Comparison of Isolated Lymphocyte and Whole Blood-Based CBMN Assays for Radiation Triage.

Following a mass-casualty nuclear/radiological event, there will be an important need for rapid and accurate estimation of absorbed dose for biological triage. The cytokinesis-block micronucleus (CBMN) assay is an established and validated cytogenetic biomarker used to assess DNA damage in irradiated peripheral blood lymphocytes. Here, we describe an intercomparison experiment between two biodosimetry laboratories, located at Columbia University (CU) and Health Canada (HC) that performed different variants of the human blood CBMN assay to reconstruct dose in human blood, with CU performing the assay on isolated lymphocytes and using semi-automated scoring whereas HC used the more conventional whole blood assay. Although the micronucleus yields varied significantly between the two assays, the predicted doses closely matched up to 4 Gy - the range from which the HC calibration curve was previously established. These results highlight the importance of a robust calibration curve(s) across a wide age range of donors that match the exposure scenario as closely as possible and that will account for differences in methodology between laboratories. We have seen that at low doses, variability in the results may be attributed to variation in the processing while at higher doses the variation is dominated by inter-individual variation in cell proliferation. This interlaboratory collaboration further highlights the usefulness of the CBMN endpoint to accurately reconstruct absorbed dose in human blood after ionizing radiation exposure.

Application of the Cytokinesis-Block Micronucleus Assay for High-Dose Exposures Using Imaging Flow Cytometry.

The cytokinesis-block micronucleus assay is a well-established method to assess radiation-induced genetic damage in human cells. This assay has been adapted to imaging flow cytometry (IFC), allowing automated analysis of many cells, and eliminating the need to create microscope slides. Furthermore, to improve the efficiency of assay performance, a small-volume method previously developed was employed. Irradiated human blood samples were cultured, stained, and analyzed by IFC to produce images of the cells. Samples were run using both manual and 96-well plate automated acquisition. Multiple parameter-based image features were collected for each sample, and the results were compared to confirm that these acquisition methods are functionally identical. This paper details the multi-parametric analysis developed and the resulting calibration curves up to 10 Gy. The calibration curves were created using a quadratic random coefficient model with Poisson errors, as well as a logistic discriminant function. The curves were then validated with blinded, irradiated samples, using relative bias and relative mean square error. Overall, the accuracy of the dose estimates was adequate for triage dosimetry (within 1 Gy of the true dose) over 90% of the time for lower doses and about half the time for higher doses, with the lowest success rate between 5 and 6 Gy where the calibration curve reached its peak and there was the smallest change in MN/BNC with dose. This work describes the application of a novel multi-parametric analysis that fits the calibration curves and allows dose estimates up to 10 Gy, which were previously limited to 4 Gy. Furthermore, it demonstrates that the results from samples acquired manually and with the autosampler are functionally similar.

Foundations of identifying individual chromosomes by imaging flow cytometry with applications in radiation biodosimetry.

Biodosimetry is an important tool for triage in the case of large-scale radiological or nuclear emergencies, but traditional microscope-based methods can be tedious and prone to scorer fatigue. While the dicentric chromosome assay (DCA) has been adapted for use in triage situations, it is still time-consuming to create and score slides. Recent adaptations of traditional biodosimetry assays to imaging flow cytometry (IFC) methods have dramatically increased throughput. Additionally, recent improvements in image analysis algorithms in the IFC software have resulted in improved specificity for spot counting of small events. In the IFC method for the dicentric chromosome analysis (FDCA), lymphocytes isolated from whole blood samples are cultured with PHA and Colcemid. After incubation, lymphocytes are treated with a hypotonic solution and chromosomes are isolated in suspension, labelled with a centromere marker and stained for DNA content with DRAQ5. Stained individual chromosomes are analyzed on the ImageStream®X (EMD-Millipore, Billerica, MA) and mono- and dicentric chromosome populations are identified and enumerated using advanced image processing techniques. Both the preparation of the isolated chromosome suspensions as well as the image analysis methods were fine-tuned in order to optimize the FDCA. In this paper we describe the method to identify and score centromeres in individual chromosomes by IFC and show that the FDCA method may further improve throughput for triage biodosimetry in the case of large-scale radiological or nuclear emergencies.

Evolution of the Health Canada astronaut biodosimetry program with a view toward international harmonization.

Biodosimetry of astronaut lymphocyte samples, taken prior to- and post-flight, provides an important in vivo measurement of radiation-induced damage incurred during space flight which can be included in the medical records of the astronauts. Health Canada has been developing their astronaut biodosimetry program since 2007 and since then has analyzed data from 7 astronauts. While multiple cytogenetic endpoints may be analyzed for the astronauts, the Fluorescent in situ hybridization (FISH) assay is considered to be key for detecting long-lasting stable damage. It is believed that this long-lasting damage is most likely to lead to an increased risk to the health of the astronauts during long-term flights (lasting 6 months or more). The complexity of damage that results from protracted, non-homogeneous radiation exposure, like that found in the space environment, requires a detailed scoring schematic to capture as much information as possible. To that end, this paper outlines the efforts to harmonize the manner in which Health Canada's FISH data are recorded to better facilitate the comparison of results with other international biodosimetry programs.

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.