Detection of partial-body exposure to ionizing radiation by the automatic detection of dicentrics

In accidental exposure to ionizing radiation, it is essential to estimate the dose received by the victims. Currently dicentric scoring is the best biological indicator of exposure. The standard biological dosimetry procedure (500 metaphases scored manually) is suitable for a few dose estimations, but the time needed for analysis can be problematic in the case of a large-scale accident. Recently, a new methodology using automatic detection of dicentrics has greatly decreased the time needed for dose estimation and preserves the accuracy of the estimation. However, the capability to detect nonhomogeneous partial-body exposures is an important advantage of dicentric scoring-based biodosimetry, and this remains to be tested with automatic scoring. Thus we analyzed the results obtained with in vitro blood dilutions and in real cases of accidental exposure (partial- or whole-body exposure) using manual scoring and automatic detection of dicentrics. We confirmed that automatic detection allows threefold quicker dicentric scoring than the manual procedure with similar dose estimations and uncertainty intervals. The results concerning partial-body exposures were particularly promising, and homogeneously exposed samples were correctly distinguished from heterogeneously exposed samples containing 5% to 75% of blood irradiated with 2 Gy. In addition, the results obtained for real accident cases were similar whatever the methodology used. This study demonstrates that automatic detection of dicentrics is a credible alternative for recent and acute cases of whole- and partial-body accidental exposures to ionizing radiation.     

Web-based scoring of the dicentric assay,a collaborative biodosimetric scoring strategy for population triage in large scale radiation accidents

In the case of a large scale radiation accident high throughput methods of biological dosimetry for population triage are needed to identify individuals requiring clinical treatment. The dicentric assay performed in web-based scoring mode may be a very suitable technique. Within the MULTIBIODOSE EU FP7 project a network is being established of 8 laboratories with expertise in dose estimations based on the dicentric assay. Here, the manual dicentric assay was tested in a web-based scoring mode. More than 23,000 high resolution images of metaphase spreads (only first mitosis) were captured by four laboratories and established as image galleries on the internet (cloud). The galleries included images of a complete dose effect curve (0–5.0 Gy) and three types of irradiation scenarios simulating acute whole body, partial body and protracted exposure. The blood samples had been irradiated in vitro with gamma rays at the University of Ghent, Belgium. Two laboratories provided image galleries from Fluorescence plus Giemsa stained slides (3 h colcemid) and the image galleries from the other two laboratories contained images from Giemsa stained preparations (24 h colcemid). Each of the 8 participating laboratories analysed 3 dose points of the dose effect curve (scoring 100 cells for each point) and 3 unknown dose points (50 cells) for each of the 3 simulated irradiation scenarios. At first all analyses were performed in a QuickScan Mode without scoring individual chromosomes, followed by conventional scoring (only complete cells, 46 centromeres). The calibration curves obtained using these two scoring methods were very similar, with no significant difference in the linear-quadratic curve coefficients. Analysis of variance showed a significant effect of dose on the yield of dicentrics, but no significant effect of the laboratories, different methods of slide preparation or different incubation times used for colcemid. The results obtained to date within the MULTIBIODOSE project by a network of 8 collaborating laboratories throughout Europe are very promising. The dicentric assay in the web based scoring mode as a high throughput scoring strategy is a useful application for biodosimetry in the case of a large scale radiation accident.     

Stable and unstable chromosome aberrations in humans and other mammals in relation to the problems of biological dosimetry

Literature data of long-term cytogenetic follow-up of people exposed to radiation as a results of different radiation accidents are considered for the purpose of discussing of some problems of biological dosimetry. The results obtained for mammals are also presented. Of particular interest is a decrease in the level of dicentrics and symmetrical translocations in peripheral blood lymphocytes with the time after acute exposure depending on the dose of irradiation. The frequency of dicentrics decreases in accordance to the exponential law passing the fast and slow phases of elimination. In different radiation situations the values of the parameter which defines the half-life period of lymphocytes characterizing 50% reduction of cells with dicentrics markedly vary. However a general regularity is a decrease in the parameter value as the exposure dose increases. The level of stable translocations estimates by the EISH method remains relatively constant at doses below 1-2 Gy. At higher doses their level in peripheral blood lymphocytes declines with time due to which the retrospective dose appears to be underestimated. The reasons of such regularity, the role of various factors affecting the scoring of translocations, criteria of analysis of the given chromosome aberrations are discussed in the context of common agreements between leading European laboratories on the use of FISH for improving biological dosimetric estimates.     

