RENEB Inter-Laboratory Comparison 2021: The Dicentric Chromosome Assay.

After large-scale radiation accidents where many individuals are suspected to be exposed to ionizing radiation, biological and physical retrospective dosimetry assays are important tools to aid clinical decision making by categorizing individuals into unexposed/minimally, moderately or highly exposed groups. Quality-controlled inter-laboratory comparisons of simulated accident scenarios are regularly performed in the frame of the European legal association RENEB (Running the European Network of Biological and Physical retrospective Dosimetry) to optimize international networking and emergency readiness in case of large-scale radiation events. In total 33 laboratories from 22 countries around the world participated in the current RENEB inter-laboratory comparison 2021 for the dicentric chromosome assay. Blood was irradiated in vitro with X rays (240 kVp, 13 mA, ∼75 keV, 1 Gy/min) to simulate an acute, homogeneous whole-body exposure. Three blood samples (no. 1: 0 Gy, no. 2: 1.2 Gy, no. 3: 3.5 Gy) were sent to each participant and the task was to culture samples, to prepare slides and to assess radiation doses based on the observed dicentric yields from 50 manually or 150 semi-automatically scored metaphases (triage mode scoring). Approximately two-thirds of the participants applied calibration curves from irradiations with γ rays and about 1/3 from irradiations with X rays with varying energies. The categorization of the samples in clinically relevant groups corresponding to individuals that were unexposed/minimally (0-1 Gy), moderately (1-2 Gy) or highly exposed (>2 Gy) was successfully performed by all participants for sample no. 1 and no. 3 and by ≥74% for sample no. 2. However, while most participants estimated a dose of exactly 0 Gy for the sham-irradiated sample, the precise dose estimates of the samples irradiated with doses >0 Gy were systematically higher than the corresponding reference doses and showed a median deviation of 0.5 Gy (sample no. 2) and 0.95 Gy (sample no. 3) for manual scoring. By converting doses estimated based on γ-ray calibration curves to X-ray doses of a comparable mean photon energy as used in this exercise, the median deviation decreased to 0.27 Gy (sample no. 2) and 0.6 Gy (sample no. 3). The main aim of biological dosimetry in the case of a large-scale event is the categorization of individuals into clinically relevant groups, to aid clinical decision making. This task was successfully performed by all participants for the 0 Gy and 3.5 Gy samples and by 74% (manual scoring) and 80% (semiautomatic scoring) for the 1.2 Gy sample. Due to the accuracy of the dicentric chromosome assay and the high number of participating laboratories, a systematic shift of the dose estimates could be revealed. Differences in radiation quality (X ray vs. γ ray) between the test samples and the applied dose effect curves can partly explain the systematic shift. There might be several additional reasons for the observed bias (e.g., donor effects, transport, experimental conditions or the irradiation setup) and the analysis of these reasons provides great opportunities for future research. The participation of laboratories from countries around the world gave the opportunity to compare the results on an international level.

Retrospective Biodosimetry of an Occupational Overexposure-Case Study.

In 2014, Health Canada was approached by the Canadian Nuclear Safety Commission to conduct biodosimetry for a possible overexposure 4 y prior to assessment. Dose estimates were determined by means of two cytogenetic assays, the dicentric chromosome assay (DCA) and translocations as measured by the fluorescent in situ hybridization (FISH). As dicentrics are considered to be unstable over time, the results of the DCA were adjusted to account for the time elapsed between the suspected exposure and sampling. The frequency of damage was then compared to Health Canada's calibration curves, respectively, to calculate dose. In addition, the translocation data were corrected for age-related increases in background. With a half-life of 36 months for dicentric chromosomes taken into consideration, the dose estimates from both assays were in agreement. Due to the uncertainty in the half-life of dicentrics, the FISH assay is considered to be more reliable as a technique for retrospective biodosimetry.

THE EFFECT OF AN OPTIMIZED IMAGING FLOW CYTOMETRY ANALYSIS TEMPLATE ON SAMPLE THROUGHPUT IN THE REDUCED CULTURE CYTOKINESIS-BLOCK MICRONUCLEUS ASSAY.

