Large-scale Radiological Event Research Articles

Validation of a blood biomarker panel for machine learning-based radiation biodosimetry in juvenile and adult C57BL/6 mice

Following a large-scale radiological event, timely collection of samples from all potentially exposed individuals may be precluded, and high-throughput bioassays capable of rapid and individualized dose assessment several days post-exposure will be essential for population triage and efficient implementation of medical treatment. The objective of this work was to validate the performance of a biomarker panel of radiosensitive intracellular leukocyte proteins (ACTN1, DDB2, and FDXR) and blood cell counts (CD19+ B-cells and CD3+ T-cells) for retrospective classification of exposure and dose estimation up to 7 days post-exposure in an in-vivo C57BL/6 mouse model. Juvenile and adult C57BL/6 mice of both sexes were total body irradiated with 0, 1, 2, 3, or 4 Gy, peripheral blood was collected 1, 4, and 7-days post-exposure, and individual blood biomarkers were quantified by imaging flow cytometry. An ensemble machine learning platform was used to identify the strongest predictor variables and combine them for biodosimetry outputs. This approach generated successful exposure classification (ROC AUC = 0.94, 95% CI: 0.90–0.97) and quantitative dose reconstruction (R2 = 0.79, RMSE = 0.68 Gy, MAE = 0.53 Gy), supporting the potential utility of the proposed biomarker assay for determining exposure and received dose in an individual.

Radiation Signature in Plasma Metabolome of Total-Body Irradiated Nonhuman Primates and Clinical Patients.

In the last decade, geopolitical instability across the globe has increased the risk of a large-scale radiological event, when radiation biomarkers would be needed for an effective triage of an irradiated population. Ionizing radiation elicits a complex response in the proteome, genome, and metabolome and hence can be leveraged as rapid and sensitive indicators of irradiation-induced damage. We analyzed the plasma of total-body irradiated (TBI) leukemia patients (n = 24) and nonhuman primates (NHPs; n = 10) before and 24 h after irradiation, and we performed a global metabolomic study aiming to provide plasma metabolites as candidate radiation biomarkers for biological dosimetry. Peripheral blood samples were collected according to the appropriate ethical approvals, and metabolites were extracted and analyzed by liquid chromatography mass spectrometry. We identified an array of metabolites significantly altered by irradiation, including bilirubin, cholesterol, and 18-hydroxycorticosterone, which were detected in leukemia patients and NHPs. Pathway analysis showed overlapping perturbations in steroidogenesis, porphyrin metabolism, and steroid hormone biosynthesis and metabolism. Additionally, we observed dysregulation in bile acid biosynthesis and tyrosine metabolism in the TBI patient cohort. This investigation is, to our best knowledge, among the first to provide valuable insights into a comparison between human and NHP irradiation models. The findings from this study could be leveraged for translational biological dosimetry.

Machine learning approach for quantitative biodosimetry of partial-body or total-body radiation exposures by combining radiation-responsive biomarkers

During a large-scale radiological event such as an improvised nuclear device detonation, many survivors will be shielded from radiation by environmental objects, and experience only partial-body irradiation (PBI), which has different consequences, compared with total-body irradiation (TBI). In this study, we tested the hypothesis that applying machine learning to a combination of radiation-responsive biomarkers (ACTN1, DDB2, FDXR) and B and T cell counts will quantify and distinguish between PBI and TBI exposures. Adult C57BL/6 mice of both sexes were exposed to 0, 2.0–2.5 or 5.0 Gy of half-body PBI or TBI. The random forest (RF) algorithm trained on ½ of the data reconstructed the radiation dose on the remaining testing portion of the data with mean absolute error of 0.749 Gy and reconstructed the product of dose and exposure status (defined as 1.0 × Dose for TBI and 0.5 × Dose for PBI) with MAE of 0.472 Gy. Among irradiated samples, PBI could be distinguished from TBI: ROC curve AUC = 0.944 (95% CI: 0.844–1.0). Mouse sex did not significantly affect dose reconstruction. These results support the hypothesis that combinations of protein biomarkers and blood cell counts can complement existing methods for biodosimetry of PBI and TBI exposures.