Biodosimetry and assessment of radiation dose

Aim

When investigating radiation accidents, it is very important to determine the exposition dose to the individuals. In the case of exposures over 1 Gy, clinicians may expect deterministic effects arising the following weeks and months, in these cases dose estimation will help physicians in the planning of therapy. Nevertheless, for doses below 1 Gy, biodosimetry data are important due to the risk of developing late stochastic effects. Finally, some accidental overexposures are lack of physical measurements and the only way of quantifying dose is by biological dosimetry.

Background

The analysis of chromosomal aberrations by different techniques is the most developed method of quantifying dose to individuals exposed to ionising radiations.^1,2 Furthermore, the analysis of dicentric chromosomes observed in metaphases from peripheral lymphocytes is the routine technique used in case of acute exposures to assess radiation doses.

Materials and methods

Solid stain of chromosomes is used to determine dicentric yields for dose estimation. Fluorescence in situ hybridization (FISH) for translocations analysis is used when delayed sampling or suspected chronically irradiation dose assessment. Recommendations in technical considerations are based mainly in the IAEA Technical Report No. 405.²

Results

Experience in biological dosimetry at Gregorio Marañón General Hospital is described, including own calibration curves used for dose estimation, background studies and real cases of overexposition.

Conclusion

Dose assessment by biological dosimeters requires a large previous standardization work and a continuous update. Individual dose assessment involves high qualification professionals and its long time consuming, therefore requires specific Centres. For large mass casualties cooperation among specialized Institutions is needed.     

The cytogenetic dosimetry of recent accidental overexposure.

When accidental exposure to ionizing radiations is suspected, optimal choice of a treatment strategy requires, in addition to information about the clinical signs and physical dosimetry, a determination by biological parameters of the dose received. The scoring of unstable chromosomal aberrations in peripheral blood lymphocytes is the current reference method. Preparation of these samples depends on the goal sought--an exact assessment of several irradiations or rapid triage in the case of a large-scale accident. Moreover, some adaptation may be necessary if the irradiation is either heterogenous or not recent. Despite the robustness and adaptability of this procedure, conventional cytogenetics remains a tedious and time-consuming technique, and it requires specialized staff. Scoring micronuclei in binucleated lymphocytes may be an easier, simpler altemative to a dicentric assay. This paper, which is based on the experience acquired by the IPSN in recent years in expert assessment of suspected radiations, has as its goal to provide a succinct technical guideline of these different approaches, as they are adapted to suspected recent irradiation and triage.     

Retrospective dosimetry after criticality accidents using low-frequency EPR: a study of whole human teeth irradiated in a mixed neutron and gamma-radiation field

In the context of accidental or intentional radiation exposures (nuclear terrorism), it is essential to separate rapidly those individuals with substantial exposures from those with exposures that do not constitute an immediate threat to health. Low-frequency electron paramagnetic resonance (EPR) spectroscopy provides the potential advantage of making accurate and sensitive measurements of absorbed radiation dose in teeth without removing the teeth from the potential victims. Up to now, most studies focused on the dose-response curves obtained for gamma radiation. In radiation accidents, however, the contribution of neutrons to the total radiation dose should not be neglected. To determine how neutrons contribute to the apparent dose estimated by EPR dosimetry, extracted whole human teeth were irradiated at the SILENE reactor in a mixed neutron and gamma-radiation field simulating criticality accidents. The teeth were irradiated in free air as well as in a paraffin head phantom. Lead screens were also used to eliminate to a large extent the contribution of the gamma radiation to the dose received by the teeth. The EPR signals, obtained with a low-frequency (1.2 GHz) spectrometer, were compared to dosimetry measurements at the same location. The contribution of neutrons to the EPR dosimetric signal was negligible in the range of 0 to 10 Gy and was rather small (neutron/gamma-ray sensitivity in the range 0-0.2) at higher doses. This indicates that the method essentially provides information on the dose received from the gamma-ray component of the radiation.     

