Ruth C. Wilkins

Interlaboratory Comparison of the Dicentric Chromosome Assay for Radiation Biodosimetry in Mass Casualty Events

Abstract Wilkins, R. C., Romm, H., Kao, T-C., Awa, A. A., Yoshida, M. A., Livingston, G. K., Jenkins, M. S., Oestreicher, U., Pellmar, T. C. and Prasanna, P. G. S. Interlaboratory Comparison of the Dicentric Chromosome Assay for Radiation Biodosimetry in Mass Casualty Events. Radiat. Res. 169, 551–560 (2008). This interlaboratory comparison validates the dicentric chromosome assay for assessing radiation dose in mass casualty accidents and identifies the advantages and limitations of an international biodosimetry network. The assays validity and accuracy were determined among five laboratories following the International Organization for Standardization guidelines. Blood samples irradiated at the Armed Forces Radiobiology Research Institute were shipped to all laboratories, which constructed individual radiation calibration curves and assessed the dose to dose-blinded samples. Each laboratory constructed a dose–effect calibration curve for the yield of dicentrics for 60Co γ rays in the 0 to 5-Gy range, using the maximum likelihood linear-quadratic model, Y = c + αD + βD2. For all laboratories, the estimated coefficients of the fitted curves were within the 99.7% confidence intervals (CIs), but the observed dicentric yields differed. When each laboratory assessed radiation doses to four dose-blinded blood samples by comparing the observed dicentric yield with the laboratorys own calibration curve, the estimates were accurate in all laboratories at all doses. For all laboratories, actual doses were within the 99.75% CI for the assessed dose. Across the dose range, the error in the estimated doses, compared to the physical doses, ranged from 15% underestimation to 15% overestimation.

The response of gamma-H2AX in human lymphocytes and lymphocytes subsets measured in whole blood cultures

Purpose: To assess the use of phosphorylated histone H2AX protein (γ-H2AX) in human blood leukocytes as a rapid screening tool for radiation biodosimetry using a method that examines the characteristics of γ-H2AX phosphorylation in a variety of lymphocyte subsets following exposure to radiation. Materials and methods: Human peripheral blood exposed to 0–10 Gy of 137Cs irradiation and cultured for 0–48 h was analysed using a rapid whole blood flow cytometry assay to measure γ-H2AX phosphorylation in different lymphocyte subpopulations. Results: Lymphocyte subsets displayed a similar linear dose response relationship, although cluster of differentiation 4+ (CD4+) and CD8+ lymphocytes were found to express H2AX phosphorylation on the order of 1.5 times higher than CD19+ lymphocytes. Phosphorylation of all lymphocyte subsets reached a maximum at 1.5 h and had essentially returned to baseline levels 24 h post-exposure. Conclusions: Differences in the expression level of γ-H2AX between lymphocyte subsets were minimal. The usefulness of this assay for radiation biodosimetry is hampered by its relatively quick lifetime kinetics and large inter-individual variation. Therefore, it could only be useful if samples were obtained within 24 h of exposure. Even in this situation, the assay could only be used as an indicator of exposure and not a dosimeter.

Comparison of a reference region model with direct measurement of an AIF in the analysis of DCE-MRI data

Models have been developed for analyzing dynamic contrast‐enhanced (DCE)‐MRI data that do not require measurements of the arterial input function (AIF). In this study, experimental results obtained from a reference region (RR) analysis are compared with results of an AIF analysis in the same set of five animals (four imaged twice, yielding nine data sets), returning estimates of the volume transfer constant (Ktrans) and the extravascular extracellular volume fraction (ve). Students t‐test values for comparisons of Ktrans and ve between the two models were 0.14 (P = 0.88) and 0.85 (P > 0.4), respectively (where the high P‐values indicate no significant difference between values derived from the two models). Linear regression analysis indicated there was a correlation between Ktrans extracted by the two methods: r2 = 0.80, P = 0.001 (where the low P‐value indicates a significant linear correlation). For ve there was no such correlation (r2 = 0.02). The mean (absolute) percent difference between the models was 22.0% for Ktrans and 28.1% for ve. However, the RR parameter values were much less precise than the AIF method. The mean SDs for Ktrans and ve for the RR analysis were 0.024 min–1 and 0.06, respectively, vs. 0.002 min–1 and 0.03 for AIF analysis. Magn Reson Med 57:353–361, 2007.

