Dose-response Calibration Curve Research Articles

Abstract ID: 230 Experimental and Monte Carlo investigation of the depth-dependent fluence perturbation of parallel-plate chambers in clinical electron beams

The electron fluence inside a parallel-plate ionization chamber positioned in a water phantom and exposed to a clinical electron beam deviates from the unperturbed fluence in water in absence of the chamber. One reason for the fluence perturbation is the "in-scattering effect" as described in ICRU 35. Aim of this work is a detailed experimental and with Monte Carlo simulations investigation of this effect. The experiments and Monte Carlo Simulations, with EGSnrc [1], were performed in a solid-water phantom (RW3) sized 30 × 30 × 30 cm3. One of the RW3 plates of height h = 0.5 cm had a hole with radius r = 1 cm which acts as the air cavity in the phantom. The phantom was placed below an electron applicator of field size 10 × 10 cm2 and irradiated with 6 MeV electrons from an Elekta Synergy accelerator. The air cavity was positioned at depths of 1.0, 1.5, 2.0 and 2.5 cm in the phantom. For the exerpiments the spatial resolved dose directly below the cavity was scored with GAFCHROMIC ETB3 films. The films were scanned with an Epson 10000xl-scanner (resolution 600 dpi, color depth 48 bit); a dose response calibration curve for the red channel was applied. At 1.0 cm depth the relative dose profile shows the well known dose increase at the air/RW3 boundary of about 4% relative to the dose at the center of the cavity and a de-crease of about 8% outside the air cavity within the RW3-phantom. The amplitude of this "dose oscillation" decreases with increasing depth of the cavity within the phantom and disappears at about 2 cm, which is less than the half value thickness of the electron beam. The results are in agreement with already published Monte Carlo results [2]. The results are partly in contradiction to the ideas summarized in ICRU 35 but confirm recent Monte Carlo simulations from Zink et al.

Biological Dosimetry Using Micronucleus Assay in Simulated Partial-Body Exposure to Ionizing Radiation

In radiation accidents, it is common that only several parts of the body are exposed to radiation. As a consequence there is a mixture of exposed and unexposed lymphocytes in peripheral blood cells of the samples. This phenomenon will cause the dose value estimated using the exposed lymphocytes to be lower than the actual dose. In this study, an assessment of partial body exposures using micronucleus assay by estimating the partial body dose and fraction of irradiated blood was conducted. An optimal D0 value also has been determined in this study to estimate the fraction of irradiated cells. Peripheral blood lymphocytes (PBLs) from three healthy donors were irradiated in vitro with 2 Gy of X-rays. Partial radiation exposure was simulated by mixing the irradiated and non-irradiated blood in different proportions. The proportions of mixtures of blood samples irradiated in vitro were 5, 10, 15, 20, and 30 %. Blood samples were then cultured and harvested based on micronuclei assay protocol. At least 2000 binucleated cells with well-preserved cytoplasm were scored for the MN frequency. Dose Estimate 5.1 software was used to calculate the dispersion index (σ2/y) and normalized unit of this index (U) in each proportion of bloods. The fractions of irradiated cells were calculated with CABAS (Chromosomal Aberration Calculation Software) for several different D0 values (2.7; 3.8; 5.4). The results showed that D0 value at 5.4 gave the closest results to the actual proportion of irradiated bloods, while for the dose estimation the estimated doses value from all proportions in all donors were higher than the actual dose. The factor that may cause this phenomenon was that the dose response calibration curve used to predict the radiation dose was not constructed in the laboratory used. Overall it can be concluded that a biodosimetry using MN assay can be used to estimate the radiation dose in partial body exposure. In order to establish a biodosimetry using MN analysis the dose-response calibration curve MN analysis should be constructed first in the laboratory used.

Biodosimetry for High-Dose Exposures Based on Dicentric Analysis in Lymphocytes Released from the G2-Block by Caffeine.

