Laboratory Intercomparison on the γ-H2AX Foci Assay

Abstract

The focus of the study is an intercomparison of laboratories' dose-assessment performances using the γ-H2AX foci assay as a diagnostic triage tool for rapid individual radiation dose assessment. Homogenously X-irradiated (240 kVp, 1 Gy/min) blood samples for establishing calibration data (0.25-4 Gy) as well as blinded test samples (0.1-6.4 Gy) were incubated at 37°C for 2 and 24 h (repair time) and sent to the participants. The foci assay was performed according to protocols individually established in participating laboratories and therefore varied. 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) of estimated doses relative to the actual doses was calculated and radiation doses were merged into four triage categories reflecting clinical relevance to calculate accuracy, sensitivity and specificity. First γ-H2AX based dose estimates were reported 7 h after sample receipt. Estimates were similarly accurate for 2 and 24 h repair times, providing scope for its use in the early phase of a radiation exposure incident. Equal accuracy was achieved by scoring 20, 30, 40 or 50 cells per sample. However, MAD values of 0.5-0.7 Gy and 1.3-1.7 Gy divided the data sets into two groups, driven mainly by the considerable differences in foci yields between calibration and blind samples. Foci yields also varied dramatically between laboratories, highlighting reproducibility issues as an important caveat of the foci assay. Nonetheless, foci counts could distinguish high- and low-dose samples in all data sets and binary dose categories of clinical significance could be discriminated with satisfactory accuracy (mean 84%, ±0.03 SEM). Overall, the results suggest that the γ-H2AX assay is a useful tool for rapidly screening individuals for significant exposures that occurred up to at least 24 h earlier, and may help to prioritize cytogenetic dosimetry follow-up.

Effect of Dose Rate on Residual γ-H2AX Levels and Frequency of Micronuclei in X-Irradiated Mouse Lymphocytes

“…During the past decade, the γ-H2AX assay has been applied to a variety of cell types and tissues to correlate γ-H2AX levels with DNA damage and repair (16). Recent studies have aimed to validate the application of the γ-H2AX protein biomarker in human population studies and for rapid triage after large-scale radiation exposure (16, 19, 20). Numerous γ-H2AX-based biodosimetry studies have established and validated a linear radiation dose response in human lymphocytes irradiated ex vivo (20–22) and in vivo (23–25) using immunofluorescent techniques.…”

The second gamma-H2AX assay inter-comparison exercise carried out in the framework of the European biodosimetry network (RENEB)

“…Timed intercomparisons have been described by Rothkamm et al (2013a) and Ainsbury et al (2014) with triage dose estimates, based on foci counts in 50 cells, reported within 7 h (10 samples) and 3 h (8 samples), respectively. With a more rapid processing technique and scoring just 20 cells it may be possible to produce dose estimates for 96 samples in 7-11 h (Moquet et al 2014).…”

“…With a more rapid processing technique and scoring just 20 cells it may be possible to produce dose estimates for 96 samples in 7-11 h (Moquet et al 2014). Past experience (Rothkamm et al 2013a;Ainsbury et al 2014) and the first inter-comparison have shown that for rapid triage purposes, i.e. detection of moderate to high doses, manual scoring of 50 cells or 200 cells scored automatically, is fully sufficient and even scoring 20 cells manually or 50 automatically, may be sufficiently accurate in a triage setting.…”

“…Several studies have shown that rapid processing time of the gamma-H2AX assay can produce dose estimates within a few hours of receiving a blood sample (Rothkamm et al 2013a;Ainsbury et al 2014). In addition, the potential for batch processing on finger prick blood samples, enabling high throughput (Moquet et al 2014), makes the assay ideal for early triage categorization to separate the 'worried well' from critically exposed individuals by quantifying radiation-induced foci in peripheral blood lymphocytes.…”

Biodosimetry versus physical dosimetry for emergency dose assessment following large-scale radiological exposures

“…Although not inclusive of all biomarkers being proposed for dosimetry the list analyzed by Ainsbury et al (2011) is comprehensive and reflective of the main proposed methods. Nevertheless, other methods have been suggested for use in triage, such as g-H2AX (Rothkamm et al, 2013;Jaworska et al, 2015;Barnard et al, 2015) or MN Jaworska et al, 2015). However, the results of the intercomparisons mentioned in these publications cannot be included in the current analysis since the mean values for the reconstructed doses are given but there is no published information about the uncertainties.…”