Radiation Research Society Journal-based Historical Review of the Use of Biomarkers for Radiation Dose and Injury Assessment: Acute Health Effects Predictions.

A multiple-parameter based approach using radiation-induced clinical signs and symptoms, hematology changes, cytogenetic chromosomal aberrations, and molecular biomarkers changes after radiation exposure is used for biodosimetry-based dose assessment. In the current article, relevant milestones from Radiation Research are documented that forms the basis of the current consensus approach for diagnostics after radiation exposure. For example, in 1962 the use of cytogenetic chromosomal aberration using the lymphocyte metaphase spread dicentric assay for biodosimetry applications was first published in Radiation Research. This assay is now complimented using other cytogenetic chromosomal aberration assays (i.e., chromosomal translocations, cytokinesis-blocked micronuclei, premature chromosome condensation, γ-H2AX foci, etc.). Changes in blood cell counts represent an early-phase biomarker for radiation exposures. Molecular biomarker changes have evolved to include panels of organ-specific plasma proteomic and blood-based gene expression biomarkers for radiation dose assessment. Maturation of these assays are shown by efforts for automated processing and scoring, development of point-of-care diagnostics devices, service laboratories inter-comparison exercises, and applications for dose and injury assessments in radiation accidents. An alternative and complementary approach has been advocated with the focus to de-emphasize dose and instead focus on predicting acute or delayed health effects. The same biomarkers used for dose estimation (e.g., lymphocyte counts) can be used to directly predict the later developing severity degree of acute health effects without performing dose estimation as an additional or intermediate step. This review illustrates contributing steps toward these developments published in Radiation Research.

Natural-history Characterization of a Murine Partial-body Irradiation Model System: Establishment of a Multiple-Parameter Based GI-ARS Severity-Scoring System.

The purpose of this investigation was to characterize the natural history of a murine total-abdominal-irradiation exposure model to measure gastrointestinal acute radiation injury. Male CD2F1 mice at 12 to 15 weeks old received total-abdominal irradiation using 4-MV linear accelerator X-rays doses of 0, 11, 13.5, 15, 15.75 and 16.5 Gy (2.75 Gy/min). Daily cage-side (i.e., in the animal housing room) observations of clinical signs and symptoms including body weights on all animals were measured up to 10 days after exposure. Jejunum tissues from cohorts of mice were collected at 1, 3, 7 and 10 days after exposure and radiation injury was assessed by histopathological analyses. Results showed time- and dose-dependent loss of body weight [for example at 7 days: 0.66 (±0.80) % loss for 0 Gy, 6.40 (±0.76) % loss at 11 Gy, 9.43 (±2.06) % loss at 13.5 Gy, 23.53 (± 1.91) % loss at 15 Gy, 29.97 (±1.16) % loss at 15.75 Gy, and 31.79 (±0.76) % loss at 16.5 Gy]. Negligible clinical signs and symptoms, except body weight changes, of radiation injury were observed up to 10 days after irradiation with doses of 11 to 15 Gy. Progressive increases in the severity of clinical signs and symptoms were found after irradiation with doses >15 Gy. Jejunum histology showed a progressive dose-dependent increase in injury. For example, at 7 days postirradiation, the percent of crypts, compared to controls, decreased to 82.3 (±9.5), 69.2 (±12.3), 45.4 (±11.9), 18.0 (±3.4), and 11.5 (± 1.8) with increases in doses from 11 to 16.5 Gy. A mucosal injury scoring system was used that mainly focused on changes in villus morphology damage (i.e., subepithelial spaces near the tips of the villi with capillary congestion, significant epithelial lifting along the length of the villi with a few denuded villus tips). Peak levels of total-abdominal irradiation induced effects on the mucosal injury score were seen 7 days after irradiation for doses ≥15 Gy, with a trend to show a decline after 7 days. A murine multiple-parameter gastrointestinal acute-radiation syndrome severity-scoring system was established based on clinical signs and symptoms that included measures of appearance (i.e., hunched and/or fluffed fur), respiratory rate, general (i.e., decreased mobility) and provoked behavior (i.e., subdued response to stimulation), weight loss, and feces/diarrhea score combined with jejunum mucosal-injury grade score. In summary, the natural-history radio-response for murine partial-body irradiation exposures is important for establishing a well-characterized radiation model system; here we established a multiple-parameter gastrointestinal acute-radiation syndrome severity-scoring system that provides a radiation injury gastrointestinal tissue-based assessment utility.