Risk perception of radiation emergency medical staff on low-dose radiation exposure: Knowledge is a critical factor

Radiation emergency medical (REM) staff respond to many types of disasters such as radiological and nuclear accidents as well as environmental radioactivity exposure. The objective of this study was to evaluate the risk perception of REM staff on radiation exposure in various situations and to analyze the factors that affect their risk perception. A questionnaire was given to 284 REM staff affiliated with various organizations, including nuclear power generation, nuclear fuel manufacturing, large-scale irradiation, and radiation-waste disposal facilities, as well as research and development institutions. To determine the substantially influential factors for risk perception, we analyzed the questionnaire responses using ordinal logistic regression, Kruskal-Wallis, and Spearman correlation analyses. It was generally perceived by REM staff that low-dose radiation exposure in daily life and work environments does not pose any health risks. A higher level of radiation knowledge was tightly associated with a lower risk perception of REM staff on extremely low-dose (several mSv) radiation exposure, thus exhibiting an inverse correlation. In contrast to radiation researchers, the work experience of REM staff was not a contributing factor to their risk perception. In our study, REM staff with a high level of radiation knowledge did not have any health concerns in their work environments. Efforts to enhance the radiation knowledge of REM staff through proper education and training would result in analytical risk evaluation, which may also improve their willingness to help meet surge capacity needs in large-scale radiological events.

The RABiT-II DCA in the Rhesus Macaque Model.

An automated platform for cytogenetic biodosimetry, the "Rapid Automated Biodosimetry Tool II (RABiT-II)," adapts the dicentric chromosome assay (DCA) for high-throughput mass-screening of the population after a large-scale radiological event. To validate this test, the U.S. Federal Drug Administration (FDA) recommends demonstrating that the high-throughput biodosimetric assay in question correctly reports the dose in an in vivo model. Here we describe the use of rhesus macaques (Macaca mulatta) to augment human studies and validate the accuracy of the high-throughput version of the DCA. To perform analysis, we developed the 17/22-mer peptide nucleic acid (PNA) probes that bind to the rhesus macaque's centromeres. To our knowledge, these are the first custom PNA probes with high specificity that can be used for chromosome analysis in M. mulatta. The accuracy of fully-automated chromosome analysis was improved by optimizing a low-temperature telomere PNA FISH staining in multiwell plates and adding the telomere detection feature to our custom chromosome detection software, FluorQuantDic V4. The dicentric frequencies estimated from in vitro irradiated rhesus macaque samples were compared to human blood samples of individuals subjected to the same ex vivo irradiation conditions. The results of the RABiT-II DCA analysis suggest that, in the lymphocyte system, the dose responses to gamma radiation in the rhesus macaques were similar to those in humans, with small but statistically significant differences between these two model systems.

A NOVEL BIOLOGICAL DOSIMETRY ASSAY AS A POTENTIAL TOOL FOR TRIAGE DOSE ASSESSMENT IN CASE OF LARGE-SCALE RADIOLOGICAL EMERGENCY

In case of mass radiological emergencies, new strategies involving biological and clinical endpoints are requested for an efficient triage classification of casualties. For this purpose, we developed a novel protocol combining the two most established cytogenetic methods used in biological dosimetry (dicentric and micronucleus assays) into a single one, in order to have a time-saving, inexpensive and potentially automatable instrument to be used for triage purposes in case of large-scale radiological events. This method could be considered as a 'three in one' assay allowing the simultaneous scoring of chromosome aberrations and micronuclei on a single slide, and also enabling to discriminate between metaphases in first and second cell division without the Fluorescence plus Giemsa staining. This method needs further validation through inter-comparisons involving biological dosimetry laboratories, to verify its reproducibility. Moreover, the possibility to apply the already existing software for automation for dicentric and micronucleus assays could be also verified.