High-dose assessments using the conventional dicentric assay are essentially restricted to doses up to 5 Gy and only to lymphocytes that succeed to proceed to first post-exposure mitosis. Since G2-checkpoint activation facilitates DNA damage recognition and arrest of damaged cells, caffeine is used to release G2-blocked lymphocytes overcoming the mitotic index and dicentric yield saturation problems, enabling thus dicentric analysis even at high-dose exposures. Using the fluorescence in situ hybridizationtechnique with telomere and centromere peptide nucleic acid probes, the released lymphocytes, identified as metaphases with decondensed chromosomes following 1.5 h caffeine treatment, show increased yield of dicentrics compared to that obtained in lymphocytes that reach metaphase without G2-checkpoint abrogation by caffeine. Here, a 3-h caffeine/colcemid co-treatment before harvesting at 55 h post-exposure is used so that the dicentric analysis using Giemsa staining is based predominantly on lymphocytes released from the G2-block, increasing thus dicentric yield and enabling construction of a dose-response calibration curve with improved precision of high-dose estimates.

Optimized automated data analysis for the cytokinesis-block micronucleus assay using imaging flow cytometry for high throughput radiation biodosimetry.

The cytokinesis‐block micronucleus (CBMN) assay is a well‐established technique that can be employed in triage radiation biodosimetry to estimate whole body doses of radiation to potentially exposed individuals through quantitation of the frequency of micronuclei (MN) in binucleated lymphocyte cells (BNCs). The assay has been partially automated using traditional microscope‐based methods and most recently has been modified for application on the ImageStreamX (ISX) imaging flow cytometer. This modification has allowed for a similar number of BNCs to be automatically scored as compared to traditional microscopy in a much shorter time period. However, the MN frequency measured was much lower than both manual and automated slide‐based methods of performing the assay. This work describes the optimized analysis template which implements newly developed functions in the IDEAS® data analysis software for the ISX that enhances specificity for BNCs and increases the frequency of scored MN. A new dose response calibration curve is presented in which the average rate of MN per BNC is of similar magnitude to those presented in the literature using automated CBMN slide scoring methods. In addition, dose estimates were generated for nine irradiated, blinded samples and were found to be within ±0.5 Gy of the delivered dose. Results demonstrate that the improved identification accuracy for MN and BNCs in the ISX‐based version of the CBMN assay will translate to increased accuracy when estimating unknown radiation doses received by exposed individuals following large‐scale radiological or nuclear emergencies. © 2016 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC

Biological dosimetry of ionizing radiation: Evaluation of the dose with cytogenetic methodologies by the construction of calibration curves

In case of a radiation accident, it is well known that in the absence of physical dosimetry biological dosimetry based on cytogenetic methods is a unique tool to estimate individual absorbed dose. Moreover, even when physical dosimetry indicates an overexposure, scoring chromosome aberrations (dicentrics and rings) in human peripheral blood lymphocytes (PBLs) at metaphase is presently the most widely used method to confirm dose assessment. The analysis of dicentrics and rings in PBLs after Giemsa staining of metaphase cells is considered the most valid assay for radiation injury. This work shows that applying the fluorescence in situ hybridization (FISH) technique, using telomeric/centromeric peptide nucleic acid (PNA) probes in metaphase chromosomes for radiation dosimetry, could become a fast scoring, reliable and precise method for biological dosimetry after accidental radiation exposures. In both in vitro methods described above, lymphocyte stimulation is needed, and this limits the application in radiation emergency medicine where speed is considered to be a high priority. Using premature chromosome condensation (PCC), irradiated human PBLs (non-stimulated) were fused with mitotic CHO cells, and the yield of excess PCC fragments in Giemsa stained cells was scored. To score dicentrics and rings under PCC conditions, the necessary centromere and telomere detection of the chromosomes was obtained using FISH and specific PNA probes. Of course, a prerequisite for dose assessment in all cases is a dose-effect calibration curve. This work illustrates the various methods used; dose response calibration curves, with 95% confidence limits used to estimate dose uncertainties, have been constructed for conventional metaphase analysis and FISH. We also compare the dose-response curve constructed after scoring of dicentrics and rings using PCC combined with FISH and PNA probes. Also reported are dose response curves showing scored dicentrics and rings per cell, combining PCC of lymphocytes and CHO cells with FISH using PNA probes after 10 h and 24 h after irradiation, and, finally, calibration data of excess PCC fragments (Giemsa) to be used if human blood is available immediately after irradiation or within 24 h.