Optimizing chemical-induced premature chromosome condensation assay for rapid estimation of high-radiation doses.

In the event of exposure to high doses of radiation, prompt dose estimation is crucial for selecting appropriate treatment modalities, such as cytokine therapy or stem cell transplantation. The chemical-induced premature chromosome condensation (PCC) method offers a simple approach for such dose estimation with significant radiation exposure, but its 48-h incubation time poses challenges for early dose assessment. In this study, we optimized the chemical-induced PCC assay for more rapid dose assessment. A sufficient number of PCC and G2/M-PCC cells were obtained after 40 h of culture for irradiated human peripheral blood up to 20 Gy. By adding caffeine (final concentration of 1 mM) at 34 h from the start of culture, G2/M-PCC index increased by 1.4-fold in 10 Gy cultures. There was also no significant difference in the G2/M-PCC ring frequency induced for doses 0 to 15 Gy between our 40-h caffeine-supplemented chemical-induced PCC method and the conventional 48-h PCC assay.

Severity scoring systems for radiation-induced GI injury - prioritization for use of GI-ARS medical countermeasures.

PURPOSE: Severity scoring systems for ionizing radiation-induced gastrointestinal injury have been used in animal radiation models, human studies involving the use of radiation therapy, and human radiation accidents. Various radiation exposure scenarios (i.e. total body irradiation, total abdominal irradiation, etc.) have been used to investigate ionizing radiation-induced gastrointestinal injury. These radiation-induced gastrointestinal severity scoring systems are based on clinical signs and symptoms and gastrointestinal-specific biomarkers (i.e. citrulline, etc.). In addition, the time course for radiation-induced changes in blood citrulline levels were compared across various animal (i.e. mice, minipigs, Rhesus Macaque, etc.) and human model systems. CONCLUSIONS: A worksheet tool was developed to prioritize individuals with severe life-threatening gastrointestinal acute radiation syndrome, based on the design of the Exposure and Symptom Tool addressing hematopoietic acute radiation syndrome, to rescue individuals from potential gastrointestinal acute radiation syndrome injury. This tool provides a triage diagnostic approach to assist first responders to assess individuals suspected of showing gastrointestinal acute radiation syndrome severity to guide medical management, hence enhancing medical readiness for managing radiological casualties.

Combined radiation injury and its impacts on radiation countermeasures and biodosimetry.

PURPOSE: Preparedness for medical responses to major radiation accidents and the increasing threat of nuclear warfare worldwide necessitates an understanding of the complexity of combined radiation injury (CI) and identifying drugs to treat CI is inevitably critical. The vital sign and survival after CI were presented. The molecular mechanisms, such as microRNA pathways, NF-κB-iNOS-IL-18 pathway, C3 production, the AKT-MAPK cross-talk, and TLR/MMP increases, underlying CI in relation to organ injury and mortality were analyzed. At present, no FDA-approved drug to protect, mitigate, or treat CI is available. The development of CI-specific medical countermeasures was reviewed. Because of the worsened acute radiation syndrome resulting from CI, diagnostic triage can be problematic. Therefore, biodosimetry and CI are bundled together with the need to establish effective triage methods with CI. CONCLUSIONS: CI mouse model studies at AFRRI are reviewed addressing molecular responses, findings from medical countermeasures, and a proposed plasma proteomic biodosimetry approach based on a panel of radiation-responsive biomarkers (i.e., CD27, Flt-3L, GM-CSF, CD45, IL-12, TPO) negligibly influenced by wounding in an algorithm used for dose predictions is described.

Mitotic index maximization with no effect on radiation-induced dicentric chromosome frequency.

