Dosimetria biologica delle radiazioni ionizzanti: valutazione della dose con metodologie citogenetiche tramite la costruzione di curve di calibrazione

The assessment of the risk for biological damage of an individual after overexposure to ionizing radiation due to an accident or a terroristic attack passes through the evaluation of the absorbed dose received from the person/s involved. This evaluation, corresponding also to how dangerous the overexposure is, using methodologies typical of the biological dosimetry, is done with the help of reference calibration curves and it presumes that all individuals exposed have the same individual radiosensitivity, which is true when the biological system into account are human lymphocytes of the peripheral blood (PBL). It supposes that a certain dose of radiation produces the same quantity of variation (chromosomal aberrations) in that biological system in all the exposed. The goal of this Ph.D. thesis is the application of various cytogenetic methodologies to detect in a reliable and possibly fast way chromosomal aberrations non stable (dicentrics and centromeric rings) in the lymphocytes of the human peripheral blood after gamma irradiation and the construction of calibration curves dose effect able to give rapidly a response of the absorbed dose in case of radiation accident. The time needed to give a reliable response on the absorbed dose is crucial and this is especially true in radiation emergency medicine. The objective was to standardize, develop and apply sophisticate, sensitive and reliable methodologies for the estimation of the absorbed doses after accidental gamma radiation overexposures for radiation protection purposes. Specifically, in the present study the following were performed: I. Application of the classic “golden standard” methodology of biological dosimetry which provides stimulation of the lymphocytes and Giemsa staining of the chromosomes at mitosis II. Development, consolidation and application of original methodologies in biological dosimetry using fluorescence in situ hybridization (FISH) and peptide nucleic acid (PNA) probes for painting specifically chromosome telomers and centromeres III. Application and consolidation of the method of premature chromosome condensation (PCC) where no stimulation at lymphocytes is needed IV. Development of a fast, sensitive and reliable methodology, absolutely original in the field of biological dosimetry for dose evaluation combining the PCC method with the FISH using PNA probes for centromers and telomers V. Construction for all the above methodologies, using in vitro experiments, of calibration curves dose-effect where unstable chromosomal aberrations where plotted against gamma dose given. The dose interval investigated was between 0.3 Gy and 8 Gy; more than 12.500 slides with metaphases and interphases were scored using Microscopic analysis of chromosome aberrations. Dose effect curves are linear quadratic and for their construction a minimum of 500 cells or 100 dicentrics per dose were analysed. The Poisson distribution of the chromosomal aberrations was tested for every dose and the coefficients alpha and beta of the linear quadratic equation, their errors and p value were calculated. Finally, all calibration curves obtained with the different methodologies were compared and the advantages of each methodology reported

nondestructive wood discrimination ftir fourier transform infrared spectroscopy in the characterization of different wood species used for artistic objects

Wooden artifacts represent a significant component of past cultures. Successful conservation of wooden artifacts depends on the knowledge of wood structure and types. It is critical that conservators know the category of wood that they are treating in order to successfully conserve it. Recently, vibrational spectroscopy has been successfully applied to determine the chemical structure of wood and to characterize wood types. FTIR (Fourier Transform Infrared) is a useful nondestructive or micro-destructive analytical technique providing information about chemical bonding and molecular structure. Its application in the discrimination between softwoods (conifers) and hardwoods (broad-leafs) has already been reported. The aim of the present study was to investigate the potential of FTIR as a tool for the discrimination between different wood types belonging to the same genus. Three different hardwood species, namely poplar (Populus spp), lime (Tilia spp) and birch (Betula spp), were investigated by means of FTIR spectroscopy. The woods were first inspected using a light microscope to certify the wood essence types through micrographic and morphoanatomical features. The FTIR spectra in the 4000 cm[Formula: see text] to 450 cm[Formula: see text] region were recorded using a Perkin-Elmer Spectrum 100 spectrometer. To enhance the qualitative interpretation of the IR spectra, second derivatives of all spectra were calculated using the Spectrum software to separate superimposed bands and to extract fine spectral details. To obtain a comprehensive characterization, the essences under investigation were also analyzed by means of Raman Spectroscopy. Clear differences were found in the spectra of the three samples confirming FTIR to be a powerful tool for wood type discrimination

Dose assessment intercomparisons within the RENEB network using G0-lymphocyte prematurely condensed chromosomes (PCC assay)

Purpose: Dose assessment intercomparisons within the RENEB network were performed for triage biodosimetry analyzing G0-lymphocyte PCC for harmonization, standardization and optimization of the PCC assay. Materials and methods: Comparative analysis among different partners for dose assessment included shipment of PCC-slides and captured images to construct dose-response curves for up to 6 Gy c-rays. Accident simulation exercises were performed to assess the suitability of the PCC assay by detecting speed of analysis and minimum number of cells required for categorization of potentially exposed individuals. Results: Calibration data based on Giemsa-stained fragments in excess of 46 PCC were obtained by different partners using galleries of PCC images for each dose-point. Mean values derived from all scores yielded a linear dose-response with approximately 4 excess-fragments/cell/Gy. To unify scoring criteria, exercises were carried out using coded PCC-slides and/or coded irradiated blood samples. Analysis of samples received 24 h post-exposure was successfully performed using Giemsa staining (1 excess-fragment/cell/Gy) or centromere/telomere FISH-staining for dicentrics. Conclusions: Dose assessments by RENEB partners using appropriate calibration curves were mostly in good agreement. The PCC assay is quick and reliable for whole- or partial-body triage biodosimetry by scoring excess-fragments or dicentrics in G0-lymphocytes. Particularly, analysis of Giemsa-stained excess PCC-fragments is simple, inexpensive and its automation could increase throughput and scoring objectivity of the PCC assay

