The response of γ-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 0-10Gy of 137Cs irradiation and cultured for 0-48h 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 1.5h and had essentially returned to baseline levels 24h post-exposure. Conclusions: Differences in the expression level of -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 24h of exposure. Even in this situation, the assay could only be used as an indicator of exposure and not a dosimeter

Report of the workshop on biological dosimetry: Increasing capacity for emergency response

Recent events have brought increased attention to the possibility and dangers of a radiological terrorist threat and its potential implication on the national capacity for radiation accident preparedness. In such an event, there is a pressing need to rapidly identify severely irradiated individuals who require prompt medical attention from those who have not been exposed or have been subject to low doses. Initial dose assessment is a key component in rapid triage and treatment, however, the development of accurate methods for rapid dose assessment remains a challenge. In this report, the authors describe a recent workshop supported by the Chemical, Biological, Radiological-Nuclear and Explosives Research and Technology Initiative regarding the international effort to increase biological dosimetry capacity to effectively mount an emergency response in a mass casualty situation. Specifically, the focus of the workshop was on the current state of biological dosimetry capabilities and capacities in North America, recent developments towards increasing throughput for biological dosimetry and to identify opportunities for developing a North American Biological Dosimetry Network and forming partnerships and collaborations within Canada and the USA

Quantitation of chromosome damage by imaging flow cytometry

Biodosimetry is a method for measuring the dose of radiation to individuals using biological markers such as chromosome damage. Following mass casualty events, it is important to provide this information rapidly in order to assist with the medical management of potentially exposed casualties. Currently, the gold standard for biodosimetry is the dicentric chromosome assay, which accurately estimates the dose from the number of dicentric chromosomes in lymphocytes. To increase throughput of analysis following a largescale mass casualty event, this assay has been adapted for use on the imaging flow cytometer. This chapter describes the methods for the identification and quantification of mono-and multicentric chromosomes using the imaging flow cytometer

Identification of gene-based responses in human blood cells exposed to alpha particle radiation

Background: The threat of a terrorist-precipitated nuclear event places humans at danger for radiological exposures. Isotopes which emit alpha (α)-particle radiation pose the highest risk. Currently, gene expression signatures are being developed for radiation biodosimetry and triage with respect to ionizing photon radiation. This study was designed to determine if similar gene expression profiles are obtained after exposures involving α-particles. Methods. Peripheral blood mononuclear cells (PBMCs) were used to identify sensitive and robust gene-based biomarkers of α-particle radiation exposure. Cells were isolated from healthy individuals and were irradiated at doses ranging from 0-1.5 Gy. Microarray technology was employed to identify transcripts that were differentially expressed relative to unirradiated cells 24 hours post-exposure. Statistical analysis identified modulated genes at each of the individual doses. Results: Twenty-nine genes were common to all doses with expression levels ranging from 2-10 fold relative to control treatment group. This subset of genes was further assessed in independent complete white blood cell (WBC) populations exposed to either α-particles or X-rays using quantitative real-time PCR. This 29 gene panel was responsive in the α-particle exposed WBCs and was shown to exhibit differential fold-changes compared to X-irradiated cells, though no α-particle specific transcripts were identified. Conclusion: Current gene panels for photon radiation may also be applicable for use in α-particle radiation biodosimetry

Radiological/nuclear human monitoring tabletop exercise: Recommendations and lessons identified

Health Canada is the lead department for coordinating the federal response to a Canadian nuclear emergency event. The framework to manage a radiological consequence is outlined in the Federal Nuclear Emergency Plan (FNEP). In 2014, a full scale exercise (FSX) was held to test the capacity of the federal government to handle a nuclear facility emergency disaster in Canada. The FSX provided a means to demonstrate the integration of various departments and agencies in response to such an event, and although a number of task teams within FNEP were tested, the capacity to monitor humans for exposure post-event was not played out fully. To address this, a table top exercise (TTX) was held in 2015 that brought together experts from human monitoring groups (HMGs) in partnership with Provincial and Municipal emergency response organizations. The TTX took the form of a facilitated discussion centered around two types of radiological/nuclear (RN) emergency scenarios that commenced post-release. The purpose of the exercise was to integrate these communities and identify knowledge gaps in policies and concepts of operations pertaining to the human monitoring aspects of RN events including biodosimetry, bioassay, portal monitors, whole body counting, and the provision of personal dosimetry. It also tested the interoperability between first responders/receivers and Federal, Provincial, and Municipal emergency response organizations. The end outcome was the identification of clear knowledge gaps in existing and newly developed concepts of operation in the human population monitoring response to an RN emergency in Canada; these and possible recommendations are captured in this report

Comparison of high dose rate, low dose rate, and high dose rate fractionated radiation for optimizing differences in radiosensitivities in vitro

