RENEB intercomparisons applying the conventional Dicentric Chromosome Assay (DCA).

PURPOSE: Two quality controlled inter-laboratory exercises were organized within the EU project 'Realizing the European Network of Biodosimetry (RENEB)' to further optimize the dicentric chromosome assay (DCA) and to identify needs for training and harmonization activities within the RENEB network. MATERIALS AND METHODS: The general study design included blood shipment, sample processing, analysis of chromosome aberrations and radiation dose assessment. After manual scoring of dicentric chromosomes in different cell numbers dose estimations and corresponding 95% confidence intervals were submitted by the participants. RESULTS: The shipment of blood samples to the partners in the European Community (EU) were performed successfully. Outside the EU unacceptable delays occurred. The results of the dose estimation demonstrate a very successful classification of the blood samples in medically relevant groups. In comparison to the 1st exercise the 2nd intercomparison showed an improvement in the accuracy of dose estimations especially for the high dose point. CONCLUSIONS: In case of a large-scale radiological incident, the pooling of ressources by networks can enhance the rapid classification of individuals in medically relevant treatment groups based on the DCA. The performance of the RENEB network as a whole has clearly benefited from harmonization processes and specific training activities for the network partners.

Radiation Dose Estimation by Automated Cytogenetic Biodosimetry.

The dose from ionizing radiation exposure can be interpolated from a calibration curve fit to the frequency of dicentric chromosomes (DCs) at multiple doses. As DC counts are manually determined, there is an acute need for accurate, fully automated biodosimetry calibration curve generation and analysis of exposed samples. Software, the Automated Dicentric Chromosome Identifier (ADCI), is presented which detects and discriminates DCs from monocentric chromosomes, computes biodosimetry calibration curves and estimates radiation dose. Images of metaphase cells from samples, exposed at 1.4-3.4 Gy, that had been manually scored by two reference laboratories were reanalyzed with ADCI. This resulted in estimated exposures within 0.4-1.1 Gy of the physical dose. Therefore, ADCI can determine radiation dose with accuracies comparable to standard triage biodosimetry. Calibration curves were generated from metaphase images in ~10 h, and dose estimations required ~0.8 h per 500 image sample. Running multiple instances of ADCI may be an effective response to a mass casualty radiation event.

Automated discrimination of dicentric and monocentric chromosomes by machine learning-based image processing.

Dose from radiation exposure can be estimated from dicentric chromosome (DC) frequencies in metaphase cells of peripheral blood lymphocytes. We automated DC detection by extracting features in Giemsa-stained metaphase chromosome images and classifying objects by machine learning (ML). DC detection involves (i) intensity thresholded segmentation of metaphase objects, (ii) chromosome separation by watershed transformation and elimination of inseparable chromosome clusters, fragments and staining debris using a morphological decision tree filter, (iii) determination of chromosome width and centreline, (iv) derivation of centromere candidates, and (v) distinction of DCs from monocentric chromosomes (MC) by ML. Centromere candidates are inferred from 14 image features input to a Support Vector Machine (SVM). Sixteen features derived from these candidates are then supplied to a Boosting classifier and a second SVM which determines whether a chromosome is either a DC or MC. The SVM was trained with 292 DCs and 3135 MCs, and then tested with cells exposed to either low (1 Gy) or high (2-4 Gy) radiation dose. Results were then compared with those of 3 experts. True positive rates (TPR) and positive predictive values (PPV) were determined for the tuning parameter, σ. At larger σ, PPV decreases and TPR increases. At high dose, for σ = 1.3, TPR = 0.52 and PPV = 0.83, while at σ = 1.6, the TPR = 0.65 and PPV = 0.72. At low dose and σ = 1.3, TPR = 0.67 and PPV = 0.26. The algorithm differentiates DCs from MCs, overlapped chromosomes and other objects with acceptable accuracy over a wide range of radiation exposures.

Evaluation of the annual Canadian biodosimetry network intercomparisons.

PURPOSE: To evaluate the importance of annual intercomparisons for maintaining the capacity and capabilities of a well-established biodosimetry network in conjunction with assessing efficient and effective analysis methods for emergency response. MATERIALS AND METHODS: Annual intercomparisons were conducted between laboratories in the Canadian National Biological Dosimetry Response Plan. Intercomparisons were performed over a six-year period and comprised of the shipment of 10-12 irradiated, blinded blood samples for analysis by each of the participating laboratories. Dose estimates were determined by each laboratory using the dicentric chromosome assay (conventional and QuickScan scoring) and where possible the cytokinesis block micronucleus (CBMN) assay. Dose estimates were returned to the lead laboratory for evaluation and comparison. RESULTS: Individual laboratories performed comparably from year to year with only slight fluctuations in performance. Dose estimates using the dicentric chromosome assay were accurate about 80% of the time and the QuickScan method for scoring the dicentric chromosome assay was proven to reduce the time of analysis without having a significant effect on the dose estimates. Although analysis with the CBMN assay was comparable to QuickScan scoring with respect to speed, the accuracy of the dose estimates was greatly reduced. CONCLUSIONS: Annual intercomparisons are necessary to maintain a network of laboratories for emergency response biodosimetry as they evoke confidence in their capabilities.

Validation of the cytokinesis-block micronucleus (CBMN) assay for use as a triage biological dosimetry tool.

Traditionally, the dicentric chromosome assay (DCA) has been used to derive biological dose estimates for unknown radiological exposures. While sensitive, this assay requires highly trained evaluators and is extremely time consuming. The cytokinesis-block micronucleus (CBMN) assay has been suggested as an alternative to the DCA, as it is much faster to evaluate samples and requires less technical expertise. In order to validate this assay for triage biodosimetry, dose-response curves were generated for six donors at eight doses of gamma-radiation (0-4.0 Gy). Each sample was evaluated by 12 individuals, among three different laboratories and the incidence of micronuclei was determined after counting 50-500 binucleated cells. This study demonstrated that the CBMN assay was capable of detecting radiation doses >or=1 Gy after scoring only 200 binucleated cells. As such, the CBMN assay may provide a sensitive and reliable technique for deployment as an initial screening tool in a large-scale radiological emergency where large numbers of biological dose estimates are required.