PURPOSE: The dicentric chromosome (Dic) assay, which is the gold standard for biological dose assessment in radiation emergency medicine, requires an analysis of at least 500 lymphocyte metaphases or 100 Dic aberrations. Therefore, peripheral blood culture conditions able to obtain a high frequency of metaphases for efficient dose evaluation should be optimized. However, the type of blood cultures [i.e. whole blood (WB) or isolated peripheral blood mononuclear cell (PBMC)-culture] and blood volume differ between biodosimetry laboratories. The purpose of this study is to investigate the blood volume at which a high mitotic index (MI) is obtained in peripheral WB-culture and isolated PBMC-culture, and to examine the possible effect of blood volume on radiation-induced Dic frequency. MATERIALS AND METHODS: Peripheral blood was collected from three healthy donors with their informed consent. The complete and differential blood counts were performed using an automated hematology analyzer. After blood count, peripheral blood was irradiated with 0 or 2 Gy X-ray. Blood was cultured with phytohemagglutinin (180 µg/ml) and demecolcine　(0.05 µg/ml) for 48 h. The MI and Dic frequency were analyzed in 5, 10, 15, 20, 25, and 30% WB-cultures and 0.6, 1.2, 1.8, 2.4, 3.0, 3.6, and 4.2 ml WB-equivalent PBMC-cultures. RESULTS: In WB-culture, MI showed the highest value (∼22%) in 5-15% WB-culture and then gradually decreased to ∼9% with 30% WB-culture. MI peaked at 36 and 31% in 1.8 and 2.4 ml-WB equivalent volumes for PMBC-cultures, respectively. MI progressively decreased as the amount of PBMCs increased. Although individual differences were observed in the MI values among the three subjects, all the subjects showed the same tendency and higher MI was seen in PBMC than WB-cultures. However, these factors had no significant impact on the yield of Dics. In all culture conditions, the estimated dose calculated based on the Dic frequency was equivalent to the absorbed dose of ex vivo X-ray-irradiated blood. CONCLUSION: While MI was affected by the blood culture type and the volume of cultured blood, Dic yield did not differ significantly between these conditions. These results could be used by relevant laboratories to optimize MI in certain circumstances.

Early-response multiple-parameter biodosimetry and dosimetry: risk predictions.

The accepted generic multiple-parameter and early-response biodosimetry and dosimetry assessment approach for suspected high-dose radiation (i.e. life-threatening) exposure includes measuring radioactivity associated with the exposed individual (if appropriate); observing and recording prodromal signs/symptoms; obtaining serial complete blood counts with white-blood-cell differential; sampling blood for the chromosome-aberration cytogenetic bioassay using the 'gold standard' dicentric assay (premature chromosome condensation assay for exposures >5 Gy photon acute doses equivalent), measurement of proteomic biomarkers and gene expression assays for dose assessment; bioassay sampling, if appropriate, to determine radioactive internal contamination; physical dose reconstruction, and using other available opportunistic dosimetry approaches. Biodosimetry and dosimetry resources are identified and should be setup in advance along with agreements to access additional national, regional, and international resources. This multifaceted capability needs to be integrated into a biodosimetry/dosimetry 'concept of operations' for use in a radiological emergency. The combined use of traditional biological-, clinical-, and physical-dosimetry should be use in an integrated approach to provide: (a) early-phase diagnostics to guide the development of initial medical-management strategy, and (b) intermediate and definitive assessment of radiation dose and injury. Use of early-phase (a) clinical signs and symptoms, (b) blood chemistry biomarkers, and (c) triage cytogenetics shows diagnostic utility to predict acute radiation injury severity.

Celebrating 60 Years of Accomplishments of the Armed Forces Radiobiology Research Institute1.

