Irradiated Blood Samples Research Articles

Calibration curve for radiation dose estimation using FDXR gene expression biodosimetry – premises and pitfalls

Purpose Radiation-induced alterations in gene expression show great promise for dose reconstruction and for severity prediction of acute health effects. Among several genes explored as potential biomarkers, FDXR is widely used due to high upregulation in white blood cells following radiation exposure. Nonetheless, the absence of a standardized protocols for gene expression-based biodosimetry is a notable gap that warrants attention to enhance the accuracy, reproducibility and reliability. The objective of this study was to evaluate the sensitivity of transcriptional biodosimetry to differences in protocols used by different laboratories and establish guidelines for the calculation of calibration curve using FDXR expression data. Material and Methods Two sets of irradiated blood samples generated during RENEB exercise were used. The first included samples irradiated with known doses including: 0, 0.25, 0.5, 1, 2, 3 and 4 Gy. The second set consisted of three ‘blind’ samples irradiated with 1.8 Gy, 0.4 Gy and a sham-irradiated sample. After irradiation, samples were incubated at 37 °C over 24 h and sent to participating laboratories, where RNA isolation and FDXR expression analysis by qPCR were performed using sets of primers/probes and reference genes specific for each laboratory. Calibration curves based on FDXR expression data were generated using non-linear and linear regression and used for dose estimation of ‘blind’ samples. Results Dose estimates for sham-irradiated sample (0.020–0.024 Gy) and sample irradiated with 0.4 Gy (0.369–0.381 Gy) showed remarkable consistency across all laboratories, closely approximating the true doses regardless variation in primers/probes and reference genes used. For sample irradiated with 1.8 Gy the dose estimates were less precise (1.198–2.011 Gy) but remained within an acceptable margin for triage within the context of high dose range. Conclusion Methodological differences in reference genes and primers/probes used for FDXR expression measurement do not have a significant impact on the dose estimates generated, provided that all reference genes performed as expected and the primers/probes target a similar set of transcript variants. The preferred method for constructing a calibration curve based on FDXR expression data involves employing linear regression to establish a function that describes the relationship between the logarithm of absorbed dose and FDXR ΔCt values. However, one should be careful with using non-irradiated sample data as these cannot be accurately represented on a logarithmic scale. A standard curve generated using this approach can give reliable dose estimations in a dose range from 50 mGy to 4 Gy at least.

LBDNet interlaboratory comparison for the dicentric chromosome assay by digitized image analysis applying weighted robust statistical methods

Purpose This interlaboratory comparison was conducted to evaluate the performance of the Latin-American Biodosimetry Network (LBDNet) in analyzing digitized images for scoring dicentric chromosomes from in vitro irradiated blood samples. The exercise also assessed the use of weighted robust algorithms to compensate the uneven expertise among the participating laboratories. Methods Three sets of coded images obtained through the dicentric chromosome assay from blood samples irradiated at 1.5 Gy (sample A) and 4 Gy (sample B), as well as a non-irradiated whole blood sample (sample C), were shared among LBDNet laboratories. The images were captured using the Metafer4 platform coupled with the AutoCapt module. The laboratories were requested to perform triage scoring, conventional scoring, and dose estimation. The dose estimation was carried out using either their laboratory calibration curve or a common calibration curve. A comparative statistical analysis was conducted using a weighted robust Hampel algorithm and z score to compensate for uneven expertise in dicentric analysis and dose assessment among all laboratories. Results Out of twelve laboratories, one had unsatisfactory estimated doses at 0 Gy, and two had unsatisfactory estimated doses at 1.5 Gy when using their own calibration curve and triage scoring mode. However, all doses were satisfactory at 4 Gy. Six laboratories had estimated doses within 95% uncertainty limits at 0 Gy, seven at 1.5 Gy, and four at 4 Gy. While the mean dose for sample C was significantly biased using robust algorithms, applying weights to compensate for the laboratory’s analysis expertise reduced the bias by half. The bias from delivered doses was only notable for sample C. Using the common calibration curve for dose estimation reduced the standard deviation (s*) estimated by robust methods for all three samples. Conclusions The results underscore the significance of performing interlaboratory comparison exercises that involve digitized and electronically transmitted images, even when analyzing non-irradiated samples. In situations where the participating laboratories possess different levels of proficiency, it may prove essential to employ weighted robust algorithms to achieve precise outcomes.

