Montserrat Ribas

Cytogenetic analysis of lymphocytes from hospital workers occupationally exposed to low levels of ionizing radiation

Cytogenetic studies were performed in lymphocytes from hospital workers exposed to low doses of radiation (1.6-42.71 mSv). When compared with controls, exposed workers showed a significant increase in structural chromosome-type aberrations, acentric fragments being the most frequent alteration. Our results suggest that acentric fragments are good indicators of exposure to very low doses of radiation, although no dose-effect correlation was observed. The incidence of numerical abnormalities (hyperdiploidy) was significantly increased.

Cytogenetic Characterization of Two Colon Cell Lines by Using Conventional G-Banding, Comparative Genomic Hybridization, and Whole Chromosome Painting

The heterogeneous nature of genetic alterations in cancer cells handicaps the full characterization of its occurrence and the analysis of their molecular bases and relation to biological processes. Although many cancer cells are highly aneuploid, in other cases, as in a subset of colorectal carcinomas displaying microsatellite instability, chromosomal aberrations are scarce. The aim of this study was to fully characterize both qualitatively and quantitatively, the karyotypes of two established colon carcinoma cell lines (LoVo and HCT 116) previously reported as being near diploid. An array of complementary cytogenetic techniques were used: G-banding, comparative genome hybridization (CGH), and whole-chromosome painting (WCP). Combinations of these techniques provided an accurate karyotype for the two cell lines: LoVo cells showed 49,XY,t(2;12)(q13;p11.2),+5,+7,+12,i(15)(q10) and HCT 116 cells showed 45,X,-Y,dup(10)(q24q26),der(16)t(8;16)(q13;p13), der(18)t(17;18)(q21;p11.3). Heterogeneity was also observed in both cell lines as shown by G-banding. Chromosomal unbalances determined by CGH (many of them related to structural reorganizations) were characterized by WCP, allowing the reliable identification of those chromosome markers that could not be completely identified by G-banding. We show that combined analysis with classical and molecular cytogenetic techniques provides an accurate map of chromosomal aberrations in these two cell lines not identified in previous investigations.

3D DVH-based metric analysis versus per-beam planar analysis in IMRT pretreatment verification

PURPOSE To evaluate methods of pretreatment IMRT analysis, using real measurements performed with a commercial 2D detector array, for clinical relevance and accuracy by comparing clinical DVH parameters. METHODS We divided the work into two parts. The first part consisted of six in-phantom tests aimed to study the sensitivity of the different analysis methods. Beam fluences, 3D dose distribution, and DVH of an unaltered original plan were compared to those of the delivered plan, in which an error had been intentionally introduced. The second part consisted of comparing gamma analysis with DVH metrics for 17 patient plans from various sites. Beam fluences were measured with the MapCHECK 2 detector, per-beam planar analysis was performed with the MapCHECK software, and 3D gamma analysis and the DVH evaluation were performed using 3DVH software. RESULTS In a per-beam gamma analysis some of the tests yielded false positives or false negatives. However, the 3DVH software correctly described the DVH of the plan which included the error. The measured DVH from the plan with controlled error agreed with the planned DVH within 2% dose or 2% volume. We also found that a gamma criterion of 3%∕3 mm was too lax to detect some of the forced errors. Global analysis masked some problems, while local analysis magnified irrelevant errors at low doses. Small hotspots were missed for all metrics due to the spatial resolution of the detector panel. DVH analysis for patient plans revealed small differences between treatment plan calculations and 3DVH results, with the exception of very small volume structures such as the eyes and the lenses. Target coverage (D(98) and D(95)) of the measured plan was systematically lower than that predicted by the treatment planning system, while other DVH characteristics varied depending on the parameter and organ. CONCLUSIONS We found no correlation between the gamma index and the clinical impact of a discrepancy for any of the gamma index evaluation possibilities (global, local, 2D, or 3D). Some of the tests yielded false positives or false negatives in a per-beam gamma analysis. However, they were correctly accounted for in a DVH analysis. We also showed that 3DVH software is reliable for our tests, and is a viable method for correlating planar discrepancies with clinical relevance by comparing the measured DVH of target and OARs with clinical tolerance.

Relationship between the DNA content of human chromosomes and their involvement in radiation-induced structural aberrations, analysed by painting.

PURPOSE To study the relationship between the DNA content of human chromosomes and their involvement in radiation-induced structural chromosome aberrations. MATERIAL AND METHODS Human lymphocytes were cultured after exposure to 5 Gy of X-rays. FISH-painting was performed for all human chromosomes. RESULTS The results indicate that with the exception of chromosome 20, there was a good fit between the DNA content and the number of exchange-type aberrations and the number of breaks. However, there was a significant tendency for short chromosomes to be more affected than expected and for long chromosomes to be less affected than expected. A better fit was observed when, instead of DNA content, the surface area of the chromosome territories obtained from a spherical model was taken into consideration. CONCLUSIONS The tendency for short chromosomes to be more involved and long chromosomes to be less involved in exchange-type aberrations could be related not only to their DNA content but also to their interphase territory surface area.

Biological dosimetry in simulated in vitro partial irradiations

The assessment by biological dosimetry of the dose received in cases of partial-body exposure to ionizing radiation can be underestimated because irradiated lymphocytes are mixed with non-irradiated ones. To determine if the exposure affects the whole body or only part of it, it may be useful to know the distribution of cells with more than one dicentric chromosome. We established a dose-effect calibration curve for X-rays by analysis of chromosome aberrations. Moreover, in the present work, 20 partial irradiations for four different doses of X-rays (2, 3, 4 and 5 Gy) have been simulated by mixing irradiated and non-irradiated blood in different proportions. In all cases, the 95% confidence intervals of the estimated dose included the real dose of irradiation. However, some difficulties were found for the estimation of the fraction of irradiated cells. In the present study, D0 = 3.8 allows to obtain the best fit between the estimated and the real fraction of irradiated cells.