Chartered by the U.S. Congress in 1961, the Armed Forces Radiobiology Research Institute (AFRRI) is a Joint Department of Defense (DoD) entity with the mission of carrying out the Medical Radiological Defense Research Program in support of our military forces around the globe. In the last 60 years, the investigators at AFRRI have conducted exploratory and developmental research with broad application to the field of radiation sciences. As the only DoD facility dedicated to radiation research, AFRRI's Medical Radiobiology Advisory Team provides deployable medical and radiobiological subject matter expertise, advising commanders in the response to a U.S. nuclear weapon incident and other nuclear or radiological material incidents. AFRRI received the DoD Joint Meritorious Unit Award on February 17, 2004, for its exceptionally meritorious achievements from September 11, 2001 to June 20, 2003, in response to acts of terrorism and nuclear/radiological threats at home and abroad. In August 2009, the American Nuclear Society designated the institute a nuclear historic landmark as the U.S.'s primary source of medical nuclear and radiological research, preparedness and training. Since then, research has continued, and core areas of study include prevention, assessment and treatment of radiological injuries that may occur from exposure to a wide range of doses (low to high). AFRRI collaborates with other government entities, academic institutions, civilian laboratories and other countries to research the biological effects of ionizing radiation. Notable early research contributions were the establishment of dose limits for major acute radiation syndromes in primates, applicable to human exposures, followed by the subsequent evolution of radiobiology concepts, particularly the importance of immune collapse and combined injury. In this century, the program has been essential in the development and validation of prophylactic and therapeutic drugs, such as Amifostine, Neupogen®, Neulasta®, Nplate® and Leukine®, all of which are used to prevent and treat radiation injuries. Moreover, AFRRI has helped develop rapid, high-precision, biodosimetry tools ranging from novel assays to software decision support. New drug candidates and biological dose assessment technologies are currently being developed. Such efforts are supported by unique and unmatched radiation sources and generators that allow for comprehensive analyses across the various types and qualities of radiation. These include but are not limited to both 60Co facilities, a TRIGA® reactor providing variable mixed neutron and γ-ray fields, a clinical linear accelerator, and a small animal radiation research platform with low-energy photons. There are five major research areas at AFRRI that encompass the prevention, assessment and treatment of injuries resulting from the effects of ionizing radiation: 1. biodosimetry; 2. low-level and low-dose-rate radiation; 3. internal contamination and metal toxicity; 4. radiation combined injury; and 5. radiation medical countermeasures. These research areas are bolstered by an educational component to broadcast and increase awareness of the medical effects of ionizing radiation, in the mass-casualty scenario after a nuclear detonation or radiological accidents. This work provides a description of the military medical operations as well as the radiation facilities and capabilities present at AFRRI, followed by a review and discussion of each of the research areas.

RENEB Inter-Laboratory comparison 2017: limits and pitfalls of ILCs.

PURPOSE: In case of a mass-casualty radiological event, there would be a need for networking to overcome surge limitations and to quickly obtain homogeneous results (reported aberration frequencies or estimated doses) among biodosimetry laboratories. These results must be consistent within such network. Inter-laboratory comparisons (ILCs) are widely accepted to achieve this homogeneity. At the European level, a great effort has been made to harmonize biological dosimetry laboratories, notably during the MULTIBIODOSE and RENEB projects. In order to continue the harmonization efforts, the RENEB consortium launched this intercomparison which is larger than the RENEB network, as it involves 38 laboratories from 21 countries. In this ILC all steps of the process were monitored, from blood shipment to dose estimation. This exercise also aimed to evaluate the statistical tools used to compare laboratory performance. MATERIALS AND METHODS: Blood samples were irradiated at three different doses, 1.8, 0.4 and 0 Gy (samples A, C and B) with 4-MV X-rays at 0.5 Gy min-1, and sent to the participant laboratories. Each laboratory was requested to blindly analyze 500 cells per sample and to report the observed frequency of dicentric chromosomes per metaphase and the corresponding estimated dose. RESULTS: This ILC demonstrates that blood samples can be successfully distributed among laboratories worldwide to perform biological dosimetry in case of a mass casualty event. Having achieved a substantial harmonization in multiple areas among the RENEB laboratories issues were identified with the available statistical tools, which are not capable to advantageously exploit the richness of results of a large ILCs. Even though Z- and U-tests are accepted methods for biodosimetry ILCs, setting the number of analyzed metaphases to 500 and establishing a tests' common threshold for all studied doses is inappropriate for evaluating laboratory performance. Another problem highlighted by this ILC is the issue of the dose-effect curve diversity. It clearly appears that, despite the initial advantage of including the scoring specificities of each laboratory, the lack of defined criteria for assessing the robustness of each laboratory's curve is a disadvantage for the 'one curve per laboratory' model. CONCLUSIONS: Based on our study, it seems relevant to develop tools better adapted to the collection and processing of results produced by the participant laboratories. We are confident that, after an initial harmonization phase reached by the RENEB laboratories, a new step toward a better optimization of the laboratory networks in biological dosimetry and associated ILC is on the way.