PUM1 and PGK1 are Favorable Housekeeping Genes over Established Biodosimetry-related Housekeeping Genes such as HPRT1, ITFG1, DPM1, MRPS5, 18S rRNA and Others after Radiation Exposure.

In gene expression (GE) studies, housekeeping genes (HKGs) are required for normalization purposes. In large-scale inter-laboratory comparison studies, significant differences in dose estimates are reported and divergent HKGs are employed by the teams. Among them, the 18S rRNA HKG is known for its robustness. However, the high abundance of 18S rRNA copy numbers requires dilution, which is time-consuming and a possible source of errors. This study was conducted to identify the most promising HKGs showing the least radiation-induced GE variance after radiation exposure. In the screening stage of this study, 35 HKGs were analyzed. This included selected HKGs (ITFG1, MRPS5, and DPM1) used in large-scale biodosimetry studies which were not covered on an additionally employed pre-designed 96-well platform comprising another 32 HKGs used for different exposures. Altogether 41 samples were examined, including 27 ex vivo X-ray irradiated blood samples (0, 0.5, 4 Gy), six X-irradiated samples (0, 0.5, 5 Gy) from two cell lines (U118, A549), as well as eight non-irradiated tissue samples to encompass multiple biological entities. In the independent validation stage, the most suitable candidate genes were examined from another 257 blood samples, taking advantage of already stored material originating from three studies. These comprise 100 blood samples from ex vivo X-ray irradiated (0-4 Gy) healthy donors, 68 blood samples from 5.8 Gy irradiated (cobalt-60) Rhesus macaques (RM) (LD29/60) collected 0-60 days postirradiation, and 89 blood samples from chemotherapy-(CTx) treated breast tumor patients. CTx and radiation-induced GE changes in previous studies appeared comparable. RNA was isolated, converted into cDNA, and GE was quantified employing TaqMan assays and quantitative RT-PCR. We calculated the standard deviation (SD) and the interquartile range (IQR) as measures of GE variance using raw cycle threshold (Ct) values and ranked the HKGs accordingly. Dose, time, age, and sex-dependent GE changes were examined employing the parametrical t-test and non-parametrical Kruskal Wallis test, as well as linear regression analysis. Generally, similar ranking results evolved using either SD or IQR GE measures of variance, indicating a tight distribution of GE values. PUM1 and PGK1 showed the lowest variance among the first ten most suitable genes in the screening phase. MRPL19 revealed low variance among the first ten most suitable genes in the screening phase only for blood and cells, but certain comparisons indicated a weak association of MRPL19 with dose (P = 0.02-0.09). In the validation phase, these results could be confirmed. Here, IQR Ct values from, e.g., X-irradiated blood samples were 0.6 raw Ct values for PUM1 and PGK1, which is considered to represent GE differences as expected due to methodological variance. Overall, when compared, the GE variance of both genes was either comparable or lower compared to 18S rRNA. Compared with the IQR GE values of PUM1 and PGKI, twofold-fivefold increased values were calculated for the biodosimetry HKG HPRT1, and comparable values were calculated for biodosimetry HKGs ITFG1, MRPS5, and DPM1. Significant dose-dependent associations were found for ITFG1 and MRPS5 (P = 0.001-0.07) and widely absent or weak (P = 0.02-0.07) for HPRT1 and DPM1. In summary, PUM1 and PGK1 appeared most promising for radiation exposure studies among the 35 HKGs examined, considering GE variance and adverse associations of GE with dose.

