Dataset Information

Gamma-H2AX biodosimetry for use in large scale radiation incidents: comparison of a rapid '96 well lyse/fix' protocol with a routine method.

ABSTRACT: Following a radiation incident, preliminary dose estimates made by ?-H2AX foci analysis can supplement the early triage of casualties based on clinical symptoms. Sample processing time is important when many individuals need to be rapidly assessed. A protocol was therefore developed for high sample throughput that requires less than 0.1 ml blood, thus potentially enabling finger prick sampling. The technique combines red blood cell lysis and leukocyte fixation in one step on a 96 well plate, in contrast to the routine protocol, where lymphocytes in larger blood volumes are typically separated by Ficoll density gradient centrifugation with subsequent washing and fixation steps. The rapid '96 well lyse/fix' method reduced the estimated sample processing time for 96 samples to about 4 h compared to 15 h using the routine protocol. However, scoring 20 cells in 96 samples prepared by the rapid protocol took longer than for the routine method (3.1 versus 1.5 h at zero dose; 7.0 versus 6.1 h for irradiated samples). Similar foci yields were scored for both protocols and consistent dose estimates were obtained for samples exposed to 0, 0.2, 0.6, 1.1, 1.2, 2.1 and 4.3 Gy of 250 kVp X-rays at 0.5 Gy/min and incubated for 2 h. Linear regression coefficients were 0.87 ± 0.06 (R (2) = 97.6%) and 0.85 ± 0.05 (R (2) = 98.3%) for estimated versus actual doses for the routine and lyse/fix method, respectively. The lyse/fix protocol can therefore facilitate high throughput processing for ?-H2AX biodosimetry for use in large scale radiation incidents, at the cost of somewhat longer foci scoring times.

SUBMITTER: Moquet J

PROVIDER: S-EPMC3961158 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Gamma-H2AX biodosimetry for use in large scale radiation incidents: comparison of a rapid '96 well lyse/fix' protocol with a routine method.

Moquet Jayne J Barnard Stephen S Rothkamm Kai K

PeerJ 20140306

Following a radiation incident, preliminary dose estimates made by γ-H2AX foci analysis can supplement the early triage of casualties based on clinical symptoms. Sample processing time is important when many individuals need to be rapidly assessed. A protocol was therefore developed for high sample throughput that requires less than 0.1 ml blood, thus potentially enabling finger prick sampling. The technique combines red blood cell lysis and leukocyte fixation in one step on a 96 well plate, in ...[more]

PMID: 24688860

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Project description:Inhalation and ingestion of 137Cs and other long-lived radionuclides can occur after large-scale accidental or malicious radioactive contamination incidents, resulting in a complex temporal pattern of radiation dose/dose rate, influenced by radionuclide pharmacokinetics and chemical properties. High-throughput radiation biodosimetry techniques for such internal exposure are needed to assess potential risks of short-term toxicity and delayed effects (e.g., carcinogenesis) for exposed individuals. Previously, we used γ-H2AX to reconstruct injected 137Cs activity in experimentally-exposed mice, and converted activity values into radiation doses based on time since injection and 137Cs-elimination kinetics. In the current study, we sought to assess the feasibility and possible advantages of combining γ-H2AX with transcriptomics to improve 137Cs activity reconstructions. We selected five genes (Atf5, Hist2h2aa2, Olfr358, Psrc1, Hist2h2ac) with strong statistically-significant Spearman's correlations with injected activity and stable expression over time after 137Cs injection. The geometric mean of log-transformed signals of these five genes, combined with γ-H2AX fluorescence, were used as predictors in a nonlinear model for reconstructing injected 137Cs activity. The coefficient of determination (R2) comparing actual and reconstructed activities was 0.91 and root mean squared error (RMSE) was 0.95 MBq. These metrics remained stable when the model was fitted to a randomly-selected half of the data and tested on the other half, repeated 100 times. Model performance was significantly better when compared to our previous analysis using γ-H2AX alone, and when compared to an analysis where genes are used without γ-H2AX, suggesting that integrating γ-H2AX with gene expression provides an important advantage. Our findings show a proof of principle that integration of radiation-responsive biomarkers from different fields is promising for radiation biodosimetry of internal emitters.

Project description:Dosimetry is an important tool for triage and treatment planning following any radiation exposure accident, and biological dosimetry, which estimates exposure dose using a biological parameter, is a practical means of determining the specific dose an individual receives. The cytokinesis-blocked micronucleus assay (CBMN) is an established biodosimetric tool to measure chromosomal damage in mitogen-stimulated human lymphocytes. The CBMN method is especially valuable for biodosimetry in triage situations thanks to simplicity in scoring and adaptability to high-throughput automated sample processing systems. While this technique produces dose-response data which fit very well to a linear-quadratic model for exposures to low linear energy transfer (LET) radiation and for doses up for 5 Gy, limitations to the accuracy of this method arise at larger doses. Accuracy at higher doses is limited by the number of cells reaching mitosis. Whereas it would be expected that the yield of micronuclei increases with the dose, in many experiments it has been shown to actually decrease when normalized over the total number of cells. This variation from a monotonically increasing dose response poses a limitation for retrospective dose reconstruction. In this study we modified the standard CBMN assay to increase its accuracy following exposures to higher doses of photons or a mixed neutron-photon beam. The assay is modified either through inhibitions of the G2/M and spindle checkpoints with the addition of caffeine and/or ZM447439 (an Aurora kinase inhibitor), respectively to the blood cultures at select times during the assay. Our results showed that caffeine addition improved assay performance for photon up to 10 Gy. This was achieved by extending the assay time from the typical 70 h to just 74 h. Compared to micronuclei yields without inhibitors, addition of caffeine and ZM447439 resulted in improved accuracy in the detection of micronuclei yields up to 10 Gy from photons and 4 Gy of mixed neutrons-photons. When the dose-effect curves were fitted to take into account the turnover phenomenon observed at higher doses, best fitting was achieved when the combination of both inhibitors was used. These techniques permit reliable dose reconstruction after high doses of radiation with a method that can be adapted to high-throughput automated sample processing systems.

Dataset Information

Gamma-H2AX biodosimetry for use in large scale radiation incidents: comparison of a rapid '96 well lyse/fix' protocol with a routine method.

Publications

Gamma-H2AX biodosimetry for use in large scale radiation incidents: comparison of a rapid '96 well lyse/fix' protocol with a routine method.

Similar Datasets

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