Improved harvest and fixation methodology for isolated human peripheral blood mononuclear cells in cytokinesis-block micronucleus assay.

PURPOSE: In suspected radiation exposures, cytokinesis-block micronucleus (CBMN) assay is used for biodosimetry by detecting micronuclei (MN) in binucleated (BN) cells in whole blood and isolated peripheral blood mononuclear cell (PBMC) cultures. Standardized harvest protocols for whole blood were published by the International Atomic Energy Agency (IAEA) in 2001 (Technical report no. 405) and 2011 (EPR-Biodosimetry). For isolated PBMC harvest, cytocentrifugation of fresh cells is recommended to preserve cytoplasmic boundaries for MN scoring. However, cytocentrifugation utilizes specialized equipment and long-term cell suspension storage is difficult. In this study, an alternative CBMN harvest protocol is proposed for laboratories interested in culturing PBMCs and storing fixed cells with routine biodosimetry methods. MATERIALS AND METHODS: Peripheral blood from 4 males (24, 34, 41, 51 y.o.) and females (26, 37, 44, 56 y.o.) was irradiated with 0 and 2 Gy X-rays. For cells harvested with IAEA 2001 and 2011 protocols, whole blood was used. For cells harvested with our protocol (CRG), isolated PBMCs were used. CRG protocol was validated in DAPI, acridine orange and Giemsa stain, and in three other laboratories. Cytoplasm status, nuclear division index (NDI) and induced MN frequency (MN frequency at 2 Gy - background MN frequency at 0 Gy) (MN/1000 BN) of Giemsa-stained BN cells were compared in IAEA 2001, IAEA 2011, IAEA 2011 + formaldehyde (FA) and CRG protocols. Effects of low and high humidity spreading were evaluated. RESULTS: >94% of 1000 BN cells were scorable with clear cytoplasmic boundaries in all donors harvested with CRG protocol. FA addition in IAEA 2011 protocol reduced cell rupture in whole blood cultures, but cell rupture was affected by age, sex and humidity. Almost all cells harvested with IAEA 2001 protocol had cytoplasm loss. PBMCs harvested with CRG protocol stained well in DAPI, acridine orange and Giemsa, and showed high scorable BN frequency in all laboratories. A higher NDI and a lower induced MN frequency were seen in 2 Gy isolated PBMC than whole blood cultures. CONCLUSION: This quick CBMN harvest protocol for isolated PBMCs is a viable alternative to cytocentrifugation, as many scorable BN cells were obtained with routine biodosimetry reagents and equipment. IAEA 2011 + FA protocol should be used to improve CBMN harvest in whole blood cultures. Humidity during spreading should be optimized depending on the harvest protocol. NDI and MN frequency should be separately evaluated for whole blood and isolated PBMC cultures.

Automated Dicentric Aberration Scoring for Triage Dose Assessment: 60Co Gamma Ray Dose-response at Different Dose Rates.

The objective of this study was to establish radiation dose-response calibration curves using automated dicentric scoring to support rapid and accurate cytogenetic triage dose-assessment. Blood was drawn from healthy human volunteers and exposed to Co gamma rays at several dose rates (i.e., 1.0, 0.6, and 0.1 Gy min). After radiation, the blood was placed for 2 h in a 37 °C incubator for repair. Blood was then cultured in complete media to which a mitogen (i.e., phytoghemagglutinin, concentration 4%) was added for 48 h. Colcemid was added to the culture at a final concentration of 0.2 µg mL after 24 h for the purpose of arresting first-division metaphase mitotics. Cells were harvested at the end of 48 h. Samples were processed using an automated metaphase harvester and automated microscope metaphase finder equipped with a suite of software including a specialized automated dicentric scoring application. The data obtained were used to create dose-response tables of dicentric yields. The null hypothesis that the data is Poisson-distributed could not be rejected at the significance level of α = 0.05 using results from a Shiny R Studio application (goodness-of-fit Poisson). Calibration curves based on linear-quadratic fits for Co gamma rays at the three different dose rates were generated using these data. The calibration curves were used to detect blind test cases. In conclusion, using the automated harvester and automated microscope metaphase finder with associated automated dicentric scoring software demonstrates high-throughput with suitable accuracy for triage radiation dose assessment.