Gene Expression in Parp1 Deficient Mice Exposed to a Median Lethal Dose of Gamma Rays.

There is a current interest in the development of biodosimetric methods for rapidly assessing radiation exposure in the wake of a large-scale radiological event. This work was initially focused on determining the exposure dose to an individual using biological indicators. Gene expression signatures show promise for biodosimetric application, but little is known about how these signatures might translate for the assessment of radiological injury in radiosensitive individuals, who comprise a significant proportion of the general population, and who would likely require treatment after exposure to lower doses. Using Parp1-/- mice as a model radiation-sensitive genotype, we have investigated the effect of this DNA repair deficiency on the gene expression response to radiation. Although Parp1 is known to play general roles in regulating transcription, the pattern of gene expression changes observed in Parp1-/- mice 24 h postirradiation to a LD50/30 was remarkably similar to that in wild-type mice after exposure to LD50/30. Similar levels of activation of both the p53 and NFκB radiation response pathways were indicated in both strains. In contrast, exposure of wild-type mice to a sublethal dose that was equal to the Parp1-/- LD50/30 resulted in a lower magnitude gene expression response. Thus, Parp1-/- mice displayed a heightened gene expression response to radiation, which was more similar to the wild-type response to an equitoxic dose than to an equal absorbed dose. Gene expression classifiers trained on the wild-type data correctly identified all wild-type samples as unexposed, exposed to a sublethal dose or exposed to an LD50/30. All unexposed samples from Parp1-/- mice were also correctly classified with the same gene set, and 80% of irradiated Parp1-/- samples were identified as exposed to an LD50/30. The results of this study suggest that, at least for some pathways that may influence radiosensitivity in humans, specific gene expression signatures have the potential to accurately detect the extent of radiological injury, rather than serving only as a surrogate of physical radiation dose.

Biomarkers of Ionizing Radiation Exposure: A Multiparametric Approach.

Humans are exposed to ionizing radiation not only through background radiation but also through the ubiquitous presence of devices and sources that generate radiation. With the expanded use of radiation in day-to-day life, the chances of accidents or misuse only increase. Therefore, a thorough understanding of the dynamic effects of radiation exposure on biological entities is necessary. The biological effects of radiation exposure on human cells depend on much variability such as level of exposure, dose rate, and the physiological state of the cells. During potential scenarios of a large-scale radiological event which results in mass casualties, dose estimates are essential to assign medical attention according to individual needs. Many attempts have been made to identify biomarkers which can be used for high throughput biodosimetry screening. In this study, we compare the results of different biodosimetry methods on the same irradiated cells to assess the suitability of current biomarkers and push forward the idea of employing a multiparametric approach to achieve an accurate dose and risk estimation.

Flow cytometry-assisted quantification of γH2AX expression has potential as a rapid high-throughput biodosimetry tool.

Large-scale radiological events require immediate and accurate estimates of doses received by victims, and possibly the first responders, to assist in treatment decisions. Although there are numerous efforts worldwide to develop biodosimetric tools to adequately handle triage needs during radiological incidents, such endeavours do not seem to actively involve sub-Saharan Africa which currently has a significant level of nuclear-related activity. To initiate a similar interest in Africa, ex vivo radiation-induced γH2AX expression in peripheral blood lymphocytes from fourteen healthy donors was assessed using flow cytometry. While the technique shows potential for use as a rapid high-throughput biodosimetric tool for radiation absorbed doses up to 5Gy, significant inter-individual differences in γH2AX expression emerged. Also, female donors exhibited higher levels of γH2AX expression than their male counterparts. To address these shortcomings, gender-based in-house dose-response curves for γH2AX induction in lymphocytes 2, 4, and 6h after X-ray irradiation are proposed for the South African population. The obtained results show that γH2AX is a good candidate biomarker for biodosimetry, but might need some refinement and validation through further studies involving a larger cohort of donors.

