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cross over. > 125 13.1.4. Slide auto-stainer This provides a rapid and consistent method of staining slides with Giemsa with minimal operator involvement. Currently available autostainers allow intelligent and flexible sample scheduling from 1 to 520 slides to be stained and rinsed unattended with identical or varied protocols. A sample priority assignment feature allows specific sample batches to be queued and processed ahead of others with no user involvement. The built-in battery backup can ensure continuation of ongoing sample processing by providing up to 40 min of run time in case of an electrical power outage. 13.2. AUTOMATED IMAGE ANALYSIS Automated analysis of images captured in a microscope is not yet routinely used by many laboratories for biological dosimetry, although it is likely to increase as systems improve. Attempts have been made to automate scoring for all four assays described in this publication. > 13.2.1. Metaphase finding and image capture While automated analysis stations with walk away reliability for scoring cytogenetic damage is still under development, high-throughput metaphase finders and satellite-scoring stations for computer assisted manual analysis can significantly improve the throughput of a technician. Metaphase-finders assist in locating metaphase spreads on slides and present them in focus, at high magnification ready for analysis [304, 305]. A traditional image-analysis based metaphase finding system may consist of a computer, a high resolution digital camera, a high-quality microscope, an automated stage with autofocus, and a robotic slide-feeder. The computer is loaded with automated metaphase-finding software and interactive automated scoring and annotation software for chromosome aberration analysis. Such metaphase finders can scan up to 150 slides per run for metaphase spreads . As it scans, the results (images and locations of potential spreads) are stored on the centralized server for subsequent automatic relocation at multiple satellite scoring stations for chromosome analysis. Alternatively, virtual
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high-resolution images of metaphase spreads acquired by metaphase finders can be digitally encrypted and transferred via a virtual private network for downstream remote analysis and assessment. This kind of ‘telescoring’ needs harmonized scoring criteria to be established to ensure comparable results. > 13.2.2. Automation of the dicentric assay Microscope analysis of dicentric chromosomes is a time consuming procedure, performed in biological dosimetry laboratories routinely by well trained and experienced scorers, who need to analyse a few hundred cells per day. At low doses a large number of metaphases must be analysed, and therefore, the primary strategy to improve the method is the automation of dicentric scoring to save time, particularly for assessing exposure to low radiation doses. Several attempts were started in the 1980s to develop automatic scoring systems [306– 308]. In the meantime, several commercial systems became available and the corresponding software modules of metaphase finding and karyotyping are now well established in many cytogenetic laboratories. This kind of computer assisted microscopy facilitates the work enormously. At first, a slide will be scanned at low magnification, the metaphases detected and their coordinates stored in an unsupervised operation. During this procedure, a gallery of the detected metaphases can be generated. As the cells are relocated and analysed manually, the individualized electronic scoring sheets are easily maintained, printed and archived as files. In rapid response to a radiation emergency, the observed aberrant cells can be captured manually, digitized and archived immediately. In total, using a metaphase finder the time of scoring might be reduced by a factor of 2 . > 126 It has long been realized that the automatic metaphase finding, image capture and other processes should then be followed by electronic image analysis to realize automatic chromosome analysis, including dicentric scoring. There are several steps on the way to
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perform automatic dicentric scoring. At first, a slide will be scanned by a metaphase finder. In a second step, the detected metaphases will be captured automatically and digitized at a high resolution. Then, the metaphase images will be segmented, to identify the chromosomes and candidate dicentrics. In the 1990s, only the images of the dicentric candidates and their coordinates were stored. Nowadays, with the progress in digital imaging, this procedure became much faster and more efficient. Furthermore the advances in hard disk technology make it possible now to store all the cells of one slide in a high resolution mode. The experience with dicentric scoring software shows, that it is very difficult, to develop hierarchical multistep algorithms, which allow the segmentation of a complete cell, resulting in 46 chromosomes [308, 309]. In general some chromosomes will not be detected, because they were overlapping or lying close together as chromosome clusters. In consequence, some dicentrics will be missed (false negatives). Also some dicentrics may be systematically ignored, because they are smaller than an X chromosome, which might be the case in less than 8%. The automatically detected candidate dicentrics have to be validated by a trained scorer, but this is a much faster and easier process than manual scoring. The dicentric candidates are displayed marked on the screen (Fig. 44a) and this allows fast evaluation. Most false positives (i.e. artefacts, overlapping chromosomes, see Fig. 44b) can easily be rejected. (a) (b) FIG. 44. a) Automatically detected dicentric candidates are identified, which makes evaluation easier and faster. b) False positive dicentric candidates can easily be recognized (i.e. overlapping chromosomes, twisted chromatids or not segmentated objects) and rejected. Because of the incomplete analysis of the cells (the manual standard is to score only complete cells with 46 centromeres) and the resulting uncertainty, automated