Construction of fluorescence in situ hybridization (FISH) translocation dose-response calibration curve with multiple donor data sets using R, based on ISO 20046:2019 recommendations.

Purpose: Dose-response curve (DRC) generation is an important aspect in cytogenetic biodosimetry for accurate dose estimation for individuals suspected of prior irradiation. DRC construction with dicentric chromosomes after acute radiation is well-established following the publication of the IAEA EPR-Biodosimetry 2011 and ISO 19238:2014. However, the short half-life of dicentrics might not be suitable for retrospective dose estimation in radiation medical workers, radiation accident clean-up workers and the general public living in areas with higher than average amount of radiation. There is an urgent need for a chromosome translocation-based DRC, which is constructed based on translocation identification with fluorescence in situ hybridization (FISH). Despite several attempts to generate such a DRC in the past 40 years, no internationally standardized protocol has been developed until 2019, resulting in possible statistical uncertainties between DRCs previously generated.Materials and methods: Using the recently published ISO 20049:2019, a DRC from five healthy donors (four males: 23, 35, 44, 55 years old, one female: 33 years old) was generated with age-adjusted translocations scored per cell equivalent (age-adjusted Tr/CE), using a modified R-script previously published in EPR-Biodosimetry, for 60Co gamma-ray doses of 0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5 and 1 Gy. The translocation data set used, based on probes used for chromosomes number 1, 2, and 4, was previously published by Abe et al. in 2018.Results: The results output from R include the DRC coefficients (C, α, ß), their p-values, the goodness-of-fit Pearson's chi square value and its corresponding p-value, and the DRC with its 95% confidence interval (CI). The equation of the DRC obtained was 0.0005 (±0.0001) +0.0178 (±0.0037) D + 0.0901 (±0.0054) D2. DRC generated with averaged Tr/CE had a wider 95% CI than DRC generated with pooled Tr/CE, resulting in a 1.3-1.5 times increase in estimated dose range. No outliers between α coefficients from previously published modified DRCs and our DRC were detected with robust Z-score.Conclusions: ISO 20046:2019 should be referenced for future FISH translocation-based DRC generation to ensure statistical reliability of dose estimation. Important considerations for FISH translocation-based DRC up to 1 Gy include scoring more than 2000 CE per dose, the use of multiple donors, age-adjustment of observed translocations, the use of a minimum of 5 dose points including 0 Gy, scoring of total simple translocations in only stable cells and the decision of using pooled or averaged age-adjusted Tr/CE.

BABOON RADIATION QUALITY (MIXED-FIELD NEUTRON AND GAMMA, GAMMA ALONE) DOSE-RESPONSE MODEL SYSTEMS: ASSESSMENT OF H-ARS SEVERITY USING HAEMATOLOGIC BIOMARKERS.

Results from archived (1986 and 1996) experiments were used to establish a baboon radiation-quality dose-response database with haematology biomarker time-course data following exposure to mixed-fields (i.e. neutron to gamma ratio: 5.5; dose: 0-8 Gy) and 60Co gamma-ray exposures (0-15 Gy). Time-course (i.e. 0-40 d) haematology changes for relevant blood-cell types for both mixed-field (neutron to gamma ratio = 5.5) and gamma ray alone were compared and models developed that showed significant differences using the maximum likehood ratio test. A consensus METREPOL-like haematology ARS (H-ARS) severity scoring system for baboons was established using these results. The data for mixed-field and the gamma only cohorts appeared similar, and so the cohorts were pooled into a single consensus H-ARS severity scoring system. These findings provide proof-of-concept for the use of a METREPOL H-ARS severity scoring system following mixed-field and gamma exposures.