Initial healthcare to pediatric population under the radiation events

The objective of this paper is to analyze the data from scientific literature and available recommendations for health professionals on healthcare providing to pediatric population in the events associated with risk of radiation exposure. Over the past sixty years there were several large scale radiological events with a large number of chil dren affected, namely the atomic bombing of Hiroshima and Nagasaki, accident at the Chornobyl nuclear power plant, contact to 137Cs radiation source unutilized at the hospital shutdown in Brazil etc. Further research has crys tallized injuries and health disorders in the survived children in all cases being much more significant vs. other pop ulations. Analysis of circumstances and features of a number of emergency situations or incidents in the nuclear industry for several decades has shown a high probability of radioactive materials release into the environment. The danger of terrorism with the use of ionizing radiation sources resulting in a considered inevitable hazard to the chil dren is estimated as serious in recent years and deserves an especial mention here.

Global Metabolomic Identification of Long-Term Dose-Dependent Urinary Biomarkers in Nonhuman Primates Exposed to Ionizing Radiation.

Due to concerns surrounding potential large-scale radiological events, there is a need to determine robust radiation signatures for the rapid identification of exposed individuals, which can then be used to guide the development of compact field deployable instruments to assess individual dose. Metabolomics provides a technology to process easily accessible biofluids and determine rigorous quantitative radiation biomarkers with mass spectrometry (MS) platforms. While multiple studies have utilized murine models to determine radiation biomarkers, limited studies have profiled nonhuman primate (NHP) metabolic radiation signatures. In addition, these studies have concentrated on short-term biomarkers (i.e., <72 h). The current study addresses the need for biomarkers beyond 72 h using a NHP model. Urine samples were collected at 7 days postirradiation (2, 4, 6, 7 and 10 Gy) and processed with ultra-performance liquid chromatography (UPLC) quadrupole time-of-flight (QTOF) MS, acquiring global metabolomic radiation signatures. Multivariate data analysis revealed clear separation between control and irradiated groups. Thirteen biomarkers exhibiting a dose response were validated with tandem MS. There was significantly higher excretion of l-carnitine, l-acetylcarnitine, xanthine and xanthosine in males versus females. Metabolites validated in this study suggest perturbation of several pathways including fatty acid β oxidation, tryptophan metabolism, purine catabolism, taurine metabolism and steroid hormone biosynthesis. In this novel study we detected long-term biomarkers in a NHP model after exposure to radiation and demonstrate differences between sexes using UPLC-QTOF-MS-based metabolomics technology.

The RABiT: high-throughput technology for assessing global DSB repair

At the Center for High-Throughput Minimally Invasive Radiation Biodosimetry, we have developed a rapid automated biodosimetry tool (RABiT); this is a completely automated, ultra-high-throughput robotically based biodosimetry workstation designed for use following a large-scale radiological event, to perform radiation biodosimetry measurements based on a fingerstick blood sample. High throughput is achieved through purpose built robotics, sample handling in filter-bottomed multi-well plates and innovations in high-speed imaging and analysis. Currently, we are adapting the RABiT technologies for use in laboratory settings, for applications in epidemiological and clinical studies. Our overall goal is to extend the RABiT system to directly measure the kinetics of DNA repair proteins. The design of the kinetic/time-dependent studies is based on repeated, automated sampling of lymphocytes from a central reservoir of cells housed in the RABiT incubator as a function of time after the irradiation challenge. In the present study, we have characterized the DNA repair kinetics of the following repair proteins: γ-H2AX, 53-BP1, ATM kinase, MDC1 at multiple times (0.5, 2, 4, 7 and 24 h) after irradiation with 4 Gy γ rays. In order to provide a consistent dose exposure at time zero, we have developed an automated capillary irradiator to introduce DNA DSBs into fingerstick-size blood samples within the RABiT. To demonstrate the scalability of the laboratory-based RABiT system, we have initiated a population study using γ-H2AX as a biomarker.