WHO 1st Consultation on the Development of a Global Biodosimetry Laboratories Network for Radiation Emergencies (BioDoseNet)

Abstract Blakely, W. F., Carr, Z., Chu, M. C-M., Dayal-Drager, R., Fujimoto, K., Hopmeir, M., Kulka, U., Lillis-Hearne, P., Livingston, G. K., Lloyd, D. C., Maznyk, N., Perez, M. D. R., Romm, H., Takashima, Y., Voisin, P., Wilkins, R. C. and Yoshida, M. A. WHO 1st Consultation on the Development of a Global Biodosimetry Laboratories Network for Radiation Emergencies (BioDoseNet). Radiat. Res. 171, 127–139 (2009). The World Health Organization (WHO) held a consultation meeting at WHO Headquarters, Geneva, Switzerland, December 17–18, 2007, to develop the framework for a global biodosimetry network. The WHO network is envisioned to enable dose assessment using multiple methods [cytogenetics, electron paramagnetic resonance (EPR), radionuclide bioassays, etc.]; however, the initial discussion focused on the cytogenetic bioassay (i.e., metaphase-spread dicentric assay). Few regional cytogenetic biodosimetry networks have been established so far. The roles and resources available from United Nations (UN) agencies that provide international cooperation in biological dosimetry after radiological emergencies were reviewed. In addition, extensive reliance on the use of the relevant International Standards Organization (ISO) standards was emphasized. The results of a WHO survey of global cytogenetic biological dosimetry capability were reported, and while the survey indicates robust global capability, there was also a clear lack of global leadership and coordination. The expert group, which had a concentrated focus on cytogenetic biodosimetry, formulated the general scope and concept of operations for the development of a WHO global biodosimetry laboratory network for radiation emergencies (BioDoseNet). Follow-on meetings are planned to further develop technical details for this network.

Biological Dosimetry by the Triage Dicentric Chromosome Assay: Potential Implications for Treatment of Acute Radiation Syndrome in Radiological Mass Casualties

Abstract Biological dosimetry is an essential tool for estimating radiation dose. The dicentric chromosome assay (DCA) is currently the tool of choice. Because the assay is labor-intensive and time-consuming, strategies are needed to increase throughput for use in radiation mass casualty incidents. One such strategy is to truncate metaphase spread analysis for triage dose estimates by scoring 50 or fewer metaphases, compared to a routine analysis of 500 to 1000 metaphases, and to increase throughput using a large group of scorers in a biodosimetry network. Previously, the National Institutes for Allergies and Infectious Diseases (NIAID) and the Armed Forces Radiobiology Research Institute (AFRRI) sponsored a double-blinded interlaboratory comparison among five established international cytogenetic biodosimetry laboratories to determine the variability in calibration curves and in dose measurements in unknown, irradiated samples. In the present study, we further analyzed the published data from this previous study to investigate how the number of metaphase spreads influences dose prediction accuracy and how this information could be of value in the triage and management of people at risk for the acute radiation syndrome (ARS). Although, as expected, accuracy decreased with lower numbers of metaphase spreads analyzed, predicted doses by the laboratories were in good agreement and were judged to be adequate to guide diagnosis and treatment of ARS. These results demonstrate that for rapid triage, a network of cytogenetic biodosimetry laboratories can accurately assess doses even with a lower number of scored metaphases.

Canadian Cytogenetic Emergency Network (CEN) for biological dosimetry following radiological/nuclear accidents

Purpose: To test the ability of the cytogenetic emergency network (CEN) of laboratories, currently under development across Canada, to provide rapid biological dosimetry using the dicentric assay for triage assessment, that could be implemented in the event of a large-scale radiation/nuclear emergency. Materials and methods: A workshop was held in May 2004 in Toronto, Canada, to introduce the concept of CEN and recruit clinical cytogenetic laboratories at hospitals across the country. Slides were prepared for dicentric assay analysis following in vitro irradiation of blood to a range of gamma-ray doses. A minimum of 50 metaphases per slide were analyzed by 41 people at 22 different laboratories to estimate the exposure level. Results: Dose estimates were calculated based on a dose response curve generated at Health Canada. There were a total of 104 dose estimates and 96 (92.3%) of them fell within the expected range using triage scoring criteria. Half of the laboratories analyzed 50 metaphases in ≤ 1 hour and the time to score them was proportional to dose. The capacity and scoring expertise of the various participating laboratories were found to be generally acceptable. Conclusions: The dose estimates generated through triage scoring by this network were acceptable for emergency biological dosimetry. When this network is fully operational, it will be the first of its kind in Canada able to respond to radiological/nuclear emergencies by providing triage quality biological dosimetry for a large number of samples. This network represents an alternate expansion of existing international emergency biological dosimetry cytogenetic networks.