Chromosomal Aberrations in Human Peripheral Blood Lymphocytes after Exposure to Ionizing Radiation.

Biological dosimetry using chromosome aberration analyses in human peripheral blood lymphocytes is suitable and useful tool for estimating the dose when a nuclear or radiological emergency is investigated. Blood samples from five healthy donors were obtained to establish dose-response calibration curves for chromosomal aberrations after exposure to ionizing radiation. In this work, dicentric assay and CBMN assay were compared considering the sensitivity and accuracy of dose estimation. In a total of 21,688 analyzed metaphase spreads, 10,969 dicentric chromosomes, 563 centric rings and 11,364 acentric chromosomes were found. The number of metaphase cells decreased with increasing radiation dose. The centric rings were not found in the non-irradiated control. There was no relationship between radiation dose and acentric ring induction. The frequency of total MN increased in a dose-dependent manner. In comparison with the control value, MN increased about 9, 32, 75, 87, and 52 fold higher after treatment with 1, 2, 3, 4, and 5 Gy, respectively. The results revealed that the mean frequency of chromosomal aberrations, both in dicentric and in micronuclei analyses increased with increasing radiation dose.

Radiation exposure dose in human blood lymphocytes as assessed by the CBMN assay

Abstract The chances of accidental exposure are augmented as the application of ionizing radiation increases in various fields. Such accidental exposures may occur at nuclear power plants, laboratories, and hospitals. Cytogenetic assays have been used for estimating radiation dose in the situation of the accidents. The micronucleus assay has several advantages over the other cytogenetic methods as it is simple and fast. The present study aimed at investigation of the micronuclei fre-quencies in cytokinesis-block cells in human blood lymphocytes after γ-irradiation and at establishment of a standard dose response relationship. The samples of peripheral blood were obtained from 6 different donors aged between 24 and 30 years old. The bloods were irradiated in vitro with 0-5 Gy. A linear quadratic dose-response equation was obtained by scoring the micronuclei in binucleated cells; y = 27.87x 2 + 46.13x + 2.08 ( r 2 = 0.99). Irradiation caused a significant decrease in the nuclear division index. Necrotic and apoptotic cells increased in number after irradiation in a dose-dependent manner. In conclusion, the conventional cytokinesis-block micronucleus assay has proven to be the great technique in biological dosimetry. Dose-response calibration curve derived from CMBN assay could be used to estimate the exposure dose during a radiological emergency.

Construction of dose response calibration curves for dicentrics and micronuclei for X radiation in a Serbian population

Biological dosimetry using chromosome damage biomarkers is a valuable dose assessment method in cases of radiation overexposure with or without physical dosimetry data. In order to estimate dose by biodosimetry, any biological dosimetry service have to have its own dose response calibration curve. This paper reveals the results obtained after irradiation of blood samples from fourteen healthy male and female volunteers in order to establish biodosimetry in Serbia and produce dose response calibration curves for dicentrics and micronuclei. Taking into account pooled data from all the donors, the resultant fitted curve for dicentrics is: Ydic=0.0009 (±0.0003)+0.0421 (±0.0042)×D+0.0602 (±0.0022)×D2; and for micronuclei: Ymn=0.0104 (±0.0015)+0.0824 (±0.0050)×D+0.0189 (±0.0017)×D2. Following establishment of the dose response curve, a validation experiment was carried out with four blood samples. Applied and estimated doses were in good agreement. On this basis, the results reported here give us confidence to apply both calibration curves for future biological dosimetry requirements in Serbia.

Effect of spectrum processing procedure on the linearity of EPR dose reconstruction in tooth enamel