Effects of genome instability under irradiation in different CT scanning modes. Results of ex vivo pilot cohort study

Introduction. Medical radiation is one of the leading sources of public exposure in the world. In recent decades, the total number of X-ray diagnostic procedures has increased significantly, and with the increase in the volume of computed tomography (CT), a significant gain in the total cumulative radiation dose is also associated. 
 The aim of the work is to compare the genotoxic effects of irradiation of human blood lymphocytes using various CT protocols. 
 Materials and methods. Among patients of different genders and ages who sought for a preventive examination, nine practically healthy volunteers (donors) who signed an informed consent, were randomly selected to participate in the ex vivo experiment. 4 venous blood samples from each donor were irradiated on various CT protocols (0.82–11.8 mSv) in an anthropometric phantom of the human chest. Aliquots of each sample were cultured under conditions of cytokinesis block, recorded and analyzed according to the protocol of cytomic analysis in a micronucleus test.
 Results. Irradiation of blood samples in the Ultra-NDCT mode (0.82 mSv) revealed a peak in the proliferation of rapidly dividing cells and the frequency of genetic damage in them, and also demonstrated a high probability of the formation and consolidation of genetic damage in generations of dividing cells. This indicates increased genotoxicity and, most likely, immunotropicity of the studied irradiation mode.
 Llimitation of the study is the inadmissibility of involving a person in an experiment or using biomaterials without obtaining his consent, as well as the inadmissibility of causing physical harm or harm to human honor and dignity during the experiment
 Conclusion. When choosing CT modes, it is necessary to take into account not only the levels of effective doses, but also the possibility of developing the effects 
 of genome instability. However, this approach requires additional genotoxic studies of CT protocols in the range from <1 to 100 mSv.

Study of light element activation in human blood during 20 MeV linac operating

A series of experiments have been performed to study the activation of chemical macronutrients in human venous blood throughout radiation treatment with the 20 MeV linac. Some elements in the human blood can be activated as positron-emitting radionuclides by (𝛾, n)-reaction. We calculated the 11C, 13N, 15O, 30P, 34𝑚Cl, and 38K activities in irradiated human venous blood samples for the first time. As a result, we concluded that the main contribution to the dose from positron-emitting nuclides activated in human blood provides three nuclides: 15O, 34𝑚Cl, and 38K.

Application of the Cytokinesis-Block Micronucleus Assay for High-Dose Exposures Using Imaging Flow Cytometry

The cytokinesis-block micronucleus assay is a well-established method to assess radiation-induced genetic damage in human cells. This assay has been adapted to imaging flow cytometry (IFC), allowing automated analysis of many cells, and eliminating the need to create microscope slides. Furthermore, to improve the efficiency of assay performance, a small-volume method previously developed was employed. Irradiated human blood samples were cultured, stained, and analyzed by IFC to produce images of the cells. Samples were run using both manual and 96-well plate automated acquisition. Multiple parameter-based image features were collected for each sample, and the results were compared to confirm that these acquisition methods are functionally identical. This paper details the multi-parametric analysis developed and the resulting calibration curves up to 10 Gy. The calibration curves were created using a quadratic random coefficient model with Poisson errors, as well as a logistic discriminant function. The curves were then validated with blinded, irradiated samples, using relative bias and relative mean square error. Overall, the accuracy of the dose estimates was adequate for triage dosimetry (within 1 Gy of the true dose) over 90% of the time for lower doses and about half the time for higher doses, with the lowest success rate between 5 and 6 Gy where the calibration curve reached its peak and there was the smallest change in MN/BNC with dose. This work describes the application of a novel multi-parametric analysis that fits the calibration curves and allows dose estimates up to 10 Gy, which were previously limited to 4 Gy. Furthermore, it demonstrates that the results from samples acquired manually and with the autosampler are functionally similar.

Establishment of ex vivo calibration curve for X-ray induced “dicentric + ring” and micronuclei in human peripheral lymphocytes for biodosimetry during radiological emergencies, and validation with dose blinded samples

In the modern developing society, application of radiation has increased extensively. With significant improvement in the radiation protection practices, exposure to human could be minimized substantially, but cannot be avoided completely. Assessment of exposure is essential for regulatory decision and medical management as applicable. Until now, cytogenetic changes have served as surrogate marker of radiation exposure and have been extensively employed for biological dose estimation of various planned and unplanned exposures. Dicentric Chromosomal Aberration (DCA) is radiation specific and is considered as gold standard, micronucleus is not very specific to radiation and is considered as an alternative method for biodosimetry. In this study dose response curves were generated for X-ray induced “dicentric + ring” and micronuclei, in lymphocytes of three healthy volunteers [2 females (age 22, 23 years) and 1 male (24 year)]. The blood samples were irradiated with X-ray using LINAC (energy 6 MV, dose rate 6 Gy/min), in the dose range of 0–5Gy. Irradiated blood samples were cultured and processed to harvest metaphases, as per standard procedures recommended by International Atomic Energy Agency. Pooled data obtained from all the three volunteers, were in agreement with Poisson distribution for “dicentric + ring”, however over dispersion was observed for micronuclei. Data (“dicentric + ring” and micronuclei) were fitted by linear quadratic model of the expression YC + αD + βD2 using Dose Estimate software, version 5.2. The data fit has resulted in linear coefficient α = 0.0006 (±0.0068) “dicentric + ring” cell−1 Gy−1 and quadratic coefficient β = 0.0619 (±0.0043) “dicentric + ring” cell−1 Gy−2 for “dicentric + ring” and linear coefficient α = 0.0459 ± (0.0038) micronuclei cell−1 Gy−1 and quadratic coefficient β = 0.0185 ± (0.0010) micronuclei cell−1 Gy−2 for micronuclei, respectively. Background frequencies for “dicentric + ring” and micronuclei were 0.0006 ± 0.0004 and 0.0077 ± 0.0012 cell−1, respectively. Established curves were validated, by reconstructing the doses of 8 dose blinded samples (4 by DCA and 4 by CBMN) using coefficients generated here. Estimated doses were within the variation of 0.9–16% for “dicentric + ring” and 21.7–31.2% for micronuclei respectively. These established curves have potential to be employed for biodosimetry of occupational, clinical and accidental exposures, for initial triage and medical management.

Effect of Low Dose Ionising Radiation on the Amount of Mitochondrial Common Deletion and D-Loop Tandem Duplication in Human Peripheral Whole Blood

The Hungarian Defence Forces assume a significant role in disaster prevention, including nuclear accident prevention tasks, so both the command and executive staff can stay in the higher-than-natural dose area. The current “gold standard” microscopic method aimed at determining the radiation dose suffered is the dicentric chromosome assay (DIC), although sensitive and accurate, it is very time-consuming. Monitoring changes in the amount of common deletion (CD) of mitochondrial DNA (mtDNA) and in the D-loop a tandem duplication (TD) of mtDNA may be a reliable marker of exposure to ionising radiation. This work used the PCR method to investigate how CD and TD change in human blood samples after X-ray irradiation. The CD appears to be a particularly useful marker, as its maximum is below the threshold for clinical symptoms. This work is the first to show the relationship between radiation and tandem duplication. The TD we investigated occurredmore frequently in irradiated human blood samples than native ones. During the future development of the diagnostic tool, both CD and TD are informative and would be used together in a PCR system to detect acute and cumulative irradiation. In recent years, more and more health institutions are dealing with molecular biological diagnostic work. In a disaster situation, if the laboratory capacity of the Hungarian Defence Forces would not be sufficient for this, more external laboratories can be involved for PCR measurements than for traditional microscopic work.

FDXR Gene Expression after in Vivo Radiation Exposure of Pediatric Patients Undergoing Interventional Cardiology Procedures.

Ferredoxin reductase (FDXR) has already been reported as a promising biomarker for estimating radiation doses in radiotherapy. This study aimed to investigate the responsiveness of FDXR on pediatric population exposed to ionizing radiation (X-rays) during pediatric interventional cardiology (IC) procedures. Peripheral blood was collected by venipuncture from 24 pediatric donors before and 24 hours after the IC procedure. To estimate the effective dose, demographic data and Air Kerma-Area Product (PKA) were recorded for each patient. The relative quantification (RQ) of the FDXR gene in irradiated patient blood samples compared to the non-irradiated blood samples was determined using qPCR analysis. The relative values of FDXR were log- transformed. The effective dose ranged from 0.002 mSv to 8.004 mSv. Over this radiation exposure range, the FDXR gene expression varied randomly with the effective dose. Up-regulation in FDXR expression was observed in 17 patients and down-regulation in 7 patients. Further studies in a larger cohort of pediatric patients along with the record of clinical data are needed to determine whether FDXR gene expression is an effective biomarker for radiation exposure estimation in pediatric imaging.

Influence of ultraviolet radiation on kinetic characteristics in erythrocytes

Erythrocytes were irradiated with an ultraviolet lamp of the VMT type (λ = 253.7 nm). The kinetic characteristics in irradiated and non-irradiated blood samples were compared by the acid (chemical) erythrogram method and by the counter transport method. Obtained:
 - by the method of acidic erythrograms it was found that in ultraviolet-irradiated blood samples there is a decrease in the time of half hemolysis.
 - ultraviolet radiation increases the speed of counter movement of ions through erythrocyte membranes.
 - a decrease in the time of half hemolysis and an increase in the rate of counter transfer of ions, irradiated blood samples, is due to a decrease in the "effective" thickness of the near-membrane diffusion layer - an immiscible layer of water adjacent to the erythrocyte membrane.
 A decrease in the "effective thickness" of the near-membrane water layer changes the rate of metabolic processes in the "cell - intercellular medium" system, changing the mode of cell functioning. The altered mode of functioning is a biological response to light radiation. Red blood cells with an altered mode of functioning are stimulus signals that force the body to mobilize resources to fight pathology. These circumstances can predict the creation of a universal phototherapeutic equipment for extracorporeal blood irradiation based on LEDs with certain exposure parameters. Figs.: 4. Tabl.: 3. Refs.: 12 titles.

Influence of sample preparation optimization on the accuracy of dose assessment of an automatic non-fluorescent MN scoring system

Purpose Automatizing the scoring of the cytokinesis-blocked micronucleus assay spares a lot of valuable time. The dose-effect relationship can be applied reliably for dose estimation if the quality of the slides is the same from the perspective of the used image processing algorithm. This aspect brings in additional requirements against the quality of the slides compared to the conventional visual scoring. Materials and methods An add-in software was created to the non-fluorescent RS-MN automatic MN scoring system which is capable of measuring quantitatively the degree of typical anomalies. The image processing is less reliable when the presence of these anomalies is more frequent. The behavior of the designed sample quality parameters (SQPs) was tested on in vitro irradiated peripheral blood samples (0, 1, and 2 Gy) obtained from a healthy donor and also on samples from patients undergoing low dose-rate brachytherapy. Results We examined 20 different SQPs and identified two that are independent and correlate significantly with the error of the fully automatic MN frequency. One is related to the size of the cells and the other reflects the homogeneity of the environment. An equation was established which presents a connection between the error of the auto MN frequency and the SQPs. By adding a fourth cleaning step to the conventional sample preparation and changing the pre-dripping temperature of the slide, the SQP can be modified, and consequently, the sample quality can be improved. The gain in accuracy is 54 ± 10 MN per 1000 binucleated cells, which corresponds to the effects of 0.5 Gy. Around the lowest limit of detection (<0.5 Gy), it means a 50–100% drop in the error of dose, which is significant. With sample quality harmonization, the positive predictive value was raised to 80–93% depending on the dose. Conclusions With the technique described in this paper, the suitability for automated scoring of a micronucleus slide can be tested quantitatively and objectively. A method is presented with which in some cases the uncertainty of the assessed doses due to variance in sample quality can be decreased or if it is not possible its bias can be predicted. The proposed protocol leads to more reliable estimation of dose. The SQPs are designed in a way that they have the potential to be adapted to similar systems.

Storage-Related Changes in Autologous Whole Blood and Irradiated Allogeneic Red Blood Cells and Their Ex Vivo Effects on Deformability, Indices, and Density of Circulating Erythrocytes in Patients Undergoing Cardiac Surgery With Cardiopulmonary Bypass

Blood-processing techniques and preservation conditions cause storage lesions, possibly leading to adverse outcomes after transfusion. The authors investigated the metabolic changes and deformability of red blood cells (RBCs) during storage and determined the effect of storage lesions on circulating RBCs during cardiac surgery. Prospective study. Tertiary care center affiliated with a university hospital. Adults who underwent elective cardiac surgery requiring cardiopulmonary bypass. The authors collected aliquots of autologous and irradiated allogeneic RBCs and blood samples from seven patients who received autologous whole blood and nine patients who received irradiated allogeneic RBCs before incision (baseline), at the start and end of cardiopulmonary bypass, and at completion of surgery. The authors analyzed RBC deformability, erythrocyte indices, and density distribution to evaluate blood banking-induced alterations of autologous and allogeneic RBCs and changes in circulating RBCs in recipients, after blood transfusion. Time-dependent biochemical changes and significant decreases in deformability during storage occurred in both groups; however, homologous RBCs had significantly lower deformability than autologous RBCs. Trends in mean corpuscular volume and mean corpuscular hemoglobin concentration differed in both groups. In the homologous transfusion group, during cardiac surgery, RBC deformability, mean corpuscular volume, and mean corpuscular hemoglobin concentration showed significant changes compared with baseline values, and a greater number of denser subpopulations was observed at surgery completion. Blood-processing techniques contribute to storage lesions, suggesting that transfusion of autologous whole blood, rather than allogeneic RBCs, could maintain the ability of circulating RBCs to deform and lead to potentially better transfusion outcomes.

Construction of dose response curve for 6 MV LINAC X-rays using Premature Chromosome Condensation assay for radiation dosimetry

Quantification of chromosomal aberrations in the exposed personnel blood samples is considered as a ‘gold standard’ and sensitive biomarker in biological dosimetry. Despite technological developments, culture of cells for 48–52 h remains an unmet need in case of triage biodosimetry. Moreover, it is difficult to get sufficient number of metaphase spreads for scoring after high doses of exposures. The technique which causes condensation of chromatin before mitosis using biological or chemical agent is named as Premature Chromosome Condensation (PCC) assay. This assay is considered as an alternative to chromosome aberration assay, particularly at high acute doses of low and high LET radiation. To establish the PCC assay, blood samples were collected from healthy non-smoking individuals (n = 3) and exposed to various doses (0–20 Gy) of 6 MV X-rays at a dose rate of 5.6 Gy/min, using a high energy Linear accelerator (LINAC). Irradiated blood samples were subjected to Calyculin-A induced PCC. About 500 cells or more than 100 Ring Chromosomes (RC) were scored at each dose. Dicentric chromosomes (DC) and acentric fragments were also scored at each dose; the number of chromosomal aberrations in G1, M, G2/M and M/A phase of cell cycle were recorded and the frequency was used to construct the dose response curve. A dose dependent increase in RC and DC frequency were observed with a slope of 0.049 ± 0.002 and 0.30 ± 0.02 respectively. This study is first of its kind to construct a dose response curve for LINAC X-rays using a PCC assay.

EFFECTS OF 532 NM AND 671 NM LASERSIRRADIATION ON ABSORPTIONCOEFFICIENTS OF NORMAL AND ANEMIC HUMAN BLOOD SAMPLES

The effects of laser irradiation on the absorption coefficients of the normal and anemic human blood samples were investigated. Two continuous wave (CW) diode-pumped solid-state lasers (DPSSLs) of different wavelengths,532 nm and 671 nm, were used. The output power of these lasers can be adjusted over the range 0-100 mW. In the present study, the output power of both lasers was fixed at 20 mW. The blood samples were irradiated at different irradiation times, 5, 15, and 30 min. The absorption coefficients of the normal and anemic human blood samples were determined from the measurements of the absorbance spectra of the unirradiated and irradiated samples. The absorbance spectra were measured using UV-Visible double-beam spectrophotometer operating over the wavelength range 190-1100 nm. Significant changes in the absorption coefficients of the irradiated blood samples were observed compared to the unirradiated blood samples. The results obtained in the present investigation showed that there are significant effects of laser irradiation on the optical properties of the human blood samples. These effects can result in noticeable changes in their absorption coefficients. The results suggested that the technique used in the present investigation has great potential to be a useful tool for the clinical diagnostics and the medical applications.

