The first gamma-H2AX biodosimetry intercomparison exercise of the developing European biodosimetry network RENEB.

In the event of a mass casualty radiation incident, the gamma-H2AX foci assay could be a useful tool to estimate radiation doses received by individuals. The rapid processing time of blood samples of just a few hours and the potential for batch processing, enabling high throughput, make the assay ideal for early triage categorisation to separate the 'worried well' from the low and critically exposed by quantifying radiation-induced foci in peripheral blood lymphocytes. Within the RENEB framework, 8 European laboratories have taken part in the first European gamma-H2AX biodosimetry exercise, which consisted of a telescoring comparison of 200 circulated foci images taken from 8 samples, and a comparison of 10 fresh blood lymphocyte samples that were shipped overnight to participating labs 4 or 24 h post-exposure. Despite large variations between laboratories in the dose-response relationship for foci induction, the obtained results indicate that the network should be able to use the gamma-H2AX assay for rapidly identifying the most severely exposed individuals within a cohort who could then be prioritised for accurate chromosome dosimetry.

Is a semi-automated approach indicated in the application of the automated micronucleus assay for triage purposes?

Within the EU MULTIBIODOSE project, the automated micronucleus (MN) assay was optimised for population triage in large-scale radiological emergencies. For MN scoring, two approaches were applied using the Metafer4 platform (MetaSystems, Germany): fully automated scoring and semi-automated scoring with visual inspection of the gallery of MN-positive objects. Dose-response curves were established for acute and protracted whole-body and partial-body exposures. A database of background MN yields was set up, allowing determination of the dose detection threshold in both scoring modes. An analysis of the overdispersion of the MN frequency distribution σ(2)/µ obtained by semi-automated scoring showed that the value of this parameter represents a reliability check of the calculated equivalent total body dose in case the accident overexposure is a partial-body exposure. The elaborated methodology was validated in an accident training exercise. Overall, the semi-automated scoring procedure represents important added value to the automated MN assay.

Validation of semi-automatic scoring of dicentric chromosomes after simulation of three different irradiation scenarios.

Large scale radiological emergencies require high throughput techniques of biological dosimetry for population triage in order to identify individuals indicated for medical treatment. The dicentric assay is the "gold standard" technique for the performance of biological dosimetry, but it is very time consuming and needs well trained scorers. To increase the throughput of blood samples, semi-automation of dicentric scoring was investigated in the framework of the MULTIBIODOSE EU FP7 project, and dose effect curves were established in six biodosimetry laboratories. To validate these dose effect curves, blood samples from 33 healthy donors (>10 donors/scenario) were irradiated in vitro with 6°Co gamma rays simulating three different exposure scenarios: acute whole body, partial body, and protracted exposure, with three different doses for each scenario. All the blood samples were irradiated at Ghent University, Belgium, and then shipped blind coded to the participating laboratories. The blood samples were set up by each lab using their own standard protocols, and metaphase slides were prepared to validate the calibration curves established by semi-automatic dicentric scoring. In order to achieve this, 300 metaphases per sample were captured, and the doses were estimated using the newly formed dose effect curves. After acute uniform exposure, all laboratories were able to distinguish between 0 Gy, 0.5 Gy, 2.0, and 4.0 Gy (p < 0.001), and, in most cases, the dose estimates were within a range of ± 0.5 Gy of the given dose. After protracted exposure, all laboratories were able to distinguish between 1.0 Gy, 2.0 Gy, and 4.0 Gy (p < 0.001), and here also a large number of the dose estimates were within ± 0.5 Gy of the irradiation dose. After simulated partial body exposure, all laboratories were able to distinguish between 2.0 Gy, 4.0 Gy, and 6.0 Gy (p < 0.001). Overdispersion of the dicentric distribution enabled the detection of the partial body samples; however, this result was clearly dose-dependent. For partial body exposures, only a few dose estimates were in the range of ± 0.5 Gy of the given dose, but an improvement could be achieved with higher cell numbers. The new method of semi-automation of the dicentric assay was introduced successfully in a network of six laboratories. It is therefore concluded that this method can be used as a high-throughput screening tool in a large-scale radiation accident.