Electron Paramagnetic Resonance (EPR) spectroscopy with tooth enamel is a widely used method of dosimetry. The accuracy of EPR tooth dosimetry depends on the spectrum processing procedure, the quality of which, in its turn, relies on instrumental noise and the signals from impurities. This is especially important in low-dose evaluation. The current paper suggests a method to estimate the accuracy of a specific spectrum processing procedure. The method is based on reconstruction of the radiation-induced signal (RIS) from a simulated spectrum with known RIS intensity. The Monte Carlo method was used for the simulations. The model of impurity and noise signals represents a composite residual spectrum (CRS) obtained by subtraction of the reconstructed RIS and the native background signal (BGS) from enamel spectra measured in HMGU (Neuherberg, Germany) and IMP (Yekaterinburg, Russia). The simulated spectra were deconvoluted using a standard procedure. The method provides an opportunity to compare the simulated “true” RIS with reconstructed values. Two modifications of the EPR method were considered: namely, with and without the use of the reference Mn2+ signals. It was observed that the spectrum processing procedure induces a nonlinear dose response of the reconstructed EPR amplitude when the height of the true RIS is comparable with the amplitudes of noise-like random splashes of CRS. The area of nonlinearity is below the limit of detection (DL). The use of reference Mn2+ signals can reduce the range of nonlinearity. However, the impact of the intensities of CRS random signals on nonlinearity is two times higher than the one observed when the reference signals were not used. The reproducibility of the software response is also dependent on both the amplitude of the CRS and the use of a reference signal, and it is also two times more sensitive to the amplitude of the CRS. In most EPR studies, all of the data are used, even those for which the dose value is lower than the DL. This study shows that low doses evaluated with the help of linear dose–response can be significantly overestimated. It is recommended that linear dose response calibration curves be constructed using only data above the DL. Data below the DL should be interpreted cautiously.

Protein biomarkers for enhancement of radiation dose and injury assessment in nonhuman primate total-body irradiation model.

Development and validation of early-response radiation injury biomarkers are critical for effective triage and medical management of irradiated individuals. Plasma protein and haematological profiles were evaluated using multivariate linear-regression analysis to provide dose-response calibration curves for photon-radiation dose assessment in 30 rhesus macaques total-body-irradiated to 1-8.5 Gy with (60)Co gamma rays (0.55 Gy min(-1)). Equations for radiation dose received were established based on different combinations of protein biomarkers [i.e. C-reactive protein (CRP), serum amyloid A (SAA), interleukin 6 (IL-6) and Flt3 Ligand (Flt3L)] at samples collection time-points 6 h, 1, 2, 3, 4 and 7 d post-total-body irradiation. Dynamic changes in the levels of CRP, SAA, IL-6 and Flt3L may function as prognostic indicators of the time course and severity of acute radiation sickness (ARS). The combination of protein biomarkers provides greater accuracy for early radiation assessment than any one biomarker alone.

Establishing cytogenetic biodosimetry laboratory in Saudi Arabia and producing preliminary calibration curve of dicentric chromosomes as biomarker for medical dose estimation in response to radiation emergencies.

In cases of public or occupational radiation overexposure and eventual radiological accidents, it is important to provide dose assessment, medical triage, diagnoses and treatment to victims. Cytogenetic bio-dosimetry based on scoring of dicentric chromosomal aberrations assay (DCA) is the “gold standard” biotechnology technique for estimating medically relevant radiation doses. Under the auspices of the National Science, Technology and Innovation Plan in Saudi Arabia, we have set up a biodosimetry laboratory and produced a national standard dose–response calibration curve for DCA, pre-required to estimate the doses received. For this, the basic cytogenetic DCA technique needed to be established. Peripheral blood lymphocytes were collected from four healthy volunteers and irradiated with radiation doses between 0 and 5 Gy of 320 keV X-rays. Then, lymphocytes were PHA stimulated, Colcemid division arrested and stained cytogenetic slides were prepared. The Metafer4 system (MetaSystem) was used for automatic and manually assisted metaphase finding and scoring of dicentric chromosomes. Results were fit to the linear-quadratic dose–effect model according to the IAEA EPR-Biodosimetry-2011 report. The resulting manually assisted dose–response calibration curve (Y = 0.0017 + 0.026 × D + 0.081 × D2) was in the range of those described in other populations. Although the automated scoring over-and-under estimates DCA at low (<1 Gy) and high (>2 Gy) doses, respectively, it showed potential for use in triage mode to segregate between victims with potential risk to develop acute radiotoxicity syndromes. In conclusion, we have successfully established the first biodosimetry laboratory in the region and have produced a preliminary national dose–response calibration curve. The laboratory can now contribute to the national preparedness plan in response to eventual radiation emergencies in addition to providing information for decision makers and public health officials who assess the magnitude of public, medical, occupational and accidental radiation exposures.