Cesium-induced chromosome aberrations analyzed by fluorescence in situ hybridization: eight years follow up of the Goiânia radiation accident victims

The radiation accident in focus here occurred in a section of Goiânia (Brazil) where more than a hundred individuals were contaminated with ¹³⁷Cesium on September 1987. In order to estimate the absorbed radiation doses, initial frequencies of dicentrics and rings were determined in 129 victims [A.T. Ramalho, PhD Thesis, Subsidios a tecnica de dosimetria citogenetica gerados a partir da analise de resultados obtidos com o acidente radiologico de Goiânia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 1992]. We have followed some of these victims cytogenetically over the years seeking for parameters that could be used as basis for retrospective radiation dosimetry. Our data on translocation frequencies obtained by fluorescence in situ hybridization (FISH) could be directly compared to the baseline frequencies of dicentrics available for those same victims. Our results provided valuable information on how precise these estimates are. The frequencies of translocations observed years after the radiation exposure were two to three times lower than the initial dicentrics frequencies, the differences being larger at higher doses (> 1 Gy). The accuracy of such dose estimates might be increased by scoring sufficient amount of cells. However, factors such as the persistence of translocation carrying lymphocytes, translocation levels not proportional to chromosome size, and inter-individual variation reduce the precision of these estimates.     

Results of cytogenetic studies of the consequences of the Chernobyl accident

The selected results of the cytogenetic studies of the Chernobyl accident consequences were summarised. The chromosomal aberrations were used as a method of biodosimetry for a dose assessment for victims during the initial period after the Chernobyl accident. A good correlation between doses calculated based on chromosomal aberrations (dicentrics) and severity of acute radiation syndrome observed in clinic was found. The biodosimetry based on conventional cytogenetic technique (dicentrics) has been unsuccessful for various groups (rehabilitation workers, evacuees, inhabitants of contaminated areas) sampled long time after the Chernobyl accident. The possible reasons of the failure are analysed. The original results of multiaberration cell yield observed in different cohorts of the Chernobyl victims are presented. The problems related to the phenomena are discussed.     

Biological dosimetry by the triage dicentric chromosome assay: potential implications for treatment of acute radiation syndrome in radiological mass casualties

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

A New Model of Biodosimetry to Integrate Low and High Doses

Biological dosimetry, that is the estimation of the dose of an exposure to ionizing radiation by a biological parameter, is a very important tool in cases of radiation accidents. The score of dicentric chromosomes, considered to be the most accurate method for biological dosimetry, for low LET radiation and up to 5 Gy, fits very well to a linear-quadratic model of dose-effect curve assuming the Poisson distribution. The accuracy of this estimation raises difficulties for doses over 5 Gy, the highest dose of the majority of dose-effect curves used in biological dosimetry. At doses over 5 Gy most cells show difficulties in reaching mitosis and cannot be used to score dicentric chromosomes. In the present study with the treatment of lymphocyte cultures with caffeine and the standardization of the culture time, metaphases for doses up to 25 Gy have been analyzed. Here we present a new model for biological dosimetry, which includes a Gompertz-type function as the dose response, and also takes into account the underdispersion of aberration-among-cell distribution. The new model allows the estimation of doses of exposures to ionizing radiation of up to 25 Gy. Moreover, the model is more effective in estimating whole and partial body exposures than the classical method based on linear and linear-quadratic functions, suggesting their effectiveness and great potential to be used after high dose exposures of radiation.     

p53 protein expression levels as bioindicator of individual exposure to ionizing radiation by flow cytometry

Ionizing radiation (IR) can cause various lesions in DNA, which induce the increase of p53 expression levels in order to repair radiation induced damage. Thus, the correlation between the increase of p53 expression and an irradiation may constitute a fast and powerful method of individual monitoring in cases of accidental or suspected exposures to IR. In this context, the aim of this research was to evaluate changes in lymphocyte p53 expression levels, based on flow cytometry, after in vitro irradiation of peripheral blood samples. For the measurement of such expression levels of p53 protein, an investigation was carried out in order to establish a methodology of analysis based on flow cytometry. Hence, relationships among levels of expression of p53 protein with the absorbed dose have been verified. The results presented in this report emphasized flow cytometry as an important tool for the fast evaluation of p53 protein expression levels as bioindicator of individual exposure to acute ionizing radiation.     