Web-based scoring of the dicentric assay, a collaborative biodosimetric scoring strategy for population triage in large scale radiation accidents.

In the case of a large scale radiation accident high throughput methods of biological dosimetry for population triage are needed to identify individuals requiring clinical treatment. The dicentric assay performed in web-based scoring mode may be a very suitable technique. Within the MULTIBIODOSE EU FP7 project a network is being established of 8 laboratories with expertise in dose estimations based on the dicentric assay. Here, the manual dicentric assay was tested in a web-based scoring mode. More than 23,000 high resolution images of metaphase spreads (only first mitosis) were captured by four laboratories and established as image galleries on the internet (cloud). The galleries included images of a complete dose effect curve (0-5.0 Gy) and three types of irradiation scenarios simulating acute whole body, partial body and protracted exposure. The blood samples had been irradiated in vitro with gamma rays at the University of Ghent, Belgium. Two laboratories provided image galleries from Fluorescence plus Giemsa stained slides (3 h colcemid) and the image galleries from the other two laboratories contained images from Giemsa stained preparations (24 h colcemid). Each of the 8 participating laboratories analysed 3 dose points of the dose effect curve (scoring 100 cells for each point) and 3 unknown dose points (50 cells) for each of the 3 simulated irradiation scenarios. At first all analyses were performed in a QuickScan Mode without scoring individual chromosomes, followed by conventional scoring (only complete cells, 46 centromeres). The calibration curves obtained using these two scoring methods were very similar, with no significant difference in the linear-quadratic curve coefficients. Analysis of variance showed a significant effect of dose on the yield of dicentrics, but no significant effect of the laboratories, different methods of slide preparation or different incubation times used for colcemid. The results obtained to date within the MULTIBIODOSE project by a network of 8 collaborating laboratories throughout Europe are very promising. The dicentric assay in the web based scoring mode as a high throughput scoring strategy is a useful application for biodosimetry in the case of a large scale radiation accident.

Comparison of established and emerging biodosimetry assays.

Rapid biodosimetry tools are required to assist with triage in the case of a large-scale radiation incident. Here, we aimed to determine the dose-assessment accuracy of the well-established dicentric chromosome assay (DCA) and cytokinesis-block micronucleus assay (CBMN) in comparison to the emerging γ-H2AX foci and gene expression assays for triage mode biodosimetry and radiation injury assessment. Coded blood samples exposed to 10 X-ray doses (240 kVp, 1 Gy/min) of up to 6.4 Gy were sent to participants for dose estimation. Report times were documented for each laboratory and assay. The mean absolute difference (MAD) of estimated doses relative to the true doses was calculated. We also merged doses into binary dose categories of clinical relevance and examined accuracy, sensitivity and specificity of the assays. Dose estimates were reported by the first laboratories within 0.3-0.4 days of receipt of samples for the γ-H2AX and gene expression assays compared to 2.4 and 4 days for the DCA and CBMN assays, respectively. Irrespective of the assay we found a 2.5-4-fold variation of interlaboratory accuracy per assay and lowest MAD values for the DCA assay (0.16 Gy) followed by CBMN (0.34 Gy), gene expression (0.34 Gy) and γ-H2AX (0.45 Gy) foci assay. Binary categories of dose estimates could be discriminated with equal efficiency for all assays, but at doses ≥1.5 Gy a 10% decrease in efficiency was observed for the foci assay, which was still comparable to the CBMN assay. In conclusion, the DCA has been confirmed as the gold standard biodosimetry method, but in situations where speed and throughput are more important than ultimate accuracy, the emerging rapid molecular assays have the potential to become useful triage tools.

Laboratory intercomparison of the cytokinesis-block micronucleus assay.