Dose-Response Curve of Chromosome Aberrations in Human Lymphocytes Induced by Gamma-Rays

Chromosome aberration is a biomarker to predict the level of cell damage caused by exposure to ionizing radiation on human body . Dicentric chromosome is a specific chromosome aberration caused by ionizing radiation and is used as a gold standard biodosimetry of individuals over exposed to ionizing radiation . In radiation accident the dicentric assays has bee n applied as biological dosimetry to estimat e radiation absorbed dose and also to confirm the radiation dose received to radiation workers. The purpose of this study was to generate a dose response curve of chromosome aberration (dicentric) in human lymphocyte induced by gamma radiation . Peripheral blood samples from three non smoking healthy volunteers aged between 25-48 year s old with informed consent were i rradiat ed with dose between 0.1 - 4.0 Gy and a control using gamma teletherapy source . The culture procedure was conducted following the IAEA standard procedures with slight modifications. Analysis of dose-response curves used was LQ model Y = a + αD + βD 2 . The result show ed that α and β values of the curve obtained were 0.018 ± 0.006 and 0.013 ± 0.002, respectively. Dose response calibration curve for dicentric chromosome aberrations in human lymphocytes induced b y gamma -ra diation fitted to linear quadratic model. I n order to apply the dose response curve of chromosome aberration disentric for biodosimetry, this standar curve still need to be validated Received: 8 November 2013; Revised: 27 December 2013; Accepted: 31 December 2013

The cytokinesis-blocked micronucleus assay: Dose estimation and inter-individual differences in the response to γ-radiation

Biological dosimetry plays an important role in case of a radiation accident or incident, either when it is the only way to estimate the dose or when it is used to complement physical dosimetry. A cytogenetic study was conducted in a group of 16 Portuguese individuals by use of the cytokinesis-blocked micronucleus (CBMN) assay. A dose–response curve for micronuclei yield was established with a linear-quadratic model: Y=(0.0122±0.0010)+(0.0241±0.0023)D+(0.0193±0.0007)D2. Also, baseline values for the micronucleus formation in the 16 donors were analyzed, with results in close agreement with those from other laboratories. A validation experiment was carried out with three individuals. The real and the estimated doses obtained with the dose–response curve were in very good agreement, allowing the use of the micronucleus dose–response calibration curve in biological dosimetry for estimation of radiation dose in case of overexposure.The results obtained for the cytogenetic endpoints, studied in the same group of 16 individuals, were also analyzed as a function of age and gender. A higher inter-variability was observed for the higher dose points and differences in response were identified between genders, above 2Gy, for all endpoints.

Implementation of a dose–response curve for γ-radiation in the Portuguese population by use of the chromosomal aberration assay

An in vitro dose–response curve following exposure to γ-radiation was determined at the IST/ITN, by use of the chromosomal aberration assay. This is the first study of this kind carried out among the Portuguese population. Un-irradiated and γ-irradiated peripheral blood lymphocytes from 16 healthy donors were cultured. A total of 22,395 metaphases were analyzed for frequency and distribution of dicentrics and centric rings, as a function of the radiation dose. The dose–response data for dicentrics and dicentrics plus centric rings were fitted by use of a linear–quadratic model: Ydic=(0.0011±0.0006)+(0.0105±0.0035)D+(0.0480±0.0019)D2 and Ydic+rings=(0.0011±0.0006)+(0.0095±0.0036)D+(0.0536±0.0020)D2. Also, calibration curves related to age and gender were determined, but no significant differences were found. Following the establishment of the dose–response curves, a validation experiment was carried out with three individuals. Real and estimated doses, obtained with the dose–response curves, were in agreement. These results give us confidence to apply both dose–response calibration curves in future biological dosimetry requirements.