In cases of overexposure to ionizing radiation, the cytokinesis-block micronucleus (CBMN) assay can be performed in order to estimate the dose of radiation to an exposed individual. However, in the event of a large-scale radiation accident with many potentially exposed casualties, the assay must be able to generate accurate dose estimates to within ±0.5 Gy as quickly as possible. The assay has been adapted to, validated and optimized on the ImageStreamX imaging flow cytometer. The ease of running this automated version of the CBMN assay allowed investigation into the accuracy of dose estimates after reducing the volume of whole blood cultured to 200 µl and reducing the culture time to 48 h. The data analysis template used to identify binucleated lymphocyte cells (BNCs) and micronuclei (MN) has since been optimized to improve the sensitivity and specificity of BNC and MN detection. This paper presents a re-analysis of existing data using this optimized analysis template to demonstrate that dose estimations from blinded samples can be obtained to the same level of accuracy in a shorter data collection time. Here, we show that dose estimates from blinded samples were obtained to within ±0.5 Gy of the delivered dose when data collection time was reduced by 30 min at standard culture conditions and by 15 min at reduced culture conditions. Reducing data collection time while retaining the same level of accuracy in our imaging flow cytometry-based version of the CBMN assay results in higher throughput and further increases the relevancy of the CBMN assay as a radiation biodosimeter.

An update of the WHO Biodosenet: Developments since its Inception.

In 2007 the World Health Organization established an international network of biodosimetry laboratories, the BioDoseNet. The goal of this network was to support international cooperation and capacity building in the area of biodosimetry around the world, including harmonisation of protocols and techniques to enable them to provide mutual assistance during a mass casualty event. In order to assess the progress and success of this network, the results of the second survey conducted in 2015 that assessed the capabilities and capacities of the members of the network, were compared to the similar first survey conducted in 2009. The results of the survey offer a unique cross-section of the global status of biodosimetry capacity and demonstrate how the BioDoseNet has brought together laboratories from around the world and strengthened the international capacity for biodosimetry.

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 ImageStream(X) (IS(X) ) 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 IS(X) 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 IS(X) -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.

Multi-parameter dose estimations in radiation biodosimetry using the automated cytokinesis-block micronucleus assay with imaging flow cytometry.

The cytokinesis-block micronucleus (CBMN) assay is an established technique in radiation biological dosimetry for estimating the dose to an individual by measuring the frequency of micronuclei (MN) in binucleated lymphocyte cells (BNCs). The assay has been partially automated using slide-scoring algorithms, but an automated multiparameter method without the need of the slide-making procedure would be advantageous to further increase throughput for application in mass casualty events. The development of the ImageStreamX (ISX) imaging flow cytometer has made it possible to adapt the CBMN assay to an automated imaging flow cytometry (FCM) method. The protocol and analysis presented in this work tailor and expand the assay to a multiparameter biodosimetry tool. Ex vivo irradiated whole blood samples were cultured, processed, and analyzed on the ISX and BNCs, MN, and mononuclear cells were imaged, identified, and enumerated automatically and simultaneously. Details on development of the method, gating strategy, and dose response curves generated for the rate of MN per BNC, percentage of mononuclear cells as well as the replication index are presented. Results indicate that adapting the CBMN assay for use in imaging FCM has produced a rapid, robust, multiparameter analysis method with higher throughput than is currently available with standard microscopy. We conclude that the ISX-CBMN method may be an advantageous tool following a radiological event where triage biodosimetry must be performed on a large number of casualties.

Automated analysis of the cytokinesis-block micronucleus assay for radiation biodosimetry using imaging flow cytometry.