The focus of the study is an intercomparison of laboratories' dose-assessment performances using the cytokinesis-block micronucleus (CBMN) assay as a diagnostic triage tool for individual radiation dose assessment. Homogenously X-irradiated (240 kVp, 1 Gy/min) blood samples for establishing calibration data (0.25-5 Gy) as well as blind samples (0.1-6.4 Gy) were sent to the participants. The CBMN assay was performed according to protocols individually established and varying among participating laboratories. The time taken to report dose estimates was documented for each laboratory. Additional information concerning laboratory organization/characteristics as well as assay performance was collected. The mean absolute difference (MAD) was calculated and radiation doses were merged into four triage categories reflecting clinical aspects to calculate accuracy, sensitivity and specificity. The earliest report time was 4 days after sample arrival. The CBMN dose estimates were reported with high accuracy (MAD values of 0.20-0.50 Gy at doses below 6.4 Gy for both manual and automated scoring procedures), but showed a limitation of the assay at the dose point of 6.4 Gy, which resulted in a clear dose underestimation in all cases. The MAD values (without 6.4 Gy) differed significantly (P = 0.03) between manual (0.25 Gy, SEM = 0.06, n = 4) or automated scoring procedures (0.37 Gy, SEM = 0.08, n = 5), but lowest MAD were equal (0.2 Gy) for both scoring procedures. Likewise, both scoring procedures led to the same allocation of dose estimates to triage categories of clinical significance (about 83% accuracy and up to 100% specificity).

Automatic scoring of dicentric chromosomes as a tool in large scale radiation accidents.

Mass casualty scenarios of radiation exposure require high throughput biological dosimetry techniques for population triage in order to rapidly identify individuals who require clinical treatment. The manual dicentric assay is a highly suitable technique, but it is also very time consuming and requires well trained scorers. In the framework of the MULTIBIODOSE EU FP7 project, semi-automated dicentric scoring has been established in six European biodosimetry laboratories. Whole blood was irradiated with a Co-60 gamma source resulting in 8 different doses between 0 and 4.5Gy and then shipped to the six participating laboratories. To investigate two different scoring strategies, cell cultures were set up with short term (2-3h) or long term (24h) colcemid treatment. Three classifiers for automatic dicentric detection were applied, two of which were developed specifically for these two different culture techniques. The automation procedure included metaphase finding, capture of cells at high resolution and detection of dicentric candidates. The automatically detected dicentric candidates were then evaluated by a trained human scorer, which led to the term 'semi-automated' being applied to the analysis. The six participating laboratories established at least one semi-automated calibration curve each, using the appropriate classifier for their colcemid treatment time. There was no significant difference between the calibration curves established, regardless of the classifier used. The ratio of false positive to true positive dicentric candidates was dose dependent. The total staff effort required for analysing 150 metaphases using the semi-automated approach was 2 min as opposed to 60 min for manual scoring of 50 metaphases. Semi-automated dicentric scoring is a useful tool in a large scale radiation accident as it enables high throughput screening of samples for fast triage of potentially exposed individuals. Furthermore, the results from the participating laboratories were comparable which supports networking between laboratories for this assay.

Heme deficiency of soluble guanylate cyclase induces gastroparesis.

BACKGROUND: Soluble guanylate cyclase (sGC) is the principal target of nitric oxide (NO) to control gastrointestinal motility. The consequence on nitrergic signaling and gut motility of inducing a heme-free status of sGC, as induced by oxidative stress, was investigated. METHODS: sGCß1 (H105F) knock-in (apo-sGC) mice, which express heme-free sGC that has basal activity, but cannot be stimulated by NO, were generated. KEY RESULTS: Diethylenetriamine NONOate did not increase sGC activity in gastrointestinal tissue of apo-sGC mice. Exogenous NO did not induce relaxation in fundic, jejunal and colonic strips, and pyloric rings of apo-sGC mice. The stomach was enlarged in apo-sGC mice with hypertrophy of the muscularis externa of the fundus and pylorus. In addition, gastric emptying and intestinal transit were delayed and whole-gut transit time was increased in the apo-sGC mice, while distal colonic transit time was maintained. The nitrergic relaxant responses to electrical field stimulation at 1-4 Hz were abolished in fundic and jejunal strips from apo-sGC mice, but in pyloric rings and colonic strips, only the response at 1 Hz was abolished, indicating the contribution of other transmitters than NO. CONCLUSIONS & INFERENCES: The results indicate that the gastrointestinal consequences of switching from a native sGC to a heme-free sGC, which cannot be stimulated by NO, are most pronounced at the level of the stomach establishing a pivotal role of the activation of sGC by NO in normal gastric functioning. In addition, delayed intestinal transit was observed, indicating that nitrergic activation of sGC also plays a role in the lower gastrointestinal tract.