The Long-Awaited Whole-Blood Thrombin Generation Test

Thrombin is a multifunctional protease that, in addition to hemostasis, supports other “nonhemostatic” mechanisms, such as the regulation of vascular permeability, vascular tone, inflammation, and angiogenesis. There is also evidence that thrombin plays a role in the pathogenesis and progression of liver fibrosis (1). Among the hemostasis functions played by thrombin, one may list the fibrinogen-to-fibrin conversion, the activation of platelets, factors V, VIII, XI, XIII, protein C (PC),2 and the thrombin-activatable fibrinolysis inhibitor. In this issue of Clinical Chemistry , Ninivaggi et al. (2) report on a new whole-blood thrombin generation test (TGT), which has been a long-awaited development in the area of coagulation testing. To understand the importance of this test to the field requires some background information on current coagulation-testing methods. Methods to assess thrombin generation from its precursor prothrombin were developed in the early 1950s, when McFarlane and Biggs triggered coagulation in whole blood or plasma by adding calcium chloride with or without tissue factor or cephalin (3). In the original TGT, the protease generated upon the triggering of coagulation was monitored by sampling a portion of the mixture at fixed time intervals and placing the sample into a second tube containing a fibrinogen solution for testing. The resulting clotting time, which was inversely proportional to the thrombin generated as a function of the coagulation factors, was eventually converted into the thrombin concentration by interpolation from a dose–response calibration curve, which was constructed by testing known amounts of thrombin. The method was too cumbersome and time-consuming for routine use, however, and was soon abandoned in favor of simpler tests, such as the prothrombin time (PT) and the activated partial thromboplastin time (APTT). Many years later, the TGT was revisited by Hemker et al. (4), who introduced important modifications. Fibrinogen as a detection system for thrombin …

Cytogenetic biodosimetry for Fukushima travelers after the nuclear power plant accident: no evidence of enhanced yield of dicentrics

Individuals who traveled to contaminated areas after the Fukushima nuclear accident have concerns about the health effects. However, medical follow-up for any adverse health effects will be difficult without personal dose measurements. Cytogenetic biodosimetry is a reasonable method of assessing absorbed doses retrospectively. We analyzed dicentric chromosomes for 265 Fukushima travelers, mostly journalists and rescue workers, who had been dispatched to northeastern Japan during the nuclear emergency. As a control group, 37 healthy volunteers who had not visited Japan since the accident were enrolled. Yields of dicentrics and absorbed doses calculated from a dose-response calibration curve for travelers and the control group were compared. The cut-off level for dicentric chromosomes in the controls was 3.5 per 1000 cells. Of the 265 travelers, 31 had elevated numbers of dicentrics (High-Dics group) while 234 were below the cut-off (Normal-Dics group). All but one of the individuals in the High-Dics group also reported a significantly higher number of medical exposures to radiation within the past three years compared with the Normal-Dics or control groups. The 225 travelers with no history of medical exposure showed no difference of dicentrics yield compared to the control group. Our data indicate that Fukushima travel alone did not enhance the yield of dicentrics.

A SERS-based immunoassay with highly increased sensitivity using gold/silver core-shell nanorods

An immunoassay based on surface enhanced Raman scattering (SERS) has been developed using immuno-gold/silver core-shell nanorods with a high sensitivity. The features of these nanoparticles were characterized by UV–vis extinction spectra, TEM images, EDX analyses and SERS spectra. It was found that the obtained gold/silver core-shell nanorods showed a much higher SERS activity than uncoated gold nanorods. After the gold/silver core-shell nanorods were modified with antibody and employed in immunoassay, the antigen concentration-dependent SERS spectra and dose-response calibration curves were obtained. By comparison, it can be concluded that the detection limit of gold/silver core-shell nanorods based immunoassay reaches 70fM, which is 104 times lower than gold nanorods based detection. As a result, SERS probes fabricated with gold/silver core-shell nanorods are demonstrated to be advantageous to those synthesized with gold nanorods due to their highly increased sensitivity in sandwich immunoassay, which also indicates the potential use of these gold/silver core-shell nanorods in other biological sensing applications.