The cytokinesis-block micronucleus (CBMN) assay is employed in biological dosimetry to determine the dose of radiation to an exposed individual from the frequency of micronuclei (MN) in binucleated lymphocyte cells. The method has been partially automated for the use in mass casualty events, but it would be advantageous to further automate the method for increased throughput. Recently, automated image analysis has been successfully applied to the traditional, slide-scoring-based method of the CBMN assay. However, with the development of new technologies such as the imaging flow cytometer, it is now possible to adapt this microscope-based assay to an automated imaging flow cytometry method. The ImageStream(X) is an imaging flow cytometer that has adequate sensitivity to quantify radiation doses larger than 1 Gy while adding the increased throughput of traditional flow cytometry. The protocol and analysis presented in this work adapts the CBMN assay for the use on the ImageStream(X). Ex vivo-irradiated whole blood samples cultured for CBMN were analyzed on the ImageStream(X), and preliminary results indicate that binucleated cells and MN can be identified, imaged and enumerated automatically by imaging flow cytometry. Details of the method development, gating strategy and the dose response curve generated are presented and indicate that adaptation of the CBMN assay for the use with imaging flow cytometry has potential for high-throughput analysis following a mass casualty radiological event.

The Internet's role in a biodosimetric response to a radiation mass casualty event.

Response to a large-scale radiological incident could require timely medical interventions to minimize radiation casualties. Proper medical care requires knowing the victim's radiation dose. When physical dosimetry is absent, radiation-specific chromosome aberration analysis can serve to estimate the absorbed dose in order to assist physicians in the medical management of radiation injuries. A mock exercise scenario was presented to six participating biodosimetry laboratories as one individual acutely exposed to Co under conditions suggesting whole-body exposure. The individual was not wearing a dosimeter and within 2-3 h of the incident began vomiting. The individual also had other medical symptoms indicating likelihood of a significant dose. Physicians managing the patient requested a dose estimate in order to develop a treatment plan. Participating laboratories in North and South America, Europe, and Asia were asked to evaluate more than 800 electronic images of metaphase cells from the patient to determine the dicentric yield and calculate a dose estimate with 95% confidence limits. All participants were blind to the physical dose until after submitting their estimates based on the dicentric chromosome assay (DCA). The exercise was successful since the mean biological dose estimate was 1.89 Gy whereas the actual physical dose was 2 Gy. This is well within the requirements for guidance of medical management. The exercise demonstrated that the most labor-intensive step in the entire process (visual evaluation of images) can be accelerated by taking advantage of world-wide expertise available on the Internet.

Analysis of chromosome damage for biodosimetry using imaging flow cytometry.

The dicentric chromosome assay (DCA), which involves counting the frequency of dicentric chromosomes in mitotic lymphocytes and converting it to a dose-estimation for ionizing radiation exposure, is considered to be the gold standard for radiation biodosimetry. Furthermore, for emergency response, the DCA has been adapted for triage by simplifying the scoring method [1]. With the development of new technologies such as the imaging flow cytometer, it may now be possible to adapt this microscope-based method to an automated cytometry method. This technology allows the sensitivity of microscopy to be maintained while adding the increased throughput of flow cytometry. A new protocol is being developed to adapt the DCA to the imaging cytometer in order to further increase the rapid determination of a biological dose. Peripheral blood mononuclear cells (PBMC) were isolated from ex vivo irradiated whole blood samples using a density gradient separation method and cultured with PHA and Colcemid. After 48h incubation, the chromosomes were isolated, stained for DNA content with propidium iodide (PI) and labelled with a centromere marker. Stained chromosomes were then analyzed on the ImageStream(×) (EMD-Millipore, Billerica, MA). Preliminary results indicate that individual chromosomes can be identified and mono- and dicentric chromosomes can be differentiated by imaging cytometry. A dose response curve was generated using this technology. The details of the method and the dose response curve are presented and compared to traditional microscope scoring. Imaging cytometry is a new technology which enables the rapid, automated analysis of fluorescently labelled chromosomes. Adapting the dicentric assay to this technology has the potential for high throughput analysis for mass casualty events.

The capacity, capabilities and needs of the WHO BioDoseNet member laboratories.

Biodosimetry is an essential tool for providing timely assessments of radiation exposure, particularly when physical dosimetry is unavailable or unreliable. For mass-casualty events involving public exposure to ionising radiation, it is paramount to rapidly provide this dose information for medical management of casualties. The dicentric chromosome assay is currently the most reliable accepted method for biodosimetry; however, in a mass-casualty scenario, the throughput of this assay will be challenged by its time-consuming nature and the specific expertise required. To address this limitation, many countries have established expertise in cytogenetic biodosimetry and started developing surge capabilities through setting up regional networks to deal with emergency situations. To capitalise on this growing expertise and organise it into an internationally coordinated laboratory network, the World Health Organization has created and launched a global biodosimetry network (BioDoseNet). In order to determine the existing capacity of BioDoseNet member laboratories, including their expertise and in vivo experience, involvement in national and international activities, problems, needs and prospects, an in-depth survey was conducted. These survey results provide significant information on the current state of emergency cytogenetic biodosimetry capabilities around the world.