RENEB accident simulation exercise

Purpose: The RENEB accident exercise was carried out in order to train the RENEB participants in coordinating and managing potentially large data sets that would be generated in case of a major radiological event. Materials and methods: Each participant was offered the possibility to activate the network by sending an alerting email about a simulated radiation emergency. The same participant had to collect, compile and report capacity, triage categorization and exposure scenario results obtained from all other participants. The exercise was performed over 27 weeks and involved the network consisting of 28 institutes: 21 RENEB members, four candidates and three non-RENEB partners. Results: The duration of a single exercise never exceeded 10 days, while the response from the assisting laboratories never came later than within half a day. During each week of the exercise, around 4500 samples were reported by all service laboratories (SL) to be examined and 54 scenarios were coherently estimated by all laboratories (the standard deviation from the mean of all SL answers for a given scenario category and a set of data was not larger than 3 patient codes). Conclusions: Each participant received training in both the role of a reference laboratory (activating the network) and of a service laboratory (responding to an activation request). The procedures in the case of radiological event were successfully established and tested

RENEB intercomparisons applying the conventional Dicentric Chromosome Assay (DCA)

Purpose: Two quality controlled inter-laboratory exercises were organized within the EU project ‘Realizing the European Network of Biodosimetry (RENEB)’ to further optimize the dicentric chromosome assay (DCA) and to identify needs for training and harmonization activities within the RENEB network. Materials and methods: The general study design included blood shipment, sample processing, analysis of chromosome aberrations and radiation dose assessment. After manual scoring of dicentric chromosomes in different cell numbers dose estimations and corresponding 95% confidence intervals were submitted by the participants. Results: The shipment of blood samples to the partners in the European Community (EU) were performed successfully. Outside the EU unacceptable delays occurred. The results of the dose estimation demonstrate a very successful classification of the blood samples in medically relevant groups. In comparison to the 1st exercise the 2nd intercomparison showed an improvement in the accuracy of dose estimations especially for the high dose point. Conclusions: In case of a large-scale radiological incident, the pooling of ressources by networks can enhance the rapid classification of individuals in medically relevant treatment groups based on the DCA. The performance of the RENEB network as a whole has clearly benefited from harmonization processes and specific training activities for the network partners

Investigation of the influence of calibration practices on cytogenetic laboratory performance for dose estimation

Purpose: In the frame of the QA program of RENEB, an inter-laboratory comparison (ILC) of calibration sources used in biological dosimetry was achieved to investigate the influence of calibration practices and protocols on the results of the dose estimation performance as a first step to harmonization and standardization of dosimetry and irradiation practices in the European biological dosimetry network. Materials and methods: Delivered doses by irradiation facilities used by RENEB partners were determined with EPR/alanine dosimetry system. Dosimeters were irradiated in the same conditions as blood samples. A short survey was also performed to collect the information needed for the data analysis and evaluate the diversity of practices. Results: For most of partners the deviation of delivered dose from the targeted dose remains below 10%. Deviations larger than 10% were observed for five facilities out of 21. Origins of the largest discrepancies were identified. Correction actions were evaluated as satisfactory. The re-evaluation of some ILC results for the fluorescence in situ hybridization (FISH) and premature chromosome condensation (PCC) assays has been performed leading to an improvement of the overall performances. Conclusions: This work has shown the importance of dosimetry in radiobiology studies and the needs of harmonization, standardization in irradiation and dosimetry practices and educational training for biologists using ionizing radiation

Nondestructive wood discrimination: FTIR – Fourier Transform Infrared Spectroscopy in the characterization of different wood species used for artistic objects

Wooden artifacts represent a significant component of past cultures. Successful conservation of wooden artifacts depends on the knowledge of wood structure and types. It is critical that conservators know the category of wood that they are treating in order to successfully conserve it. Recently, vibrational spectroscopy has been successfully applied to determine the chemical structure of wood and to characterize wood types. FTIR (Fourier Transform Infrared) is a useful nondestructive or micro-destructive analytical technique providing information about chemical bonding and molecular structure. Its application in the discrimination between softwoods (conifers) and hardwoods (broad-leafs) has already been reported. The aim of the present study was to investigate the potential of FTIR as a tool for the discrimination between different wood types belonging to the same genus. Three different hardwood species, namely poplar (Populus spp), lime (Tilia spp) and birch (Betula spp), were investigated by means of FTIR spectroscopy. The woods were first inspected using a light microscope to certify the wood essence types through micrographic and morphoanatomical features. The FTIR spectra in the 4000 cm[Formula: see text] to 450 cm[Formula: see text] region were recorded using a Perkin-Elmer Spectrum 100 spectrometer. To enhance the qualitative interpretation of the IR spectra, second derivatives of all spectra were calculated using the Spectrum software to separate superimposed bands and to extract fine spectral details. To obtain a comprehensive characterization, the essences under investigation were also analyzed by means of Raman Spectroscopy. Clear differences were found in the spectra of the three samples confirming FTIR to be a powerful tool for wood type discrimination