Differential apoptotic response to ionizing radiation in subpopulations of human white blood cells

The purpose of this paper is to characterize the apoptotic response of various subpopulations of human white blood cells after in vitro exposure to ionizing radiation using the modified neutral comet assay (MNCA). White blood cells, isolated from human whole blood, were fractionated into granulocytes and mononuclear cells which were further separated into B-cells, natural killer (NK) cells, and CD4(+) and CD8(+) T-cells. The separated fractions were exposed to low doses of X-rays and then MNCA was used to measure the apoptotic fraction (AF) at different time points in irradiated and unirradiated aliquots of sorted cultures. The spontaneous AF in unirradiated control cells was the most critical determinant of whether an apoptotic response could be detected in irradiated cells. When cultured in isolation granulocytes and B-cells had the highest background AF, with NK cells having the next highest. CD4(+) and CD8(+) T-cells had a low, stable, spontaneous AF which gave them the highest signal-to-noise ratio. Although B-cells demonstrated the highest radiation-induced apoptotic response to 1Gy of X-rays, CD8(+) T-cells were the most radiation-responsive lymphocytes due to their low spontaneous AF. By generating dose response curves for CD4(+) and CD8(+) T-cells, the sensitivity of the MNCA for detecting apoptosis in these two cell types was also examined.

QUICKSCAN DICENTRIC CHROMOSOME ANALYSIS FOR RADIATION BIODOSIMETRY

The dicentric chromosome assay (DCA) is the gold-standard assay for accurately estimating unknown radiological doses to individuals following radiological or nuclear accidents. However in a mass-casualty scenario, this assay is not well suited for providing timely dose estimates due to its time- and expertise-intensive nature. In Canada, two approaches are being developed in an attempt to increase triage-quality biological dosimetry throughput. These are 1) increasing the number of trained personnel capable of conducting the DCA, and 2) evaluating alternative biodosimetry approaches or DCA variations. In a recent exercise, a new scoring technique (termed DCA QuickScan) was evaluated as an alternative rapid-scoring approach. Triage-quality conventional DCA and DCA QuickScan analysis were based upon scoring a minimum of 50 metaphase cells or 30 dicentrics by 9–15 scorers across four laboratories. Dose estimates for the conventional DCA were found to be within 0.5 Gy of the actual dose for 83% of the unknown samples, while DCA QuickScan dose estimates were within 0.5 Gy for 80% of the samples. Of the dose estimates falling 0.5 Gy or more outside the actual dose, the majority were dose over-estimates. It was concluded that the DCA QuickScan approach can provide critical dose information at a much faster rate than the conventional DCA without sacrificing accuracy. Future studies will further evaluate the accuracy of the DCA QuickScan method.

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.

Validation of QuickScan dicentric chromosome analysis for high throughput radiation biological dosimetry.

Currently, the dicentric chromosome assay (DCA) is used to estimate radiation doses to individuals following accidental radiological and nuclear overexposures when traditional dosimetry methods are not available. While being an exceptionally sensitive method for estimating doses by radiation, conventional DCA is time-intensive and requires highly trained expertise for analysis. For this reason, in a mass casualty situation, triage-quality conventional DCA struggles to provide dose estimations in a timely manner for triage purposes. In Canada, a new scoring technique, termed DCA QuickScan, has been devised to increase the throughput of this assay. DCA QuickScan uses traditional DCA sample preparation methods while adapting a rapid scoring approach. In this study, both conventional and QuickScan methods of scoring the DCA assay were compared for accuracy and sensitivity. Dose response curves were completed on four different donors based on the analysis of 1,000 metaphases or 200 events at eight to nine dose points by eight different scorers across two laboratories. Statistical analysis was performed on the data to compare the two methods within and across the laboratories and to test their respective sensitivities for dose estimation. This study demonstrated that QuickScan is statistically similar to conventional DCA analysis and is capable of producing dose estimates as low as 0.1 Gy but up to six times faster. Therefore, DCA QuickScan analysis can be used as a sensitive and accurate method for scoring samples for radiological biodosimetry in mass casualty situations or where faster dose assessment is required.