Influence of age and gender in response to γ-radiation in Portuguese individuals using chromosomal aberration assay – Preliminary findings

Abstract Cytogenetic indicators are widely used in radiobiology to evaluate effects of ionizing radiation since dicentric chromosomes (Dic) are almost exclusively induced by ionizing radiation, and spontaneous frequency of Dic is very low in the healthy general population (about one Dic per 1000 cells). A particular interest of biodosimetry has been not only to obtain absorbed dose estimates using adequate calibration curves, under the assumption that all individuals respond equally to radiation-induced chromosome aberrations, but also to find a way to demonstrate inter-individual radiosensitivity and a possible correlation with age and gender. Thus, the objective of this preliminary work was the evaluation of the influence of age and gender on the outcome of cytogenetic biomarkers after γ-irradiation. Samples of peripheral blood lymphocytes from six healthy, non-smoker, donors from both genders (three men and three women), in the range of 20 to 49 years, were irradiated with doses from 0 Gy to 3 Gy air kerma, using a 60 Co gamma rays source with a dose rate from 170–180 mGy/min. A clear dose-dependent increase in terms of aberrant cells excluding gaps (ACEG) and Dic was observed for all donors. Our preliminary results suggest, in the higher dose level evaluated (3 Gy), a larger intervariability among individuals for Dic, with females apparently more sensitive than males (P Future work will consider the study of more individuals, from both genders and different ages, in order to verify if this tendency persists and to enable the implementation of a dose-response calibration curve at Instituto Tecnologico e Nuclear for the Portuguese population, to quantify the biological dose in case of a radiological accident or emergency.

CYTOKINESIS-BLOCK MICRONUCLEUS (CBMN) ASSAY/CBMN CYTOME ASSAY IN HUMAN LYMPHOCYTES AFTER IN VITRO IRRADIATION AND ITS USE IN BIODOSIMETRY

Summary Aim: Cytokinesis-block micronucleus (CBMN) assay and its comprehensive variant CBMN cytome assay are cytogenetic methods. CBMN is based on assessment of micronuclei in nucleated cells that have completed only one nuclear division. Besides micronuclei, CBMN cytome assay analyzes additional genotoxic (nucleoplasmic bridges and nuclear buds), cytostatic (nuclear division index), and cytotoxic (amount of necrotic and apoptotic cells) parameters. The aim of this study is to evaluate these parameters in human blood lymphocytes after in vitro irradiation and to assess its contribution to biodosimetry. Material and methods: Human blood from 6 donors was in vitro irradiated by 0, 1, 2, 3, 4, or 5 Gy and cultivated for 72 hours. Blood lymphocytes were stimulated with phytohemagglutinin and their cytokinesis was blocked by cytochalasin B. After cultivation, cultures were hypotonically treated, dropped onto glass slides and stained with Giemsa. Slides were evaluated by microscope. Results: We observed significantly increased amount of micronuclei, nucleoplasmic bridges, and nuclear buds measured in binucleated cells, significantly increased amount of micronuclei measured in mononucleated cells and significantly decreased nuclear division index after irradiation by 1, 2, 3, 4, and 5 Gy. Amount of death cells (apoptotic and necrotic) significantly increased after irradiation by 4 and 5 Gy. Conclusion: Although all parameters assessed by CBMN cytome assay have biodosimetric potential, practically feasible is only evaluation of micronuclei in binucleated cells. This parameter was used to construct in vitro linear-quadratic dose-response calibration curve which could be used as a biodosimetric tool for triage of radiation casualties.

Qγ−H2AX, an analysis method for partial-body radiation exposure using γ-H2AX in non-human primate lymphocytes

We previously used the γ-H2AX assay as a biodosimeter for total-body-irradiation (TBI) exposure (γ-rays) in a rhesus macaque (Macaca mulatta) model. Utilizing peripheral blood lymphocytes and plucked hairs, we obtained statistically significant γ-H2AX responses days after total-body exposure to 1-8.5 Gy ((60)Co γ-rays at 55 cGy min(-1)). Here, we introduce a partial-body exposure analysis method, Q(γ-H2AX), which is based on the number of γ-H2AX foci per damaged cells as evident by having one or more γ-H2AX foci per cell. Results from the rhesus monkey - TBI study were used to establish Q(γ-H2AX) dose-response calibration curves to assess acute partial-body exposures. γ-H2AX foci were detected in plucked hairs for several days after in vivo irradiation demonstrating this assay's utility for dose assessment in various body regions. The quantitation of γ-H2AX may provide a robust biodosimeter for analyzing partial body exposures to ionizing radiation in humans.