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.

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.

RENEB Inter-Laboratory Comparison 2021: The Gene Expression Assay.

Early and high-throughput individual dose estimates are essential following large-scale radiation exposure events. In the context of the Running the European Network for Biodosimetry and Physical Dosimetry (RENEB) 2021 exercise, gene expression assays were conducted and their corresponding performance for dose-assessment is presented in this publication. Three blinded, coded whole blood samples from healthy donors were exposed to 0, 1.2 and 3.5 Gy X-ray doses (240 kVp, 1 Gy/min) using the X-ray source Yxlon. These exposures correspond to clinically relevant groups of unexposed, low dose (no severe acute health effects expected) and high dose exposed individuals (requiring early intensive medical health care). Samples were sent to eight teams for dose estimation and identification of clinically relevant groups. For quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microarray analyses, samples were lysed, stored at 20°C and shipped on wet ice. RNA isolations and assays were run in each laboratory according to locally established protocols. The time-to-result for both rough early and more precise later reports has been documented where possible. Accuracy of dose estimates was calculated as the difference between estimated and reference doses for all doses (summed absolute difference, SAD) and by determining the number of correctly reported dose estimates that were defined as ±0.5 Gy for reference doses <2.5 Gy and ±1.0 Gy for reference doses >3 Gy, as recommended for triage dosimetry. We also examined the allocation of dose estimates to clinically/diagnostically relevant exposure groups. Altogether, 105 dose estimates were reported by the eight teams, and the earliest report times on dose categories and estimates were 5 h and 9 h, respectively. The coefficient of variation for 85% of all 436 qRT-PCR measurements did not exceed 10%. One team reported dose estimates that systematically deviated several-fold from reported dose estimates, and these outliers were excluded from further analysis. Teams employing a combination of several genes generated about two-times lower median SADs (0.8 Gy) compared to dose estimates based on single genes only (1.7 Gy). When considering the uncertainty intervals for triage dosimetry, dose estimates of all teams together were correctly reported in 100% of the 0 Gy, 50% of the 1.2 Gy and 50% of the 3.5 Gy exposed samples. The order of dose estimates (from lowest to highest) corresponding to three dose categories (unexposed, low dose and highest exposure) were correctly reported by all teams and all chosen genes or gene combinations. Furthermore, if teams reported no exposure or an exposure >3.5 Gy, it was always correctly allocated to the unexposed and the highly exposed group, while low exposed (1.2 Gy) samples sometimes could not be discriminated from highly (3.5 Gy) exposed samples. All teams used FDXR and 78.1% of correct dose estimates used FDXR as one of the predictors. Still, the accuracy of reported dose estimates based on FDXR differed considerably among teams with one team's SAD (0.5 Gy) being comparable to the dose accuracy employing a combination of genes. Using the workflow of this reference team, we performed additional experiments after the exercise on residual RNA and cDNA sent by six teams to the reference team. All samples were processed similarly with the intention to improve the accuracy of dose estimates when employing the same workflow. Re-evaluated dose estimates improved for half of the samples and worsened for the others. In conclusion, this inter-laboratory comparison exercise enabled (1) identification of technical problems and corrections in preparations for future events, (2) confirmed the early and high-throughput capabilities of gene expression, (3) emphasized different biodosimetry approaches using either only FDXR or a gene combination, (4) indicated some improvements in dose estimation with FDXR when employing a similar methodology, which requires further research for the final conclusion and (5) underlined the applicability of gene expression for identification of unexposed and highly exposed samples, supporting medical management in radiological or nuclear scenarios.

Inter-laboratory comparison of gene expression biodosimetry for protracted radiation exposures as part of the RENEB and EURADOS WG10 2019 exercise.

Large-scale radiation emergency scenarios involving protracted low dose rate radiation exposure (e.g. a hidden radioactive source in a train) necessitate the development of high throughput methods for providing rapid individual dose estimates. During the RENEB (Running the European Network of Biodosimetry) 2019 exercise, four EDTA-blood samples were exposed to an Iridium-192 source (1.36 TBq, Tech-Ops 880 Sentinal) at varying distances and geometries. This resulted in protracted doses ranging between 0.2 and 2.4 Gy using dose rates of 1.5-40 mGy/min and exposure times of 1 or 2.5 h. Blood samples were exposed in thermo bottles that maintained temperatures between 39 and 27.7 °C. After exposure, EDTA-blood samples were transferred into PAXGene tubes to preserve RNA. RNA was isolated in one laboratory and aliquots of four blinded RNA were sent to another five teams for dose estimation based on gene expression changes. Using an X-ray machine, samples for two calibration curves (first: constant dose rate of 8.3 mGy/min and 0.5-8 h varying exposure times; second: varying dose rates of 0.5-8.3 mGy/min and 4 h exposure time) were generated for distribution. Assays were run in each laboratory according to locally established protocols using either a microarray platform (one team) or quantitative real-time PCR (qRT-PCR, five teams). The qRT-PCR measurements were highly reproducible with coefficient of variation below 15% in ≥ 75% of measurements resulting in reported dose estimates ranging between 0 and 0.5 Gy in all samples and in all laboratories. Up to twofold reductions in RNA copy numbers per degree Celsius relative to 37 °C were observed. However, when irradiating independent samples equivalent to the blinded samples but increasing the combined exposure and incubation time to 4 h at 37 °C, expected gene expression changes corresponding to the absorbed doses were observed. Clearly, time and an optimal temperature of 37 °C must be allowed for the biological response to manifest as gene expression changes prior to running the gene expression assay. In conclusion, dose reconstructions based on gene expression measurements are highly reproducible across different techniques, protocols and laboratories. Even a radiation dose of 0.25 Gy protracted over 4 h (1 mGy/min) can be identified. These results demonstrate the importance of the incubation conditions and time span between radiation exposure and measurements of gene expression changes when using this method in a field exercise or real emergency situation.