Overview of the principles and practice of biodosimetry

The principle of biodosimetry is to utilize changes induced in the individual by ionizing radiation to estimate the dose and, if possible, to predict or reflect the clinically relevant response, i.e., the biological consequences of the dose. Ideally, the changes should be specific for ionizing radiation, and the response should be unaffected by prior medical or physiological variations among subjects, including changes that might be caused by the stress and trauma from a radiation event. There are two basic types of biodosimetry with different and often complementary characteristics: those based on changes in biological parameters such as gene activation or chromosomal abnormalities and those based on physical changes in tissues (detected by techniques such as EPR). In this paper, we consider the applicability of the various techniques for different scenarios: small- and large-scale exposures to levels of radiation that could lead to the acute radiation syndrome and exposures with lower doses that do not need immediate care, but should be followed for evidence of long-term consequences. The development of biodosimetry has been especially stimulated by the needs after a large-scale event where it is essential to have a means to identify those individuals who would benefit from being brought into the medical care system. Analyses of the conventional methods officially recommended for responding to such events indicate that these methods are unlikely to achieve the results needed for timely triage of thousands of victims. Emerging biodosimetric methods can fill this critically important gap.     

Cytogenetic evaluation of occupational exposure to external γ-rays and internal

Single cell measurements of electrophoretic mobility showed a linear dose-response of human erythrocytes after exposure to ionizing radiation. To establish a biological indicator- or dosimeter-system for application to dose-estimations after accidental exposures, we tried to measure dose dependent EPM-changes by the free flow electrophoresis technique, a rapid and efficient method which supplies the capacity needed for application. The in vitro irradiated erythrocytes of most donors showed an oscillating dose-response but erythrocytes from several other individuals responded in a contradictory manner whereby the EPM of erythrocytes from radiotherapy patients increased linearly with the amount of administered dose. However, the wide interindividual EPM-range of unirradiated erythrocytes inhibits the application of this technique in biological dosimetry.     

Assessment of electrophoretic mobility changes of human erythrocytes by free flow electrophoresis after in vitro and in vivo irradiation

Single cell measurements of electrophoretic mobility showed a linear dose-response of human erythrocytes after exposure to ionizing radiation. To establish a biological indicator- or dosimeter-system for application to dose-estimations after accidental exposures, we tried to measure dose dependent EPM-changes by the free flow electrophoresis technique, a rapid and efficient method which supplies the capacity needed for application. The in vitro irradiated erythrocytes of most donors showed an oscillating dose-response but erythrocytes from several other individuals responded in a contradictory manner whereby the EPM of erythrocytes from radiotherapy patients increased linearly with the amount of administered dose. However, the wide interindividual EPM-range of unirradiated erythrocytes inhibits the application of this technique in biological dosimetry.     

The mouse splenocyte assay, an in vivo/in vitro system for biological monitoring: studies with X-rays, fission neutrons and bleomycin.

A modified mouse splenocyte culture system was standardized after testing different mitogens (i.e., phytohemagglutinin (PHA), concanavalin A (Con A)). The mitotic index was determined for comparison between different mitogens. Following selection of appropriate mitogen (PHA 16, Flow), a series of experiments were conducted to evaluate the application of a cytokinesis-block for scoring micronuclei and assays for chromosomal aberrations produced by treatment in G0 and G2 for the purposes of biological dosimetry following in vivo and/or in vitro exposure to X-rays, fission neutrons and bleomycin. In the X-irradiation studies, the frequencies of micronuclei and chromosomal aberrations (i.e., dicentrics and rings) increased in a dose-dependent manner. These data could be fitted to a linear-quadratic model. No difference was observed between irradiation in vivo and in vitro, suggesting that measurement of dicentrics and micronuclei in vitro after X-irradiation can be used as an in vivo dosimeter. Following in vivo irradiation with 1 MeV fission neutrons and in vitro culturing of mouse splenocytes, linear dose-response curves were obtained for induction of micronuclei and chromosomal aberrations. The lethal effects of neutrons were shown to be significantly greater than for a similar dose of X-rays. The relative biological effectiveness (RBE) was 6-8 in a dose range of 0.25-3 Gy for radiation-induced asymmetrical exchanges (dicentrics and rings), and about 8 for micronuclei in a dose range of 0.25-2 Gy. Furthermore, the induction of chromosomal aberrations by bleomycin was investigated in mouse G0 splenocytes (in vitro) and compared with X-ray data. Following bleomycin treatment (2 h) a similar pattern of dose-response curve was obtained as with X-rays. In this context a bleomycin rad equivalent of 20 micrograms/ml = 0.50 Gy was estimated.     