Validation of QuickScan dicentric chromosome analysis for high throughput radiation biological dosimetry.

Currently, the dicentric chromosome assay (DCA) is used to estimate radiation doses to individuals following accidental radiological and nuclear overexposures when traditional dosimetry methods are not available. While being an exceptionally sensitive method for estimating doses by radiation, conventional DCA is time-intensive and requires highly trained expertise for analysis. For this reason, in a mass casualty situation, triage-quality conventional DCA struggles to provide dose estimations in a timely manner for triage purposes. In Canada, a new scoring technique, termed DCA QuickScan, has been devised to increase the throughput of this assay. DCA QuickScan uses traditional DCA sample preparation methods while adapting a rapid scoring approach. In this study, both conventional and QuickScan methods of scoring the DCA assay were compared for accuracy and sensitivity. Dose response curves were completed on four different donors based on the analysis of 1,000 metaphases or 200 events at eight to nine dose points by eight different scorers across two laboratories. Statistical analysis was performed on the data to compare the two methods within and across the laboratories and to test their respective sensitivities for dose estimation. This study demonstrated that QuickScan is statistically similar to conventional DCA analysis and is capable of producing dose estimates as low as 0.1 Gy but up to six times faster. Therefore, DCA QuickScan analysis can be used as a sensitive and accurate method for scoring samples for radiological biodosimetry in mass casualty situations or where faster dose assessment is required.

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

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

Quickscan dicentric chromosome analysis for radiation biodosimetry.

The dicentric chromosome assay (DCA) is the gold-standard assay for accurately estimating unknown radiological doses to individuals following radiological or nuclear accidents. However in a mass-casualty scenario, this assay is not well suited for providing timely dose estimates due to its time- and expertise-intensive nature. In Canada, two approaches are being developed in an attempt to increase triage-quality biological dosimetry throughput. These are 1) increasing the number of trained personnel capable of conducting the DCA, and 2) evaluating alternative biodosimetry approaches or DCA variations. In a recent exercise, a new scoring technique (termed DCA QuickScan) was evaluated as an alternative rapid-scoring approach. Triage-quality conventional DCA and DCA QuickScan analysis were based upon scoring a minimum of 50 metaphase cells or 30 dicentrics by 9-15 scorers across four laboratories. Dose estimates for the conventional DCA were found to be within 0.5 Gy of the actual dose for 83% of the unknown samples, while DCA QuickScan dose estimates were within 0.5 Gy for 80% of the samples. Of the dose estimates falling 0.5 Gy or more outside the actual dose, the majority were dose over-estimates. It was concluded that the DCA QuickScan approach can provide critical dose information at a much faster rate than the conventional DCA without sacrificing accuracy. Future studies will further evaluate the accuracy of the DCA QuickScan method.

Evaluating the biological effects of intermittent 1.9 GHz pulse-modulated radiofrequency fields in a series of human-derived cell lines.

Several recent studies have suggested that radiofrequency (RF) fields may cause changes in a variety of cellular functions that may eventually lead to potential long-term health effects. In the present study, we have assessed the ability of non-thermal RF-field exposure to affect a variety of biological processes (including apoptosis, cell cycle progression, viability and cytokine production) in a series of human-derived cell lines (TK6, HL60 and Mono-Mac-6). Exponentially growing cells were exposed to intermittent (5 min on, 10 min off) 1.9 GHz pulse-modulated RF fields for 6 h at mean specific absorption rates (SARs) of 0, 1 and 10 W/kg. Concurrent negative (incubator) and positive (heat shock for 1 h at 43 degrees C) controls were included in each experiment. Immediately after the 6-h exposure period and 18 h after exposure, cell pellets were collected and analyzed for cell viability, the incidence of apoptosis, and alterations in cell cycle kinetics. The cell culture supernatants were assessed for the presence of a series of human inflammatory cytokines (TNFA, IL1B, IL6, IL8, IL10, IL12) using a cytometric bead array assay. No detectable changes in cell viability, cell cycle kinetics, incidence of apoptosis, or cytokine expression were observed in any of RF-field-exposed groups in any of the cell lines tested, relative to the sham controls. However, the positive (heat-shock) control samples displayed a significant decrease in cell viability, increase in apoptosis, and alteration in cell cycle kinetics (G(2)/M block). Overall, we found no evidence that non-thermal RF-field exposure could elicit any detectable biological effect in three human-derived cell lines.

