Validation of semi-automatic scoring of dicentric chromosomes after simulation of three different irradiation scenarios.

Large scale radiological emergencies require high throughput techniques of biological dosimetry for population triage in order to identify individuals indicated for medical treatment. The dicentric assay is the "gold standard" technique for the performance of biological dosimetry, but it is very time consuming and needs well trained scorers. To increase the throughput of blood samples, semi-automation of dicentric scoring was investigated in the framework of the MULTIBIODOSE EU FP7 project, and dose effect curves were established in six biodosimetry laboratories. To validate these dose effect curves, blood samples from 33 healthy donors (>10 donors/scenario) were irradiated in vitro with 6°Co gamma rays simulating three different exposure scenarios: acute whole body, partial body, and protracted exposure, with three different doses for each scenario. All the blood samples were irradiated at Ghent University, Belgium, and then shipped blind coded to the participating laboratories. The blood samples were set up by each lab using their own standard protocols, and metaphase slides were prepared to validate the calibration curves established by semi-automatic dicentric scoring. In order to achieve this, 300 metaphases per sample were captured, and the doses were estimated using the newly formed dose effect curves. After acute uniform exposure, all laboratories were able to distinguish between 0 Gy, 0.5 Gy, 2.0, and 4.0 Gy (p < 0.001), and, in most cases, the dose estimates were within a range of ± 0.5 Gy of the given dose. After protracted exposure, all laboratories were able to distinguish between 1.0 Gy, 2.0 Gy, and 4.0 Gy (p < 0.001), and here also a large number of the dose estimates were within ± 0.5 Gy of the irradiation dose. After simulated partial body exposure, all laboratories were able to distinguish between 2.0 Gy, 4.0 Gy, and 6.0 Gy (p < 0.001). Overdispersion of the dicentric distribution enabled the detection of the partial body samples; however, this result was clearly dose-dependent. For partial body exposures, only a few dose estimates were in the range of ± 0.5 Gy of the given dose, but an improvement could be achieved with higher cell numbers. The new method of semi-automation of the dicentric assay was introduced successfully in a network of six laboratories. It is therefore concluded that this method can be used as a high-throughput screening tool in a large-scale radiation accident.

A novel peptide (Thx) homing to non-small cell lung cancer identified by ex vivo phage display

AIM: To identify linear peptide homing to non-small cell lung cancer (NSCLC) tumor cells using ex vivo phage display method. MATERIALS AND METHODS: Twenty-six clinical patient samples were used to identify linear homing peptide, which was exposed to NSCLC cell cultures and control cell lines to determine cell binding affinity and cell localization. Also, ex vivo biodistribution was analyzed using tumor-bearing mice. RESULTS: The panning yielded peptide enrichment with a core motif (A)/SRXPXXX. Based on this, an amino acid sequence, ARRPKLD, was selected for characterization and named Thx-peptide. The in vitro binding properties of Thx-peptide demonstrated selectivity toward NSCLC. Internalization assays showed that Thx-Alexa and fluorescein conjugates were located in a subset of perinuclearly located lysosomes of tumor cells. Thx-peptide appeared with fluorescein-labeled peptide and peptide-DTPA-chelator complex in adenocarcinoma xenografts in mice. CONCLUSION: Thx shows promise for targeted imaging and drug delivery (AU)

Automatic scoring of dicentric chromosomes as a tool in large scale radiation accidents.

Mass casualty scenarios of radiation exposure require high throughput biological dosimetry techniques for population triage in order to rapidly identify individuals who require clinical treatment. The manual dicentric assay is a highly suitable technique, but it is also very time consuming and requires well trained scorers. In the framework of the MULTIBIODOSE EU FP7 project, semi-automated dicentric scoring has been established in six European biodosimetry laboratories. Whole blood was irradiated with a Co-60 gamma source resulting in 8 different doses between 0 and 4.5Gy and then shipped to the six participating laboratories. To investigate two different scoring strategies, cell cultures were set up with short term (2-3h) or long term (24h) colcemid treatment. Three classifiers for automatic dicentric detection were applied, two of which were developed specifically for these two different culture techniques. The automation procedure included metaphase finding, capture of cells at high resolution and detection of dicentric candidates. The automatically detected dicentric candidates were then evaluated by a trained human scorer, which led to the term 'semi-automated' being applied to the analysis. The six participating laboratories established at least one semi-automated calibration curve each, using the appropriate classifier for their colcemid treatment time. There was no significant difference between the calibration curves established, regardless of the classifier used. The ratio of false positive to true positive dicentric candidates was dose dependent. The total staff effort required for analysing 150 metaphases using the semi-automated approach was 2 min as opposed to 60 min for manual scoring of 50 metaphases. Semi-automated dicentric scoring is a useful tool in a large scale radiation accident as it enables high throughput screening of samples for fast triage of potentially exposed individuals. Furthermore, the results from the participating laboratories were comparable which supports networking between laboratories for this assay.

A novel peptide (Thx) homing to non-small cell lung cancer identified by ex vivo phage display.

AIM: To identify linear peptide homing to non-small cell lung cancer (NSCLC) tumor cells using ex vivo phage display method. MATERIALS AND METHODS: Twenty-six clinical patient samples were used to identify linear homing peptide, which was exposed to NSCLC cell cultures and control cell lines to determine cell binding affinity and cell localization. Also, ex vivo biodistribution was analyzed using tumor-bearing mice. RESULTS: The panning yielded peptide enrichment with a core motif (A)/SRXPXXX. Based on this, an amino acid sequence, ARRPKLD, was selected for characterization and named Thx-peptide. The in vitro binding properties of Thx-peptide demonstrated selectivity toward NSCLC. Internalization assays showed that Thx-Alexa and fluorescein conjugates were located in a subset of perinuclearly located lysosomes of tumor cells. Thx-peptide appeared with fluorescein-labeled peptide and peptide-DTPA-chelator complex in adenocarcinoma xenografts in mice. CONCLUSION: Thx shows promise for targeted imaging and drug delivery.

Search for clastogenic factors in the plasma of locally irradiated individuals.

In studies reported in the 1960s and in several investigations since, plasma from irradiated individuals was shown to induce chromosomal aberrations when transferred into normal blood cultures. In the present study, the aim was to investigate the occurrence of these clastogenic factors (CF) using markers representing DNA damage produced in reporter lymphocytes that are treated with plasma from locally exposed individuals. Blood plasma was obtained from clinical patients with benign conditions before and after they had received radiation to small treatment volumes. Three patient groups were studied: (I) marginal resected basal cell carcinoma, (II) painful osteoarthritis of the knee, and (III) painful tendinitis of the elbow or the heel. Patients in each treatment group obtained the same fractionated treatment regimen, ranging from a total dose of 40 Gy (8 × 5 Gy, 2 factions/week) to a very small volume (1-3.5 cm³) in group I to a total dose of 6 Gy (6 × 1 Gy, 2 fractions/week) for groups II and III (treatment volumes 800-1150 cm³ and 80-160 cm³, respectively). The presence of CF in the plasma was investigated through cytogenetic (chromosomal aberrations, micronuclei) assays and kinetics of early DNA damage (γ-H2AX foci) in reporter cells. With the experimental settings applied, local radiation exposure had no apparent effect on the induction of CF in patient plasma; no deviations in chromosomal aberrations or micronucleus or focus induction were observed in reporter cells treated with postexposure plasma with respect to pre-exposure samples when the mean values of the groups were compared. However, there was a large interindividual variation in the plasma-induced DNA-damaging effects. Steroid treatment of patients was demonstrated to be the most influential factor affecting the occurrence of plasma factors; plasma from patients treated with steroids led to significant reductions of γ-H2AX foci and reduced numbers of chromatid aberrations in reporter cells. In addition to the locally exposed patients, newly obtained plasma samples from three radiological accident victims exposed in 1994 were examined. In contrast to the patient data, a significant increase in chromosomal aberrations was induced with plasma from two accident victims.

Domestic radon exposure and the frequency of stable or unstable chromosomal aberrations in lymphocytes.

PURPOSE: To clarify the relationship between domestic radon exposure and the occurrence of chromosomal aberrations, stable translocations especially, in peripheral blood lymphocytes. MATERIALS AND METHODS: The study comprised a total of 84 nonsmoking individuals, divided into three groups according to radon concentration measurements performed in their homes: low radon concentration (<100Bq/m3, mean 67Bq/m3), medium (200-400Bq/m3, mean 293Bq/m3) or high (>800Bq/m3, mean 1737Bq/m3). Minimum residence in the present low-rise house was 10 years. The groups were matched with regard to age, gender and medical exposure. Fluorescence in-situ hybridization (FISH) was performed using chromosome paints for chromosomes 1, 2 and 4; 1500 metaphases were scored from each individual. RESULTS: Equal frequencies of translocations and also other aberrations, e.g. dicentrics and complex rearrangements, were obtained in each group. Significant correlation of translocations with age was observed, and due to the high mean age (50 years) the genome-corrected frequency of translocations was high: about one translocation in 100 metaphases. CONCLUSIONS: Chronic exposure to high concentrations of domestic radon did not increase the rate of stable or unstable chromosomal aberrations in peripheral blood lymphocytes detected by FISH chromosome painting. A strong age effect was observed.

Comparison of dose-response curves for chromosomal aberrations established by chromosome painting and conventional analysis.

PURPOSE: To establish 60Co gamma-ray dose-response curves for dicentrics and translocations visualized by chromosome painting and for dicentrics analysed after conventional solid staining. MATERIALS AND METHODS: Analysis of chromosomal aberrations was performed on peripheral blood lymphocytes obtained from 48 h old cultures of irradiated whole blood. Dicentrics were scored from Giemsa-stained preparations, and bi-coloured dicentrics and translocations after FISH painting of chromosomes 1, 2 and 4. RESULTS: Equal frequencies of complete dicentrics and translocations, where both members of the exchanges were seen, were observed in the chromosome painting analysis at all doses, resulting in similar calibration curves. Due to differences in scoring criteria, dicentrics scored in conventionally Giemsa-stained slides agreed better with data for total than for complete exchanges. Donor differences for translocations at the control level and at low doses were seen and large uncertainty surrounds the linear component of the dose-response for total translocations. CONCLUSIONS: Dose reconstruction of past exposures in cases of low doses is very dependent on the linear coefficient of the curve. Results indicate that total translocations would give less reliable dose estimates and therefore complete translocations are preferred.

Biodosimetry after accidental radiation exposure by conventional chromosome analysis and FISH.

A 137Cs source was stolen from a radioactive waste depository in Estonia on 21 October 1994 and kept in a private house for 4 weeks. This resulted in the death of one person, acute radiation injuries to four people and exposure of several other people to lower doses of radiation. Analysis of chromosomal aberrations in peripheral blood lymphocytes was used in the assessment of radiation exposure of 18 people involved in the accident. Dose estimation assessment based on the frequencies of dicentric chromosomes was performed both by the standard method and by considering possible dose protraction and non-uniform exposure. Considerable differences in dose estimates were obtained depending on the approach used, ranging from about 1 Gy to almost 3 Gy in the patients most heavily exposed. In view of the deterministic health effects observed in some of the subjects, it was concluded that the dose estimates involving information on dose protraction were more realistic than those obtained by comparison with the standard high dose-rate calibration curve. Chromosome painting analyses using fluorescence in situ hybridization, with a probe cocktail for chromosomes 1, 2 and 4 and centromere detection, were performed in parallel. Good agreement on dicentric chromosome frequencies was observed between the conventional and painting analyses. The frequencies of complete translocations were comparable with the frequencies of dicentric chromosomes. In addition to the complete translocations, a pronounced increase in the frequency of incomplete translocations was observed. Dose estimates performed on the basis of FISH translocation frequencies were consistent with the dicentric analysis.