Infrastructure to support ultra high throughput biodosimetry screening after a radiological event

Purpose: After a large-scale radiological event, there will be a pressing need to assess, within a few days, the radiation doses received by tens or hundreds of thousands of individuals. This is for triage, to prevent treatment locations from being overwhelmed, in what is sure to be a resource limited scenario, as well as to facilitate dose-dependent treatment decisions. In addition there are psycho-social considerations, in that active reassurance of minimal exposure is a potentially effective antidote to mass panic, as well as long-term considerations, to facilitate later studies of cancer and other long-term disease risks.Materials and methods: As described elsewhere in this issue, we are developing a Rapid Automated Biodosimetry Tool (RABiT). The RABiT allows high throughput analysis of thousands of blood samples per day, providing a dose estimate that can be used to support clinical triage and treatment decisions.Results: Development of the RABiT has motivated us to consider the logistics of incorporating such a system into the existing emergency response scenarios of a large metropolitan area. We present here a view of how one or more centralized biodosimetry readout devices might be incorporated into an infrastructure in which fingerstick blood samples are taken at many distributed locations within an affected city or region and transported to centralized locations.Conclusions: High throughput biodosimetry systems offer the opportunity to perform biodosimetric assessments on a large number of persons. As such systems reach a high level of maturity, emergency response scenarios will need to be tweaked to make use of these powerful tools. This can be done relatively easily within the framework of current scenarios.

Optimization of a Microfluidic Mixing Process for Gene Expression–Based Bio-Dosimetry

ABSTRACT In recent decades advances in radiation imaging and radiation therapy have led to a dramatic increase in the number of people exposed to radiation. Consequently, there is a clear need for personalized biodosimetry diagnostics in order to monitor the dose of radiation received and adapt it to each patient depending on their sensitivity to radiation exposure (Hall and Brenner 2008). Similarly, after a large-scale radiological event such as a dirty bomb attack, there will be a major need to assess, within a few days, the radiation doses received by tens of thousands of individuals. Current high-throughput devices can handle only a few hundred individuals per day. Hence, there is a great need for a very fast, self-contained, noninvasive biodosimetric device based on a rapid blood test. This article presents a case study where regression methods and designed experiments are used to arrive at the optimal settings for various factors that impact the kinetics in a biodosimetric device. We use ridge regression to initially identify a set of potentially important variables in the mixing process, which is one of the critical subsystems of the device. This was followed by a series of designed experiments to arrive at the optimal setting of the significant microfluidic cartridge and piezoelectric disk (PZT; Sadler and Zenhausern 2006; Lee et al. 2005) related factors. This statistical approach has been utilized to study the microfluidic mixing to mix water and dye mixtures of 70 μL volume. The outcome of the statistical design, experimentation, and analysis was then exploited for optimizing the design, fabrication, and assembly of microfluidic devices. As a result of the experiments performed, the system was fine-tuned and the mixing time was reduced from 5.5 to 2 minutes.

The Use of Gamma-H2AX as a Biodosimeter for Total-Body Radiation Exposure in Non-Human Primates

BackgroundThere is a crucial shortage of methods capable of determining the extent of accidental exposures of human beings to ionizing radiation. However, knowledge of individual exposures is essential for early triage during radiological incidents to provide optimum possible life-sparing medical procedures to each person.Methods and FindingsWe evaluated immunocytofluorescence-based quantitation of γ-H2AX foci as a biodosimeter of total-body radiation exposure (60Co γ-rays) in a rhesus macaque (Macaca mulatta) model. Peripheral blood lymphocytes and plucked hairs were collected from 4 cohorts of macaques receiving total body irradiation doses ranging from 1 Gy to 8.5 Gy. Each cohort consisted of 6 experimental and 2 control animals. Numbers of residual γ-H2AX foci were proportional to initial irradiation doses and statistically significant responses were obtained until 1 day after 1 Gy, 4 days after 3.5 and 6.5 Gy, and 14 days after 8.5 Gy in lymphocytes and until 1 day after 1 Gy, at least 2 days after 3.5 and 6.5 Gy, and 9 days after 8.5 Gy in plucked hairs.ConclusionThese findings indicate that quantitation of γ-H2AX foci may make a robust biodosimeter for analyzing total-body exposure to ionizing radiation in humans. This tool would help clinicians prescribe appropriate types of medical intervention for optimal individual outcome. These results also demonstrate that the use of a high throughput γ-H2AX biodosimeter would be useful for days post-exposure in applications like large-scale radiological events or radiation therapy. In addition, this study validates a possibility to use plucked hair in future clinical trials investigating genotoxic effects of drugs and radiation treatments.