Relative biological effectiveness of 280 keV neutrons for apoptosis in human lymphocytes.

The relative biological effectiveness (RBE) of neutrons varies from unity to greater than ten depending upon neutron energy and the biological endpoint measured. In our study, we examined apoptosis in human lymphocytes to assess the RBE of low energy 280 keV neutrons compared to Cs gamma radiation and found the RBE to be approximately one. Similar results have been observed for high energy neutrons using the same endpoint. As apoptosis is a major process that influences the consequences of radiation exposure, our results indicate that biological effect and the corresponding weighting factors for 280 keV neutrons may be lower in some cell types and tissues.

Increased FITC fluorescence on LPS stimulated neutrophils cultured in whole blood.

Recently, it has been observed that Annexin V labelling of phosphatidylserine (PS) on non-apoptotic cells can vary in different leukocyte populations and with the activation of cells, due to differences in the absolute level of exposed PS. We have also observed changes in the absolute level of Annexin V-FITC intensity, but under conditions where absolute PS expression did not change. In the present study, we have explored the effect of neutrophil cell activation on Annexin V-FITC fluorescence intensity by comparing alternatively labelled matched antibodies against Annexin V. Human venous whole blood was cultured with and without stimulation with lipopolysaccharide (LPS). Apoptosis in the neutrophil and lymphocyte populations was analyzed by flow cytometry and the intensity of FITC labelling was compared to matched fluorochromes conjugated to the same cell surface markers. There was an increase in the intensity of Annexin V-FITC in non-apoptotic neutrophils when stimulated with LPS, which did not correlate with increased apoptosis. Furthermore, CD65-FITC intensity also increased on activated neutrophils. Activated neutrophils exhibited higher amounts of FITC fluorescence that were not associated with changes in extracellular PS expression. This effect appears to be fluorochrome related, likely due to an increase in the pH surrounding activated neutrophils.

Effect of pro-inflammatory cytokines on spontaneous apoptosis in leukocyte sub-sets within a whole blood culture.

Pro-inflammatory cytokines are known to affect apoptosis in human peripheral blood cells. Neutrophils, which are an essential component of the immune response and usually undergo apoptosis rapidly, are greatly affected by these cytokines. In this study, the effect of varying concentrations of TNF-alpha, IL-1beta and IL-6 on the apoptotic response of leukocytes and their sub-sets in cultured whole blood were studied over a 48 h culture period. At clinically relevant concentrations, it was found that these pro-inflammatory cytokines reduced the amount of spontaneous apoptosis in neutrophils in culture, but had little effect on the lymphocyte population. Distinct differences in the sensitivity of neutrophils to cytokine-mediated protection against spontaneous apoptosis were apparent when compared to previous studies conducted using purified or enriched neutrophil cultures. IL-1beta, at a dose of 0.01 pg/mL, was observed to significantly inhibit spontaneous neutrophil apoptosis by approximately 90% and 65% at 24 and 48 h of culturing, respectively. This concentration used in whole blood is dramatically lower than that required to elicit similar protection in neutrophil-enriched cell cultures. Higher concentrations of TNF-alpha (1.0 pg/mL) and IL-6 (125 pg/mL) were also found to significantly inhibit neutrophil apoptosis, at levels much lower than previously published using neutrophil-enriched cultures. Furthermore, each cytokine displayed a unique signature with respect to the optimal applied doses required to elicit maximal protection against spontaneous neutrophil apoptosis. These results demonstrate the dramatic differences in cellular responses that exist between neutrophil-enriched cultures and whole blood culture systems, where multiple blood cell types provide a much more complex environment.

