RENEB Inter-Laboratory Comparison 2021: The Gamma-H2AX Foci Assay.

The Running the European Network of biological and retrospective dosimetry (RENEB) network of laboratories has a range of biological and physical dosimetry assays that can be deployed in the event of a radiation incident to provide exposure assessment. To maintain operational capability and provide training, RENEB runs regular inter-laboratory comparison (ILC) exercises. The RENEB ILC2021 was carried out with all the biological and physical dosimetry assays employed in the network. The focus of this paper is to evaluate the results from 6 laboratories that took part using the gamma-H2AX radiation-induced foci assay. For two laboratories this was their first RENEB ILC. Blood samples were homogenously exposed to 240 kVp X rays (1 Gy/min) to provide calibration data, (0-4 Gy), and a few weeks later three blind coded test samples, (0, 1.2 and 3.5 Gy) were prepared. All samples were allowed a 2 h repair time at 37°C before being transported, on ice packs, to the participating laboratories. On arrival, the samples were processed, scored either manually or automatically for gamma-H2AX foci and dose estimates for the 3 blind coded samples sent to the organizing laboratory. The temperature of samples during transit and the time taken to report the dose estimates were recorded. Subsequent examination of the data from each laboratory used the doses estimates to assign triage categories to the samples. After receipt of the samples, the quickest report of dose estimates was 4.6 h. Analysis of variance revealed that the laboratory carrying out the assay had a significant effect on the foci yield (P < 0.001) for the calibration data, but not on the dose estimates of the blind coded samples (P = 0.101). All laboratories correctly identified the unirradiated and irradiated samples, although the dose estimates for the latter tended to under-estimate the dose. Two participants seriously under-estimated the dose for the highly exposed sample, which resulted in the sample being placed in the lowest triage category not the highest. However, this under-estimation resulted from the samples not remaining cold during shipment, due to a delay in transit and was not related to the experience of the participating laboratory. Overall, the RENEB network laboratories have demonstrated it is possible to quickly identify a recent whole-body acute exposure using the gamma-H2AX assay within the conditions of the ILC. In addition, an ILC provides a useful training and harmonization exercise for laboratories.

Radiation field and dose inhomogeneities using an X-ray cabinet in radiation biology research.

PURPOSE: X-ray cabinets are replacing 137 Cs/60 Co sources in radiation biology research due to advantages in size, handling, and radiation protection. However, because of their different physical properties, X-ray cabinets are more susceptible to experimental influences than conventional sources. The aim of this study was to examine the variations related to the experimental setups typically used to investigate biological radiation effects with X-ray cabinets. MATERIALS AND METHODS: A combined approach of physical dose measurements by thermoluminescence dosimetry and detection of biological effects by quantification of γH2AX and 53BP1 foci was used to analyze field inhomogeneity and evaluate the influence of the components of the experimental setup. RESULTS: Irradiation was performed using an X-ray tube (195 kV, 10 mA, 0.5-mm-thick copper filter, dose rate of 0.59 Gy/min). Thermoluminescence dosimetry revealed inhomogeneity and a dose decrease of up to 42.3% within the beam area (diameter 31.1 cm) compared to the dose at the center. This dose decrease was consistent with the observed decline in the number of radiation-induced foci by up to 55.9 %. Uniform dose distribution was measured after reducing the size of the radiation field (diameter 12.5 cm). However, when using 15-ml test tubes placed at different positions within this field, the dose decreased by up to 17% in comparison to the central position. Analysis of foci number revealed significant differences between the tubes for γH2AX (1 h) and 53BP1 (4 h) at different time points after irradiation. Neither removal of some tubes nor of the caps improved the dose decrease significantly. By contrast, when using 1.5-ml tubes, dose differences were less than 4%, and no significant differences in foci number were detected. CONCLUSION: X-ray cabinets are user-friendly irradiation units for investigating biological radiation effects. However, field inhomogeneities and experimental setup components considerably affect the delivered irradiation doses. For this reason, strict dosimetric monitoring of experimental irradiation setups is mandatory for reliable studies.

Analysis of chromosomal aberrations and γH2A.X foci to identify radiation-sensitive ataxia-telangiectasia patients.

Ataxia-telangiectasia (AT) is a rare inherited recessive disorder which is caused by a mutated Ataxia-telangiectasia mutated (ATM) gene. Hallmarks include chromosomal instability, cancer predisposition and increased sensitivity to ionizing radiation. The ATM protein plays an important role in signaling of DNA double-strand breaks (DSB), thereby phosphorylating the histone H2A.X. Non-functional ATM protein leads to defects in DNA damage response, unresolved DSBs and genomic instability. The aim of this study was to evaluate chromosomal aberrations and γH2A.X foci as potential radiation sensitivity biomarkers in AT patients. For this purpose, lymphocytes of 8 AT patients and 10 healthy controls were irradiated and induced DNA damage and DNA repair capacity were detected by the accumulation of γH2A.X foci. The results were heterogeneous among AT patients. Evaluation revealed 2 AT patients with similar γH2A.X foci numbers as controls after 1 h while 3 patients showed a lower induction. In regard to DNA repair, 3 of 5 AT patients showed poor damage repair. Therefore, DNA damage induction and DNA repair as detected by H2A.X phosphorylation revealed individual differences, seems to depend on the underlying individual mutation and thus appears not well suited as a biomarker for radiation sensitivity. In addition, chromosomal aberrations were analyzed by mFISH. An increased frequency of spontaneous chromosomal breakage was characteristic for AT cells. After irradiation, significantly increased rates for non-exchange aberrations, translocations, complex aberrations and dicentric chromosomes were observed in AT patients compared to controls. The results of this study suggested, that complex aberrations and dicentric chromosomes might be a reliable biomarker for radiation sensitivity in AT patients, while non-exchange aberrations and translocations identified both, spontaneous and radiation-induced chromosomal instability.

Comparison of inexperienced operators and experts in γH2A.X and 53BP1 foci assay for high-throughput biodosimetry approaches in a mass casualty incident.

PURPOSE: In case of population exposure by ionizing radiation, a fast and reliable dose assessment of exposed and non-exposed individuals is crucial important. In initial triage, physicians have to take fast decisions whom to treat with adequate medical care. In addition, worries about significant exposure can be taken away from hundreds to thousands non- or low exposed individuals. Studies have shown that the γH2A.X radiation-induced foci assay is a promising test for fast triage decisions. However, in a large-scale scenario most biodosimetry laboratories will quickly reach their capacity limit. The aim of this study was to evaluate the benefit of inexperienced experimenters to speed up the foci assay and manual foci scoring. MATERIALS AND METHODS: The participants of two training courses performed the radiation-induced foci assay (γH2A.X) under the guidance of experts and scored foci (γH2A.X and 53BP1) on sham-irradiated and irradiated blood samples (0.05-1.5 Gy). The outcome of laboratory experiments and manual foci scoring by 26 operators with basic experience in laboratory work was statistically analyzed in comparison to the results from experts. RESULTS: Inexperienced operators prepared slides with significant dose-effects (0, 0.1 and 1.0 Gy) for semi-automatic microscopic analyses. Manual foci scoring by inexperienced scorer resulted in a dose-effect curve for γH2A.X, 53BP1 and co-localized foci. In addition, inexperienced scorers were able to distinguish low irradiation doses from unirradiated cells. While 53BP1 foci scoring was in accordance to the expert counting, differences between beginners and expert increased for γH2A.X or co-localized foci. CONCLUSIONS: In case of a large-scale radiation event, inexperienced staff is useful to support laboratories in slide preparation for semi-automatic foci counting as well as γH2A.X and 53BP1 manual foci scoring for triage-mode biodosimetry. Slides can be clearly classified in the non-, low- or high-exposed category.

EPI-CT: in vitro assessment of the applicability of the γ-H2AX-foci assay as cellular biomarker for exposure in a multicentre study of children in diagnostic radiology.

PURPOSE: To conduct a feasibility study on the application of the γ-H2AX foci assay as an exposure biomarker in a prospective multicentre paediatric radiology setting. MATERIALS AND METHODS: A set of in vitro experiments was performed to evaluate technical hurdles related to biological sample collection in a paediatric radiology setting (small blood sample volume), processing and storing of blood samples (effect of storing blood at 4°C), the reliability of foci scoring for low-doses (merge γ-H2AX/53BP1 scoring), as well as the impact of contrast agent administration as potential confounding factor. Given the exploratory nature of this study and the ethical constraints related to paediatric blood sampling, blood samples from adult volunteers were used for these experiments. In order to test the feasibility of pooling the γ-H2AX data when different centres are involved in an international multicentre study, two intercomparison studies in the low-dose range (10-500 mGy) were performed. RESULTS: Determination of the number of X-ray induced γ-H2AX foci is feasible with one 2 ml blood sample pre- and post-computed tomography (CT) scan. Lymphocyte isolation and fixation on slides is necessary within 5 h of blood sampling to guarantee reliable results. The possible enhancement effect of contrast medium on the induction of DNA DSB in a patient study can be ruled out if radiation doses and the contrast agent concentration are within diagnostic ranges. The intercomparison studies using in vitro irradiated blood samples showed that the participating laboratories, executing successfully the γ-H2AX foci assay in lymphocytes, were able to rank blind samples in order of lowest to highest radiation dose based on mean foci/cell counts. The dose response of all intercomparison data shows that a dose point of 10 mGy could be distinguished from the sham-irradiated control (p = 0.006). CONCLUSIONS: The results demonstrate that it is feasible to apply the γ-H2AX foci assay as a cellular biomarker of exposure in a multicentre prospective study in paediatric CT imaging after validating it in an in vivo international pilot study on paediatric patients.