THE RABIT: A RAPID AUTOMATED BIODOSIMETRY TOOL FOR RADIOLOGICAL TRIAGE

In response to the recognized need for high throughput biodosimetry methods for use after large-scale radiological events, a logical approach is complete automation of standard biodosimetric assays that are currently performed manually. The authors describe progress to date on the RABIT (Rapid Automated BIodosimetry Tool), designed to score micronuclei or gamma-H2AX fluorescence in lymphocytes derived from a single drop of blood from a fingerstick. The RABIT system is designed to be completely automated, from the input of the capillary blood sample into the machine to the output of a dose estimate. Improvements in throughput are achieved through use of a single drop of blood, optimization of the biological protocols for in situ analysis in multi-well plates, implementation of robotic-plate and liquid handling, and new developments in high-speed imaging. Automating well-established bioassays represents a promising approach to high-throughput radiation biodosimetry, both because high throughputs can be achieved, but also because the time to deployment is potentially much shorter than for a new biological assay. Here the authors describe the development of each of the individual modules of the RABIT system and show preliminary data from key modules. System integration is ongoing, followed by calibration and validation.

Biological dosimetry for triage of casualties in a large-scale radiological emergency:capacity of the EU member states

In recent years, a number of events have occurred that highlight the necessity of being prepared for a possible large-scale radiological event. An important question is how well are European Union (EU) Member States prepared to cope with mass radiological casualties. A survey to establish the current status of biological dosimetry across the EU was carried out with the aim of assessing capacity to perform biodosimetric triage of accident victims. Information was sought from the radiation protection authorities of the 27 Member States plus Switzerland and Norway. Biology dosimetry is established in 15 EU countries. Their total capacity for dosimetric triage is about 1500 cases per week analysed with a dicentric assay or about 800 cases analysed with a micronucleus assay. Although these numbers appear encouraging, there is not much collaboration between the laboratories and what is required is a network at the EU level.

Predicting Individual Radiation Sensitivity: Current and Evolving Technologies

In 2005, the National Institute of Allergy and Infectious Diseases (NIAID) of the U.S. National Institutes of Health established a Radiation/Nuclear Program to develop radioprotectors, mitigators and therapeutic agents to facilitate an effective medical response against radiological and nuclear threats. This program also supports development of biomarker/biodosimetry techniques and devices for rapid triage and treatment of radiation exposed individuals after any radiological event. In support of this program, NIAID in collaboration with the Columbia University Medical Center organized a workshop entitled Predicting Individual Radiation Sensitivity: Current and Evolving Technologies. The meeting was held at Columbia University in New York on March 17-18, 2008, and was attended by 86 participants from eight countries. The background to the Workshop relates to the need for mass biodosimetry and mitigation/therapy after a large-scale radiological event, both in regard to short-term sequelae and also in terms of long-term end points such as carcinogenesis and heart disease. While retrospective dose estimates provide information about average risks, it is known from studies of higher-dose radiotherapy that there is considerable person-to-person variability in response to a given radiation dose. Thus the goals of this Workshop were to assess the significance of interindividual radiation sensitivity in terms of the aftermath of a large-scale radiological event and to assess whether the approaches used at clinical doses can be translated to lower doses or whether different approaches will be needed. Session themes were: