RENEB Inter-Laboratory Comparison 2021: The FISH-Based Translocation Assay

Abstract

Translocation analysis using fluorescence in situ hybridization (FISH) is the method of choice for dose assessment in case of chronic or past exposures to ionizing radiation. Although it is a widespread technique, unlike dicentrics, the number of FISH-based inter-laboratory comparisons is small. For this reason, although the current Running the European Network of Biological and Physical retrospective Dosimetry (RENEB) inter-laboratory comparison 2021 was designed as a fast response to a real emergency scenario, it was considered a good opportunity to perform an inter-laboratory comparison using the FISH technique to gain further experience. The Bundeswehr Institute of Radiobiology provided peripheral blood samples from one healthy human volunteer. Three test samples were irradiated with blinded doses of 0, 1.2, and 3.5 Gy, respectively. Samples were then sent to the seven participating laboratories. The FISH technique was applied according to the standard procedure of each laboratory. Both, the frequency of translocations and the estimated dose for each sample were sent to the coordinator using a special scoring sheet for FISH. All participants sent their results in due time. However, although it was initially requested to send the results based on the full analysis, evaluating 500 equivalent cells, most laboratories only sent the results based on triage, with a smaller number of analyzed cells. In the triage analysis, there was great heterogeneity in the number of equivalent cells scored. On the contrary, for the full analysis, this number was more homogeneous. For all three samples, one laboratory showed outlier yields compared to the other laboratories. Excluding these results, in the triage analysis, the frequency of translocations in sample no. 1 ranged from 0 to 0.013 translocations per cell, and for samples no. 2 and no. 3 the genomic mean frequency were 0.27 ± 0.03 and 1.47 ± 0.14, with a coefficient of variation of 0.29 and 0.23 respectively. Considering only results obtained in the triage analysis for sample no. 1, all laboratories, except one, classified this sample as the non-irradiated one. For sample no. 2, excluding the outlier value, the mean reported dose was 1.74 ± 0.16 Gy indicating a mean deviation of about 0.5 Gy to the delivered dose of 1.2 Gy. For sample no. 3 the mean dose estimated was 4.21 ± 0.21 Gy indicating a mean deviation of about 0.7 Gy to the delivered dose of 3.5 Gy. In the frame of RENEB, this is the second FISH-based inter-laboratory comparison. The whole exercise was planned as a response to an emergency, therefore, a triage analysis was requested for all the biomarkers except for FISH. Although a full analysis was initially requested for FISH, most of the laboratories reported only a triage-based result. The main reason is that it was not clearly stated what was required before starting the exercise. Results show that most of the laboratories successfully discriminated unexposed and irradiated samples from each other without any overlap. A good agreement in the observed frequencies of translocations was observed but there was a tendency to overestimate the delivered doses. Efforts to improve the harmonization of this technique and subsequent exercises to elucidate the reason for this trend should be promoted.

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

“…Also, at higher doses (1.2 Gy and 3.5 Gy), a systematic shift towards higher dose estimates by cytogenetic-based assays was observed. Detailed discussions of these unexpected results and possible reasons are presented in the following series of manuscripts, each focusing on one assay ( 47–51 ). Furthermore, for GE and gH2AX assays, another fraction of dose estimates lying below or above the accepted uncertainty dose interval was reported by most teams (Fig.…”

“…The performance of quality controlled ILC exercises using ex vivo irradiated, blind coded samples are a common measure in RENEB to validate assay performances as well as laboratories. The RENEB ILC 2021 presented here in this inter-assay comparison article and followed by assay specific publications (47)(48)(49)(50)(51) has several unique features: 1. Comparing, in parallel, established cytogenetic assays (DCA, CBMN, PCC, FISH) with molecular biology (GE, gH2AX) and physical dosimetry assays (EPR, LUM); 2.…”

“…The analysis of cytogenetic assays revealed a systematic shift towards higher dose estimates, while dose estimates of other assays were partly biased by methodological issues and limits. These challenges will be addressed in subsequent publications within this special issue (47)(48)(49)(50)(51), with each focusing on results of a specific assay, except the PCC assay where only two teams participated and the physical dosimetry assays (EPR, OSL, TL), which will be presented in later publications. However, within this inter-assay comparison manuscript, these first dose estimates were analyzed unfiltered (except for elimination of redundantly reported dose estimates) to simulate a scenario, where early (possibly partly biased) dose estimates are required to support immediate medical management.…”

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

“…Using a conversion factor between enamel/air kerma of 6.5 ( 28 ), the results agreed well with the air kerma references. Moreover, for cytogenetic assays (DCA, CBMN, FISH and PCC) the dose estimates were systematically higher than the reference doses ( 26–29 ). This systematic shift can partly be attributed to differences in the biological effectiveness between the irradiation sources used for the establishment of calibration curves (mostly 60 Co or 137 Cs gamma rays) and for the exposure of the samples of this ILC (X rays, 240 kVp).…”

“…In total 46 organizations from 27 countries participated in the RENEB ILC 2021. Several assays for biological dosimetry (DCA, CBMN, FISH, GE, yH2AX, PCC) and physical retrospective dosimetry (EPR, OSL, TL) were applied (Table 2) and the results for most of the assays are published in a number of papers in this special issue ( 26–31 ). Blood was irradiated in terms of dose in water and various different materials (electronic components and glass from mobile phones, watch glass and mini-biopsies of tooth enamel) were irradiated in terms of air kerma with 0, 1.2 and 3.5 Gy X-ray doses (240 kVp, 1 Gy/min, ∼75 keV, 13 mA, HVL = 0.63) at the Bundeswehr Institute of Radiobiology, Munich, Germany and sent to the participating laboratories.…”