Ex vivo radiosensitivity is increased in non-cancer patients taking valproate

BackgroundValproate (VPA) is a commonly prescribed antiepileptic drug for patients experiencing epileptic seizures due to brain tumors. VPA increases radiation sensitivity in various tumor cells in vitro due to complex mechanisms. This could make tumors more vulnerable to ionizing radiation or overcome radioresistance. Yet, clinical data on possible improvement of tumor control by adding VPA to tumor therapy is controversial. Potentially radiosensitizing effects of VPA on healthy tissue remain unclear. To determine individual radiosensitivity, we analyzed blood samples of individuals taking VPA.MethodsEx vivo irradiated blood samples of 31 adult individuals with epilepsy were studied using 3-color fluorescence in situ hybridization. Aberrations in chromosomes 1, 2 and 4 were analyzed. Radiosensitivity was determined by the mean breaks per metaphase (B/M) and compared to age-matched (2:1) healthy donors.ResultsThe patient cohort (n = 31; female: 38.7%) showed an increase of their average B/M value compared to healthy individuals (n = 61; female: 56.9%; B/M: 0.480 ± 0.09 vs. 0.415 ± 0.07; p = .001). The portion of radiosensitive (B/M > 0.500) and distinctly radiosensitive individuals (B/M > 0.600) was increased in the VPA group (54.9% vs. 11.3 and 9.7% vs. 0.0%; p < .001). In 3/31 patients, radiosensitivity was determined prior to and after VPA treatment and radiosensitivity was increased by VPA-treatment.ConclusionsIn our study, we confirmed that patients treated with VPA had an increased radiosensitivity compared to the control group. This could be considered in patients taking VPA prior to the beginning of radiotherapy to avoid toxic side effects of VPA-treatment.

EFFECTS OF LASER IRRADIATION ON NORMAL AND ANEMIC HUMAN BLOOD

The effects of laser irradiation on the whole human blood were studied. The blood samples were taken from healthy donors with normal blood and donors with anemic blood. The blood samples were exposed to laser radiation. Two lasers of the same types, continuous wave (CW) diode pumped solid-state lasers (DPSSL’ s) were used to irradiate the blood samples. One of these laser has a wavelength 532 nm (green laser beam), while the other laser has a wavelength 671 nm (red laser beam). The output power of the two lasers can be varied over the range 0 - 100 mW. In the present study, the output power was fixed at 20 mW for both lasers. The blood samples were exposed to different irradiation times, 5, 15, and 30 min. Significant changes in the optical parameters of the irradiated blood samples were observed in comparison with that of the un-irradiated blood samples (the control samples). These optical parameters were determined from the measured absorbance spectra of the un-irradiated and irradiated samples. The absorbance spectra were measured using a UV-Visible double-beam spectrophotometer operating over the wavelength range 190 - 1100 nm.&#x0D; The results of the present study revealed that the laser irradiation can produce considerable effects on the human blood samples, suggesting the suitability of the laser beam irradiation for the biological and medical applications.

Comparison of inexperienced operators and experts in γH2A.X and 53BP1 foci assay for high-throughput biodosimetry approaches in a mass casualty incident

Purpose In case of population exposure by ionizing radiation, a fast and reliable dose assessment of exposed and non-exposed individuals is crucial important. In initial triage, physicians have to take fast decisions whom to treat with adequate medical care. In addition, worries about significant exposure can be taken away from hundreds to thousands non- or low exposed individuals. Studies have shown that the γH2A.X radiation-induced foci assay is a promising test for fast triage decisions. However, in a large-scale scenario most biodosimetry laboratories will quickly reach their capacity limit. The aim of this study was to evaluate the benefit of inexperienced experimenters to speed up the foci assay and manual foci scoring. Materials and methods The participants of two training courses performed the radiation-induced foci assay (γH2A.X) under the guidance of experts and scored foci (γH2A.X and 53BP1) on sham-irradiated and irradiated blood samples (0.05–1.5 Gy). The outcome of laboratory experiments and manual foci scoring by 26 operators with basic experience in laboratory work was statistically analyzed in comparison to the results from experts. Results Inexperienced operators prepared slides with significant dose-effects (0, 0.1 and 1.0 Gy) for semi-automatic microscopic analyses. Manual foci scoring by inexperienced scorer resulted in a dose–effect curve for γH2A.X, 53BP1 and co-localized foci. In addition, inexperienced scorers were able to distinguish low irradiation doses from unirradiated cells. While 53BP1 foci scoring was in accordance to the expert counting, differences between beginners and expert increased for γH2A.X or co-localized foci. Conclusions In case of a large-scale radiation event, inexperienced staff is useful to support laboratories in slide preparation for semi-automatic foci counting as well as γH2A.X and 53BP1 manual foci scoring for triage-mode biodosimetry. Slides can be clearly classified in the non-, low- or high-exposed category.

