Validation of Semi-automatic Scoring of Dicentric Chromosomes after Simulation of Three Different Irradiation Scenarios

Abstract

Large scale radiological emergencies require high throughput techniques of biological dosimetry for population triage in order to identify individuals indicated for medical treatment. The dicentric assay is the "gold standard" technique for the performance of biological dosimetry, but it is very time consuming and needs well trained scorers. To increase the throughput of blood samples, semi-automation of dicentric scoring was investigated in the framework of the MULTIBIODOSE EU FP7 project, and dose effect curves were established in six biodosimetry laboratories. To validate these dose effect curves, blood samples from 33 healthy donors (>10 donors/scenario) were irradiated in vitro with ⁶⁰Co gamma rays simulating three different exposure scenarios: acute whole body, partial body, and protracted exposure, with three different doses for each scenario. All the blood samples were irradiated at Ghent University, Belgium, and then shipped blind coded to the participating laboratories. The blood samples were set up by each lab using their own standard protocols, and metaphase slides were prepared to validate the calibration curves established by semi-automatic dicentric scoring. In order to achieve this, 300 metaphases per sample were captured, and the doses were estimated using the newly formed dose effect curves. After acute uniform exposure, all laboratories were able to distinguish between 0 Gy, 0.5 Gy, 2.0, and 4.0 Gy (p < 0.001), and, in most cases, the dose estimates were within a range of ± 0.5 Gy of the given dose. After protracted exposure, all laboratories were able to distinguish between 1.0 Gy, 2.0 Gy, and 4.0 Gy (p < 0.001), and here also a large number of the dose estimates were within ± 0.5 Gy of the irradiation dose. After simulated partial body exposure, all laboratories were able to distinguish between 2.0 Gy, 4.0 Gy, and 6.0 Gy (p < 0.001). Overdispersion of the dicentric distribution enabled the detection of the partial body samples; however, this result was clearly dose-dependent. For partial body exposures, only a few dose estimates were in the range of ± 0.5 Gy of the given dose, but an improvement could be achieved with higher cell numbers. The new method of semi-automation of the dicentric assay was introduced successfully in a network of six laboratories. It is therefore concluded that this method can be used as a high-throughput screening tool in a large-scale radiation accident.

Integration of new biological and physical retrospective dosimetry methods into EU emergency response plans – joint RENEB and EURADOS inter-laboratory comparisons

“…The only notable difference was that lab DCA_1, scored both dicentrics and centric rings in manual mode, whereas all the other labs scored only dicentrics. All labs scored only complete cells with exactly 46 centromeres in manual scoring mode but for semi-automatic mode, scoring was carried out according to each labs' chosen classifier, according to standard practice (Romm et al 2014). For statistical analysis of the dicentric results, the variance to mean ratio and u-test (the normalised unit of the standardised normal distribution) were applied to test for departure of the distribution of aberrations from Poisson and the Chisquared test was used to give an initial indication of the homogeneity of scoring results between laboratories.…”

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

“…The number of cells scored can have a huge impact on the precision of the dose estimate where an assay is robust but events scarce, e.g. the dicentric assay (Romm et al 2014). However, this is much less of an issue for the gamma-H2AX assay where, in comparison, there are many events (foci) per cell and many other factors contributing to dose uncertainty (Rothkamm & Horn 2009;Rothkamm et al 2015).…”

“…The following experimental factors were tested against the dose estimates: post-exposure time (4 or 24 h); scoring method (manual or automatic); temperature during transit; donor nested in postexposure time (1-5); laboratory (1-8; omitting lab 2 as they were unable to take part in the second exercise); number of cells scored nested in scoring method (10-50 for manual and 50-200 for automated scoring); and sample type (whole blood or lymphocytes). In addition, a z-score of the dose was calculated for each dose estimate in order to determine a laboratory's results as satisfactory (jzj 2), questionable (2 < jzj < 3), and unsatisfactory (jzj > 3) (Romm et al 2014). It should be noted that, throughout the paper, the statistical power of the data is in some cases fairly weak due to the small numbers of participants.…”

Comparing Seven Mitogens With Pha-M for Improved Lymphocyte Stimulation in Dicentric Chromosome Analysis for Biodosimetry

“…Because of the time expenditure and labour intensity of these methods, several strategies are pursued to increase the biodosimetry capacity such as progressive automation (3) , validation of new fast scoring protocols (4 -6) and network formation of cooperating biodosimetry laboratories (7 -9) . The authors have already implemented several approaches to increase their lab's DCA capacity: (1) they automated single working steps such as fully automatic lymphocyte fixation using an automatic cell harvester (Hanabi PII, Transgenomic, UK) and automatic metaphase (MP) acquisition followed by semi-automatic dicentric (dic) scoring with the metafer4 platform (Metasystems, Germany) (10) . (2) A basis for mutual assistance during a large-scale radiation incident was established by an inter-comparison with another German biodosimetry lab aimed at the validation of the DCA as a cytogenetic triage tool (11) .…”