RENEB Inter-Laboratory Comparison 2021: The Gene Expression Assay

Abstract

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.

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

“…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)(27)(28)(29)(30)(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 ¼ 0.63) at the Bundeswehr Institute of Radiobiology, Munich, Germany and sent to the participating laboratories.…”

“…For yH2AX, the highest dose point was underestimated, due to saturation of the foci signal (yH2AX). In the assay specific papers (26,27,29,30,31) of this special issue and the inter-assay comparison paper (28) these observations are discussed in detail. In most cases it was possible to identify methodological problems which can help the participants with problematic results to improve their workflow in future.…”

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

“…This can be achieved by using high throughput automated systems, (2,3), or adaptation of protocols (4)(5)(6), or laboratories combining their efforts within a network (7)(8)(9). Standardization and validation of exposure biomarkers/ physical dosimetry within a network are essential to ensure that dose assessment from different laboratories is consistent and all networks carry out regular inter-comparisons to evaluate their performance (e.g., [10][11][12][13][14][15][16].…”

The DNA damage response to radiological imaging: from ROS and γH2AX foci induction to gene expression responses in vivo

“…Numerous validation efforts have been published for the γH2AX assay (Ainsbury et al 2014 ; Barnard et al 2015 ; Rothkamm et al 2013 ) and gene expression (Abend et al 2021 , 2023 , 2016 ; Badie et al 2013 ; Biolatti et al 2021 ; Manning et al 2017 ) as IR biomarkers sensitive to doses in the mGy range (Schule et al 2023 ). γH2AX was tested as a biomarker of DNA damage and repair in human population studies related to diagnostic procedures (Brand et al 2012 ; Halm et al 2014 ; Kuefner et al 2009 ; Lobrich et al 2005 ; Pathe et al 2011 ; Rothkamm et al 2007 ; Vandevoorde et al 2015a ), chemotherapy (Halicka et al 2009 ; Karp et al 2007 ; Sak et al 2009 ) and/or radiotherapy (Sak et al 2007 ; Zahnreich et al 2015 ; Zwicker et al 2011 ), as previously reviewed (Valdiglesias et al 2013 ).…”