Decreased sensitivity to the cytogenetic effects of bleomycin in individuals occupationally exposed to ionizing radiation

In the present work, 12 individuals occupationally exposed to ionizing radiation and 11 unexposed ones were studied to determine the cytogenetic effect of a challenge dose of bleomycin on their phytohemagglutinin stimulated lymphocytes. After bleomycin treatment, the frequencies of chromatid breaks and gaps were significantly lower in the exposed population (p < 0.025 for both types of chromatid alterations). These results could indicate that occupational exposure to ionizing radiation can induce an adaptive response that can be detected by a subsequent treatment with bleomycin.

Suitability of FISH painting techniques for the detection of partial-body irradiations for biological dosimetry.

Abstract Duran, A., Barquinero, J. F., Caballín, M. R., Ribas, M., Puig, P., Egozcue, J. and Barrios, L. Suitability of FISH Painting Techniques for the Detection of Partial-Body Irradiations for Biological Dosimetry. Radiat. Res. 157, 461–468 (2002). Peripheral blood was irradiated with 2, 3, 4 or 5 Gy of X rays and was mixed with nonirradiated blood at five different dilutions to simulate partial-body irradiations. Analysis by FISH was performed using whole-chromosome painting probes for chromosomes 1, 4 and 11 in combination with a pancentromeric probe. Chromosome aberrations affecting the painted fraction were classified according to the PAINT nomenclature; other unstable aberrations affecting the unpainted material were also recorded. To evaluate the suitability of painting for dose assessment in partial-body irradiations, the ability of the u test and a proposed s test to detect the expected overdispersion and the similarity between the real doses and the doses estimated using Dolphins approach were considered. For short-term biodosimetry, compared with solid-stained dicentric analyses, the suitability of FISH painting techniques for the detection of partial-body exposures is reduced, because of the decrease in the frequency of aberrations detected by FISH and in the number of cells with two or more aberrations. For reconstruction of past doses, when only complete apparently simple translocations in cells free of unstable aberrations were considered, the detection of the overdispersion and the accuracy of dose estimations were dramatically reduced. In a partial-body exposure, as the original dose increased, the whole-body dose estimated a long time after irradiation would tend to be lower, and the difference from the original dose would tend to be greater.

Establishment and validation of a dose-effect curve for γ-rays by cytogenetic analysis

Abstract A dose-effect curve obtained by analysis of dicentric chromosomes after irradiation of peripheral blood samples, from one donor, at 11 different doses of γ-rays is presented. For the elaboration of this curve, more than 18 000 first division metaphases have been analyzed. The results fit very well to the linear-quadratic model. To validate the curve, samples from six individuals (three controls and three occupationally exposed persons) were irradiated at 2 Gy. The results obtained, when compared with the curve, showed that in all cases the 95% confidence interval included the 2 Gy dose, with estimated dose ranges from 1.82 to 2.19 Gy.

Cytogenetic Analyses by Fluorescence In Situ Hybridization (FISH) in Hospital Workers Occupationally Exposed to Low Levels of Ionizing Radiation

Abstract Cigarrán, S., Barquinero, J. F., Barrios, L., Ribas, M., Egozcue, J. and Caballín, M. R. Cytogenetic Analyses by Fluorescence In Situ Hybridization (FISH) in Hospital Workers Occupationally Exposed to Low Levels of Ionizing Radiation. An occupationally exposed population has been studied to evaluate the suitability of FISH painting techniques to detect chronic exposures to very low doses of ionizing radiation by the analysis of translocations. Whole-chromosome painting probes for chromosomes 1, 4 and 11 in combination with a pancentromeric probe have been employed. For comparison, a matched control population has also been studied. The mean genomic frequencies per 100 cells of total translocations in the control and exposed populations were 0.90 ± 0.12 and 1.04 ± 0.11, respectively. In the occupationally exposed population, no correlation between the frequencies of translocations and the doses received was found. When the two populations were compared, no significant differences were observed for the frequencies of the different chromosomal abnormalities examined. The absence of differences between control and exposed populations could be attributed to the very low-dose exposures recorded in the occupationally exposed population and to the wide range of individual frequencies of translocations observed.

Comparison of X-ray dose-response curves obtained by chromosome painting using conventional and PAINT nomenclatures

PURPOSE The compare the suitability of PAINT and conventional nomenclature systems for the construction of chromosome aberration dose-effect curves for X-rays using FISH techniques, and to compare these curves with those based on solid-stained dicentrics analysed in first division metaphases by the FPG technique. MATERIALS AND METHODS Blood samples were irradiated at 0.1, 0.25, 0.50, 0.75, 1, 1.5, 2, 3, 4 and 5 Gy 180 kV X-rays. FISH painting was performed using probes for chromosomes 1, 4 and 11 in combination with a pan-centromeric probe. RESULTS Translocations showed a higher background frequency than dicentrics. This influences the ratio of translocations:dicentrics at the lower doses and the uncertainties of dose-effect curves for translocations. The dose-effect curves for dicentrics obtained by FISH and solid stain were in close agreement. CONCLUSION For short-term biological dosimetry purposes by FISH, the use of dic(BA) (PAINT nomenclature) or total dicentrics (conventional nomenclature) should give similar dose estimates. For dose reconstruction, the use of total or complete translocations result in similar uncertainties.