CONCEPTS OF OPERATIONS FOR A US DOSIMETRY AND BIODOSIMETRY NETWORK.

The USA must be prepared to provide a prompt, coordinated and integrated response for radiation dose and injury assessment for suspected radiation exposure, whether it involves isolated cases or mass casualties. Dose estimation for radiation accidents typically necessitates a multiple parameter diagnostics approach that includes clinical, biological and physical dosimetry to provide an early-phase radiation dose. A US Individual Dosimetry and Biodosimetry Network (US-IDBN) will increase surge capacity for civilian and military populations in a large-scale incident. The network's goal is to leverage available resources and provide an integrated biodosimetry capability, using multiple parameter diagnostics. Initial operations will be to expand an existing functional integration of two cytogenetic biodosimetry laboratories by developing Standard Operating Procedures, cross-training laboratorians, developing common calibration curves, supporting inter-comparison exercises and obtaining certification to process clinical samples. Integration with certified commercial laboratories will increase surge capacity to meet the needs of a mass-casualty incident.

System for Scoring Severity of Acute Radiation Syndrome Response in Rhesus Macaques (Macaca mulatta).

We developed a clinical assessment tool for use in an NHP radiation model to 1) quantify severity responses for subsyndromes of the acute radiation syndrome (ARS; that is, hematopoietic and others) and 2) identify animals that required enhanced monitoring. Our assessment tool was based primarily on the MEdical TREatment ProtocOLs for Radiation Accident Victims (METREPOL) scoring system but was adapted for NHP to include additional indices (for example, behaviors) for use in NHP studies involving limited medical intervention. Male (n = 16) and female (n = 12) rhesus macaques (Macaca mulatta; 5 groups: sham and 1.0, 3.5, 6.5, and 8.5 Gy; n = 6 per group) received sham- or bilateral 60Co γ-irradiation at approximately 0.6 Gy/mn. Clinical signs of ARS and blood analysis were obtained before and serially for clinical assessment during the period of 6 h to 60 d after sham or 60Co irradiation. Minimal supportive care (that is, supplemental nutrition, subcutaneous fluid, loperamide, acetaminophen, and topical antibiotic ointment) was prescribed based on clinical observations. Results from clinical signs and assays for assessment of relevant organ systems in individual animals were stratified into ARS severity scores of normal (0), mild (1), moderate (2), and severe (3 or 4). Individual NHP were scored for maximal subsyndrome ARS severity in multiple organ systems by using the proposed ARS scoring system to obtain an overall ARS response category. One NHP died unexpectedly. The multiple-parameter ARS severity scoring tool aided in the identification of animals in the high-dose (6.5 and 8.5 Gy) groups that required enhanced monitoring.

Use of Proteomic and Hematology Biomarkers for Prediction of Hematopoietic Acute Radiation Syndrome Severity in Baboon Radiation Models.

Use of plasma proteomic and hematological biomarkers represents a promising approach to provide useful diagnostic information for assessment of the severity of hematopoietic acute radiation syndrome. Eighteen baboons were evaluated in a radiation model that underwent total-body and partial-body irradiations at doses of Co gamma rays from 2.5 to 15 Gy at dose rates of 6.25 cGy min and 32 cGy min. Hematopoietic acute radiation syndrome severity levels determined by an analysis of blood count changes measured up to 60 d after irradiation were used to gauge overall hematopoietic acute radiation syndrome severity classifications. A panel of protein biomarkers was measured on plasma samples collected at 0 to 28 d after exposure using electrochemiluminescence-detection technology. The database was split into two distinct groups (i.e., "calibration," n = 11; "validation," n = 7). The calibration database was used in an initial stepwise regression multivariate model-fitting approach followed by down selection of biomarkers for identification of subpanels of hematopoietic acute radiation syndrome-responsive biomarkers for three time windows (i.e., 0-2 d, 2-7 d, 7-28 d). Model 1 (0-2 d) includes log C-reactive protein (p < 0.0001), log interleukin-13 (p < 0.0054), and procalcitonin (p < 0.0316) biomarkers; model 2 (2-7 d) includes log CD27 (p < 0.0001), log FMS-related tyrosine kinase 3 ligand (p < 0.0001), log serum amyloid A (p < 0.0007), and log interleukin-6 (p < 0.0002); and model 3 (7-28 d) includes log CD27 (p < 0.0012), log serum amyloid A (p < 0.0002), log erythropoietin (p < 0.0001), and log CD177 (p < 0.0001). The predicted risk of radiation injury categorization values, representing the hematopoietic acute radiation syndrome severity outcome for the three models, produced least squares multiple regression fit confidences of R = 0.73, 0.82, and 0.75, respectively. The resultant algorithms support the proof of concept that plasma proteomic biomarkers can supplement clinical signs and symptoms to assess hematopoietic acute radiation syndrome risk severity.