Когерентне та монохроматичне випромінювання змінює час гемолізу еритроцитів, а також швидкість зустрічного переміщення іонів крізь еритроцитарну мембрану

The erythrocytes of human blood were sequentially irradiated with a low-intensity laser (λ = 640 nm), a violet LED (λ = 400 nm), a green LED (λ = 540 nm), and a yellow LED (λ = 592 nm). The method of acid (chemical) erythrograms and the method of counter ion transport compared the kinetic characteristics in irradiated and unirradiated blood samples. Received: - by the method of acid erythrograms it was found that in the irradiated blood samples there is a decrease in the time of hemolysis; - low intensity laser radiation, as well as the emission of LEDs, increase the rate of counter ion transport through red blood cells. - a decrease in the time of hemolysis and an increase in the rate of counter ion transport of irradiated blood samples is due to a decrease in the "effective" thickness of the near-membrane diffusion layer - an immiscible layer of water adjoined to the erythrocyte membrane. A decrease in the "effective” thickness of the near-membrane water layer (minimal in the wavelength range of 570-590 nm and 630-640 nm) changes the rate of metabolic processes in the "cell - intercellular medium" system, changing the mode of cell functioning. The altered mode of functioning is a biological response to light radiation. Red blood cells with an altered mode of functioning are signals - stimuli that cause the body to mobilize resources to fight pathology. These circumstances can predict the creation of a universal phototherapeutic equipment for extracorporeal blood irradiation based on light-emitting diodes with certain exposure parameters.

Probing the Effects of Ionizing Radiation on Young's Modulus of Human Erythrocytes Cytoskeleton using Atomic Force Microscopy.

Purpose/Aim:In this work, we examined the possible effects of ionizing radiation (IR) on biomechanical properties of the membrane–cytoskeleton of human erythrocytes, after X-ray irradiation.Materials and Methods:Whole human blood from three healthy middle-aged volunteers was drawn by venipuncture and stored in tubes containing anticoagulant. Six blood samples were collected for each volunteer. Five of them were irradiated in the range of 0.1 Gy–2.0 Gy doses and one was used as control. The morphology and the elastic modulus of the erythrocytes were examined using atomic force microscopy and just few drops of whole blood.Results:No morphological changes appeared according to the shape and the morphology of the erythrocytes. The elastic modulus of the irradiated samples was reduced with the increase of radiation dose. The findings indicate that X-ray irradiation affects the biomechanical properties of erythrocyte cytoskeleton. The mean value of Young's modulus of all the irradiated blood samples was significant difference from the control at a level, P < 0.01.Conclusions:The elastic modulus of the erythrocytes could be an indicator of the adverse effect in the human blood generated by IR exposure through a radiotherapy treatment.

Extension of lymphocyte viability for radiation biodosimetry: Potential implications for radiological/nuclear mass casualty incidents.

Dicentric chromosome assay (DCA) is routinely used for estimating the absorbed radiation dose in exposed humans. Optimal lymphocyte viability is crucial for reliable dose estimation and most cytogenetic laboratories prefer the receipt of blood samples within 24 to 36 hours after collection. Delays in the shipment/receipt of samples can occur sometimes under certain unforeseen circumstances: (1) Adverse weather conditions, (2) distant location of blood collection sites, and (3) shipping and handling of a large number of samples after radiological/nuclear mass casualty incident(s). To circumvent some of these limitations, we evaluated the suitability of ex vivo irradiated blood samples stored in the presence of phytohemagglutinin (PHA) for 7 days at ambient temperature (22-24°C) for radiation biodosimetry. Blood samples stored in the presence of PHA for up to 7 days showed a higher mitotic index than blood samples stored without PHA. To verify the use of stored blood samples for DCA, frequencies of X-rays induced dicentric chromosomes were analyzed in the blood samples that were cultured either 24 hours after exposure or 7 days later after storage. Our results indicate that storage of ex vivo irradiated blood samples in the presence of PHA at ambient temperature was found optimal for DCA and that the radiation doses estimated by dicentric chromosome frequencies were grossly similar between the fresh and stored blood samples. Our study suggests that reliable and accurate biodosimetry results can be obtained for triage using blood samples stored for up to a week at ambient temperature in the presence of PHA.