Topographic and quantitative relationship between prostate inflammation, proliferative inflammatory atrophy and low-grade prostate intraepithelial neoplasia: a biopsy study in chronic prostatitis patients.

Inflammatory processes are important components in the pathogenesis of many human cancers. According to the 'injury and regeneration' model for prostate carcinogenesis, injury caused by pathogens or pro-inflammatory cytotoxic agents would trigger proliferation of prostatic glandular cells, leading to the appearance of epithelial lesions named 'Proliferative Inflammatory Atrophy' (PIA). Inflammatory cells infiltrating the prostate would release genotoxic reactive oxygen species, leading atrophic cells to neoplastic progression. The hypothesis pointing to PIA as risk-lesion for prostate cancer has been extensively investigated at the cellular and molecular levels, but few morphological data are available linking PIA or prostatic intraepithelial neoplasia (PIN) to inflammation or clinical prostatitis. We investigated at the morphological level 1367 prostate biopsies from 98 patients with a recent history of chronic prostatitis, and 32 patients with biopsies positive for carcinoma. Our results show that i) PIA is found more frequently in biopsy cores containing a severe or moderate inflammatory focus, compared to NON-PIA lesions (partial or cystic atrophy); ii) the PIA lesion post-atrophic hyperplasia is more frequently found in tissues showing mild or no inflammation; iii) the extent of PIA per patient correlates with the burden of moderate or severe inflammation, whereas NON-PIA lesions do not; iv) low-grade PIN is in over 90% of cases emerging from normal, non-atrophic glands and is more frequently found in biopsy cores with absent or mild inflammatory burden; v) the inverse relationship between the prevalence of low-grade PIN and the extent of PIA lesions per patient is described by a power law function, suggesting the low likelihood of the concomitant presence of these lesions in the same tissue; vi) NON-PIA lesions correlate inversely with neoplasia in patients with prostate cancer; vii) the total scores of the NIH-CPSI questionnaire correlate with both PIA and inflammation burdens at diagnosis of prostatitis but not after pharmacological intervention. These results point to a positive association between tissue inflammation, clinical prostatitis and the putative cancer risk-lesion PIA, but do not support a model whereby low-grade PIN would arise from PIA.

Realising the European Network of Biodosimetry (RENEB).

In Europe, a network for biological dosimetry has been created to strengthen the emergency preparedness and response capabilities in case of a large-scale nuclear accident or radiological emergency. Through the RENEB (Realising the European Network of Biodosimetry) project, 23 experienced laboratories from 16 European countries will establish a sustainable network for rapid, comprehensive and standardised biodosimetry provision that would be urgently required in an emergency situation on European ground. The foundation of the network is formed by five main pillars: (1) the ad hoc operational basis, (2) a basis of future developments, (3) an effective quality-management system, (4) arrangements to guarantee long-term sustainability and (5) awareness of the existence of RENEB. RENEB will thus provide a mechanism for quick, efficient and reliable support within the European radiation emergency management. The scientific basis of RENEB will concurrently contribute to increased safety in the field of radiation protection.

Pulpal regeneration and root development after subcutaneous transplantation of cryopreserved immature teeth in rats.