Comparison of cytogenic response of human lymphocytes to in vivo and in vitro exposure to low doses of gamma-rays. Unstable chromosomal exchanges detected by FPG techniques

On peripheral lymphocytes of eight cancer patients undergone whole-body therapeutic irradiation (at daily dose of 10 cGy up to total dose of 50 cGy of 60Co gamma-rays) the dose-response of unstable chromosome exchanges (dicentrics and centric rings) was studied. This dose response fitted well linear function. The lower slope of dose-response curve was found for in vivo irradiated lymphocytes as compared to the dose response curve obtained for in vitro irradiated lymphocytes of the same patients. This finding seems to provide evidence that in case of protracted irradiation of individuals an absorbed dose could be underestimated if for biological dosimetry an in vitro dose response curve for unstable chromosome aberrations is used as referent one.     

Biological dosimetry for astronauts: a real challenge

Manned space missions recently increased in number and duration, thus it became important to estimate the biological risks encountered by astronauts. They are exposed to cosmic and galactic rays, a complex mixture of different radiations. In addition to the measurements realized by physical dosimeters, it becomes essential to estimate real biologically effective doses and compare them to physical doses. Biological dosimetry of radiation exposures has been widely performed using cytogenetic analysis of chromosomes. This approach has been used for many years in order to estimate absorbed doses in accidental or chronic overexposures of humans. In addition to conventional techniques (Giemsa or FPG staining, R- or G-banding), faster and accurate means of analysis have been developed (fluorescence in situ hybridization [FISH] painting). As results accumulate, it appears that strong interindividual variability exists in the basal level of aberrations. Moreover, some aberrations such as translocations exhibit a high background level. Radiation exposures seem to induce variability between individual responses. Its extent strongly differs with the mode of exposure, the doses delivered, the kind of radiation, and the cytogenetic method used. This paper aims to review the factors that may influence the reliability of cytogenetic dosimetry. The emphasis is on the exposure to high linear energy transfer (LET) particles in space as recent studies demonstrated interindividual variations in doses estimated from aberration analysis after long-term space missions. In addition to the problem of dose estimates, the heterogeneity of cosmic radiation raises questions relating to the real numbers of damaged cells in an individual, and potential long-term risks. Actually, densely ionizing particles are extremely potent to induce late chromosomal instability, and again, interindividual variability exists in the expression of damage.     

Directional genomic hybridization: inversions as a potential biodosimeter for retrospective radiation exposure

Chromosome aberrations in blood lymphocytes provide a useful measure of past exposure to ionizing radiation. Despite the widespread and successful use of the dicentric assay for retrospective biodosimetry, the approach suffers substantial drawbacks, including the fact that dicentrics in circulating blood have a rather short half-life (roughly 1–2 years by most estimates). So-called symmetrical aberrations such as translocations are far more stable in that regard, but their high background frequency, which increases with age, also makes them less than ideal for biodosimetry. We developed a cytogenetic assay for potential use in retrospective biodosimetry that is based on the detection of chromosomal inversions, another symmetrical aberration whose transmissibility (stability) is also ostensibly high. Many of the well-known difficulties associated with inversion detection were circumvented through the use of directional genomic hybridization, a method of molecular cytogenetics that is less labor intensive and better able to detect small chromosomal inversions than other currently available approaches. Here, we report the dose-dependent induction of inversions following exposure to radiations with vastly different ionization densities [i.e., linear energy transfer (LET)]. Our results show a dramatic dose-dependent difference in the yields of inversions induced by low-LET gamma rays, as compared to more damaging high-LET charged particles similar to those encountered in deep space.     

Whole-blood immunoassay for γH2AX as a radiation biodosimetry assay with minimal sample preparation

The current state of the art in high-throughput minimally invasive radiation biodosimetry involves the collection of samples in the field and analysis at a centralized facility. We have developed a simple biological immunoassay for radiation exposure that could extend this analysis out of the laboratory into the field. Such a forward placed assay would facilitate triage of a potentially exposed population. The phosphorylation and localization of the histone H2AX at double-stranded DNA breaks has already been proven to be an adequate surrogate assay for reporting DNA damage proportional to radiation dose. Here, we develop an assay for phosphorylated H2AX directed against minimally processed sample lysates. We conduct preliminary verification of H2AX phosphorylation using irradiated mouse embryo fibroblast cultures. Additional dosimetry is performed using human blood samples irradiated ex vivo. The assay reports H2AX phosphorylation in human blood samples in response to ionizing radiation over a range of 0–5 Gy in a linear fashion, without requiring filtering, enrichment, or purification of the blood sample.