Prediction of radiosensitivity by measurement of X-ray induced apoptosis in human blood using the comet assay.

BACKGROUND: The aim of this work was to determine whether levels of radiation-induced apoptosis in human peripheral leukocytes could be used as a predictor of radiosensitivity. MATERIALS AND METHODS: Peripheral blood was obtained from venous blood and exposed to 0-3 Gy of X-rays. Apoptosis levels were measured at 4, 24, 48 and 72 hours after exposure using the neutral comet assay. Intra-individual apoptotic response was measured using repeated blood samples from four healthy individuals. Inter-individual variation was investigated in whole blood, granulocytes and mononuclear cells from 8 radiotherapy patients (4 demonstrating a radiosensitive response and 4 demonstrating a normal response to radiation exposures). RESULTS: Amongst the four healthy individuals there was both inter- and intra-individual variation of about the same magnitude. However, when comparing the apoptotic response of the radiosensitive and normal patients, consistent trends were observed at all X-ray doses for all of the patients. CONCLUSION: This indicates that apoptosis has some potential as a predictive assay, however, large intra-individual variation exists. More studies are required to investigate the causes of intra-individual variation and how it might be minimized.

The effect of the ratio of CD4+ to CD8+ T-cells on radiation-induced apoptosis in human lymphocyte subpopulations.

PURPOSE: Apoptosis occurs spontaneously in cultured human peripheral blood lymphocytes but is enhanced by exposure to ionizing radiation. Subpopulations of lymphocytes are known to have varying radiosensitivities to radiation-induced apoptosis. The purpose of this study was to examine the radiation-induced apoptotic response of CD4(+) and CD8(+) T-cells incubated as a complete lymphocyte population. MATERIALS AND METHODS: Using a four-colour flow-cytometry method, which measures annexin-V binding to phosphatidyl serine and propidium iodide, spontaneous and radiation-induced apoptosis was measured in the total lymphocyte fraction and in CD4(+) and CD8(+) T-cell subpopulations. RESULTS: It was found that CD8(+) T-cells were more sensitive to radiation-induced apoptosis than CD4(+) T-cells at doses up to 2 Gy. The yield of radiation-induced apoptosis in the total lymphocyte fraction decreased with increasing ratios of CD4(+) to CD8(+) T-cells (CD4/CD8 ratio). By manipulating the CD4/CD8 ratio within lymphocyte cultures, it was found that the CD4/CD8 ratio had a dramatic effect on the yield of spontaneous apoptosis of total lymphocytes fraction and CD4(+) T-cells but not CD8(+) T-cells. CONCLUSION: The CD4/CD8 ratio affects the apoptotic response of human lymphocytes and CD4(+) T-cells.

Differential apoptotic response to ionizing radiation in subpopulations of human white blood cells.

The purpose of this paper is to characterize the apoptotic response of various subpopulations of human white blood cells after in vitro exposure to ionizing radiation using the modified neutral comet assay (MNCA). White blood cells, isolated from human whole blood, were fractionated into granulocytes and mononuclear cells which were further separated into B-cells, natural killer (NK) cells, and CD4(+) and CD8(+) T-cells. The separated fractions were exposed to low doses of X-rays and then MNCA was used to measure the apoptotic fraction (AF) at different time points in irradiated and unirradiated aliquots of sorted cultures. The spontaneous AF in unirradiated control cells was the most critical determinant of whether an apoptotic response could be detected in irradiated cells. When cultured in isolation granulocytes and B-cells had the highest background AF, with NK cells having the next highest. CD4(+) and CD8(+) T-cells had a low, stable, spontaneous AF which gave them the highest signal-to-noise ratio. Although B-cells demonstrated the highest radiation-induced apoptotic response to 1Gy of X-rays, CD8(+) T-cells were the most radiation-responsive lymphocytes due to their low spontaneous AF. By generating dose response curves for CD4(+) and CD8(+) T-cells, the sensitivity of the MNCA for detecting apoptosis in these two cell types was also examined.