The purpose of this in vivo study was to investigate revascularization and root growth after autotransplantation of cryopreserved immature teeth. Immature molar teeth were extracted in 4-week-old Wistar rats. In the test group, teeth were cryopreserved for 1 week and transplanted subcutaneously to the abdomen. In the control group, teeth were transplanted subcutaneously immediately after extraction. Material was collected in test and control animals at intervals of 1, 2, 4 and 10 weeks post-transplantation and histological and microradiographical examination was performed. Results showed that during the first weeks after transplantation, pulpal repair was similar in both groups although degenerated pulpal tissue was replaced slower in cryopreserved teeth and some differences in types of hard tissue formation were found between test and control teeth. After 10 weeks, the differences in the regenerated pulpal tissue between cryopreserved and control teeth observed during the first weeks were no longer detectable. No root growth was detected microradiographically 10 weeks after transplantation in any of the transplanted teeth. The presence of dentin-like tissue in the pulp cavity of some autotransplanted cryopreserved teeth, suggests survival of pulpal tissue after cryopreservation.

Combined effect of polymorphisms in Rad51 and Xrcc3 on breast cancer risk and chromosomal radiosensitivity.

Enhanced in vitro chromosomal radiosensitivity (CRS) has been proposed as a marker for low-penetrance gene mutations predisposing to breast cancer (BC). Since the double strand break (DSB) is the most detrimental form of DNA damage induced by ionizing radiation, it is possible that mutations in genes encoding proteins involved in DSB repair affect breast cancer risk. The purpose of the present study was to examine whether five single nucleotide polymorphisms (SNPs) in Rad51 and Xrcc3 (rs1801320, rs1801321, rs1799796, rs861539 and rs1799794) exhibited an association with breast cancer susceptibility in a Belgian population of BC patients with a known or putative genetic predisposition. We also ascertained whether a relationship exists between the occurrence of the variant alleles of these variations and in vitro CRS. Blood samples were obtained from BC patients and from the control population that included healthy female individuals. Variations in the 5' UTR of Rad51 and Xrcc3 were genotyped, and statistical analysis was performed. The results showed that low-penetrant variations in Rad51 and Xrcc3, two proteins belonging to the homologous recombination DSB repair pathway, may modify BC risk in patients already carrying a pathological mutation in the highly penetrant BC genes BRCA1 and BRCA2. Combined risk genotype analysis revealed that Rad51 SNPs enhance BC risk in BRCA2 patients, whereas Xrcc3 SNPs significantly enhance BC risk in carriers of BRCA1 mutations and in patients with hereditary BC. When four putative risk genotypes of Rad51 and Xrcc3 were combined, positive significant odds ratios were obtained in the entire patient population and in patients with a hereditary history of disease. Although obtained from a limited number of patients, our data are supportive of a polygenic model whereby combinations of weak variations are responsible for an enhanced BC risk by acting jointly with high-penetrant mutations in BRCA1 or BRCA2.

Review of retrospective dosimetry techniques for external ionising radiation exposures.

The current focus on networking and mutual assistance in the management of radiation accidents or incidents has demonstrated the importance of a joined-up approach in physical and biological dosimetry. To this end, the European Radiation Dosimetry Working Group 10 on 'Retrospective Dosimetry' has been set up by individuals from a wide range of disciplines across Europe. Here, established and emerging dosimetry methods are reviewed, which can be used immediately and retrospectively following external ionising radiation exposure. Endpoints and assays include dicentrics, translocations, premature chromosome condensation, micronuclei, somatic mutations, gene expression, electron paramagnetic resonance, thermoluminescence, optically stimulated luminescence, neutron activation, haematology, protein biomarkers and analytical dose reconstruction. Individual characteristics of these techniques, their limitations and potential for further development are reviewed, and their usefulness in specific exposure scenarios is discussed. Whilst no single technique fulfils the criteria of an ideal dosemeter, an integrated approach using multiple techniques tailored to the exposure scenario can cover most requirements.