Radiotherapy is administered with the assumption that all patients respond similarly to radiation although radiosensitivity does vary from patient to patient, resulting in different degrees of early and late effects. Because the dose given to a patient is limited by the response of normal tissue in the treatment field, it would be beneficial to determine the sensitivity of this normal tissue prior to therapy. Previous studies to predict radiosensitivity have used surviving fractions after a single dose given in vitro, however, differences in cell survival at this low level of kill are not easy to resolve. In this study, we set out to evaluate the use of alternative dose regimens which may better resolve differences in radiosensitivity. We have examined several radiation protocols for predictive value, including survival after high doses (6 Gy) at both high (112 cGy/min) and low (.882 cGy/min) dose rates and after fractionated doses of 2 Gy (6 fractions). A sensitive human fibroblast line (S11358) cultured from a patient showing severe effects after therapy is compared with a cell line (OMB1) cultured from an apparently normal subject. Differences between these cell lines have been compared with those between two human melanoma cell lines (SKMEL3 and HT144) which have shown resistant and sensitive response to radiation in vitro respectively. In both fibroblast and melanoma cell lines, the difference in the survival of normal and sensitive cells increased with increasing dose regardless of whether irradiation was delivered as low dose rate, high dose rate, or as fractionated doses. We propose that radiation doses which more closely mimic clinical treatment are more suitable than surviving fraction after 2 Gy (SF2) for in vitro evaluation of relative radiosensitivities of cell populations

Pilot website to support international collaboration for dose assessments in a radiation emergency

Nuclear terrorism has emerged as a significant threat which could require timely medical interventions to reduce potential radiation casualties. Early dose assessments are critical since optimal care depends on knowing a victim's radiation dose. The dicentric chromosome aberration assay is considered the "gold standard" to estimate the radiation dose because the yield of dicentrics correlates positively with the absorbed dose. Dicentrics have a low background frequency, are independent of age and gender and are relatively easy to identify. This diagnostic test for radiation exposure, however, is labor intensive and any single or small group of laboratories could easily be overwhelmed by a mass casualty event. One solution to this potential problem is to link the global WHO BioDoseNet members via the Internet so multiple laboratories could work cooperatively to screen specimens for dicentric chromosomes and generate timely dose estimates. Inter-laboratory comparison studies have shown that analysis of electronic chromosome images viewed on the computer monitor produces scoring accuracy equivalent to viewing live images in the microscope. This functional equivalence was demonstrated during a comparative study involving five laboratories constructing 60Co gamma ray calibration curves and was further confirmed when comparing results of blind dose estimates submitted by each laboratory. It has been further validated in two recent WHO BioDoseNet trial exercises where 20 metaphase images were shared by e-mail and 50 images were shared on a test website created for this purpose. The Internet-based exercise demonstrated a high level of concordance among 20 expert scorers who evaluated the same 50 metaphase spreads selected to exhibit no, low, moderate and severe radiation damage. Nineteen of 20 scorers produced dicentric equivalent counts within the 95% confidence limits of the mean. The Chi-squared test showed strong evidence of homogeneity in the data (p = 0.999). Altogether, data obtained from these studies support the conclusion that Internet-based scoring is likely to overcome the "bottleneck" in workflow, reduce turn-a-round time for dose estimates and ultimately strengthen surge capacity. Use of the Internet for biodosimetry would obviously leverage the human and equipment resources throughout the world. As part of radiation emergency planning, we conclude that a global IT network/infrastructure is needed to serve the needs of an expanding biodosimetry community and should be given high priority to meet the growing threat of radiological and nuclear terrorism

The application of imaging flow cytometry to high-throughput biodosimetry

Biodosimetry methods, including the dicentric chromosome assay, the cytokinesis-block micronucleus assay and the γH2AX marker of DNA damage are used to determine the dose of ionizing radiation. These techniques are particularly useful when physical dosimetry is absent or questioned. While these assays can be very sensitive and specific, the standard methods need to be adapted to increase sample throughput in the case of a large-scale radiological/nuclear event. Recent modifications to the microscope-based assays have resulted in some increased throughput, and a number of biodosimetry networks have been, and continue to be, established and strengthened. As the imaging flow cytometer (IFC) is a technology that can automatically image and analyze processed blood samples for markers of radiation damage, the microscope-based biodosimetry techniques can be modified for the IFC for high-throughput biological dosimetry. Furthermore, the analysis templates can be easily shared between networked biodosimetry laboratories for increased capacity and improved standardization. This review describes recent advances in IFC methodology and their application to biodosimetry

Validation of the cytokinesis-block micronucleus assay using imaging flow cytometry for high throughput radiation biodosimetry

The cytokinesis-block micronucleus assay can be employed in triage radiation biodosimetry to determine the dose of radiation to an exposed individual by quantifying the frequency of micronuclei in binucleated lymphocyte cells. Partially automated analysis of the assay has been applied to traditional microscope-based methods, and most recently, the assay has been adapted to an automated imaging flow cytometry method. This method is able to automatically score a larger number of binucleated cells than are typically scored by microscopy. Whole blood samples were irradiated, divided into 2 mL and 200 mL aliquots, cultured for 48 h and 72 h, and processed to generate calibration curves from 0-4 Gy. To validate the method for use in radiation biodosimetry, nine separate whole blood samples were then irradiated to known doses, blinded, and processed. Results indicate that dose estimations can be determined to within ±0.5 Gy of the delivered dose after only 48 h of culture time with an initial blood volume of 200 mL. By performing the cytokinesis-block micronucleus assay using imaging flow cytometry, a significant reduction in the culture time and volume requirements is possible, which greatly increases the applicability of the assay in high throughput triage radiation biodosimetry