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.

U.S. Department of Defense Multiple-Parameter Biodosimetry Network.

The U.S. Department of Defense (USDOD) service members are at risk of exposure to ionizing radiation due to radiation accidents, terrorist attacks and national defense activities. The use of biodosimetry is a standard of care for the triage and treatment of radiation injuries. Resources and procedures need to be established to implement a multiple-parameter biodosimetry system coupled with expert medial guidance to provide an integrated radiation diagnostic system to meet USDOD requirements. Current USDOD biodosimetry capabilities were identified and recommendations to fill the identified gaps are provided. A USDOD Multi-parametric Biodosimetry Network, based on the expertise that resides at the Armed Forces Radiobiology Research Institute and the Naval Dosimetry Center, was designed. This network based on the use of multiple biodosimetry modalities would provide diagnostic and triage capabilities needed to meet USDOD requirements. These are not available with sufficient capacity elsewhere but could be needed urgently after a major radiological/nuclear event.

Non-human Primate Total-body Irradiation Model with Limited and Full Medical Supportive Care Including Filgrastim for Biodosimetry and Injury Assessment.

An assessment of multiple biomarkers from radiation casualties undergoing limited- or full-supportive care including treatment with filgrastim is critical to develop rapid and effective diagnostic triage strategies. The efficacy of filgrastim with full-supportive care was compared with results with limited-supportive care by analyzing survival, necropsy, histopathology and serial blood samples for hematological, serum chemistry and protein profiles in a non-human primate (Macaca mulatta, male and female) model during 60-d post-monitoring period following sham- and total-body irradiation with 6.5 Gy 60Co gamma-rays at 0.6 Gy min-1 Filgrastim (10 µg kg-1) was administered beginning on Day 1 post-exposure and continued daily until neutrophil counts were ≥2,000 µL-1 for two consecutive days. Filgrastim and full-supportive care significantly decreased the pancytopenia duration and resulted in improved animal survival and recovery compared to animals with a limited-supportive care. These findings also identified and validated a multiparametric biomarker panel to support radiation diagnostic device development.

MODELING H-ARS USING HEMATOLOGICAL PARAMETERS: A COMPARISON BETWEEN THE NON-HUMAN PRIMATE AND MINIPIG.

Multiple hematological biomarkers (i.e. complete blood counts and serum chemistry parameters) were used in a multivariate linear-regression fit to create predictive algorithms for estimating the severity of hematopoietic acute radiation syndrome (H-ARS) using two different species (i.e. Göttingen Minipig and non-human primate (NHP) (Macacca mulatta)). Biomarker data were analyzed prior to irradiation and between 1-60 days (minipig) and 1-30 days (NHP) after irradiation exposures of 1.6-3.5 Gy (minipig) and 6.5 Gy (NHP) 60Co gamma ray doses at 0.5-0.6 Gy min-1 and 0.4 Gy min-1, respectively. Fitted radiation risk and injury categorization (RRIC) values and RRIC prediction percent accuracies were compared between the two models. Both models estimated H-ARS severity with over 80% overall predictive power and with receiver operating characteristic curve area values of 0.884 and 0.825. These results based on two animal radiation models support the concept for the use of a hematopoietic-based algorithm for predicting the risk of H-ARS in humans.