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dicentric scoring > 127 has not yet been established as a routine method. Furthermore data are needed to decide if the detected frequency of dicentrics should be related to the number of detected chromosomes, or if it may be counted as dicentrics per cell bearing in mind that it is not a constant number of chromosomes being evaluated in every cell. This raises questions about using the dicentric overdispersion index (Section 9.7.4.3) and thus the potential of this method to detect partial body irradiations. Another aspect of interest might be the influence of the preparation quality of the slides. Good metaphase spreads increase the number of detected chromosomes. Here more investigation is needed and may be optimized by software training of the dicentric classifier. The automatic dicentric detection gives very reproducible results. Comparisons between dose effect curves, established by manual and semiautomatic scoring demonstrate a very good correlation between both methods. The detection efficiency of dicentrics by automated systems has been reported to be about 50–70% [310, 311]. The automation of dicentric scoring has the potential to improve the dicentric assay as a helpful tool to screen large numbers of blood samples in case of a large scale radiation emergency. One workstation can be supplemented by satellite stations, where the cells and dicentrics will be evaluated, which increase the capacity and throughput of the system. For triage mode the automation of dicentric scoring significantly reduces the time for analysis and results correlate well with manual scoring [300, 310, 311]. > 13.2.3. Automated scoring of micronuclei Several algorithms for automated image analysis of the CBMN assay were already developed in the 1990s [312, 313]. These systems however showed limitations such as a relative high inaccuracy in classification of the BN cells. More recently, new and better automated image analysis systems for
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the CBMN assay have been developed. The MN software module integrated in the metaphase finder system MSearch, developed and commercialized by Metasystems (a manufacturer of microscopic imaging systems) automatically identifies by morphological criteria BN cells by the occurrence of two adjacent similarly DAPI stained nuclei. In a second step, MN are counted automatically in a circular area defined around the two nuclei of the BN cell [314, 315] (see gallery of BN cells with MN, Fig. 45). A further evaluation of the detected yield of MN by a scorer is not necessary. It is important to note that, unlike visual scoring criteria for the CBMN assay, the Metasystems software does not use the cytoplasmic boundary to identify binucleated cells but simply assumes that close proximity of 2 nuclei (meeting specified parameters in the software pattern recognition classifiers) is sufficient to accurately identify a binucleated cell; if required the cytoplasmic boundary can be visualized using phase-contrast microscopy as recommended by Eastmond and Tucker (1989) to verify the accuracy of binucleated cell detection. > 128 FIG. 45. Gallery of BN cells with and without MN captured by an automated system. A system developed by Decordier et al . for use in biomonitoring of in vivo exposure to mutagenic agents, uses a capture station and two MN analysis workstations. This system identifies firstly the cytoplasm of Giemsa stained cells, then detects the number of nuclei in the cell thus allowing identification of BN cells and in a third step it scores the MN. A study performed by Willems et al . demonstrated the suitability and advantage of automated MN scoring for population triage in case of large scale radiation emergencies, where it is important to distinguish severely exposed individuals ( > 1 Gy), who require early medical follow up and
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treatment, from those less exposed. The fully automated MN scores obtained in the latter study were highly correlated with the manual MN scores (r 2 = 0.917) and demonstrated that a visual validation was not needed . The reference dose response curve obtained for automated MN scoring, based on MN data of 10 individuals, showed that the uncertainty on a dose determination of 1 Gy amounts to 0.2 Gy. The 95 % confidence intervals of the 0 Gy and 1 Gy doses did not overlap. Accurate dose estimations were also achieved at the higher doses of 2 and 3 Gy. Therefore, the MN scoring system is able to distinguish exposures with doses of 1, 2 or 3 Gy. In this study it was estimated that 2 scorers can process at least 60 blood samples (120 slides) in a 12 hours shift. In general, the number of blood samples analysed can be increased extensively by using more automated working units. Here, a network of trained laboratories with similar equipment and MN classifiers, using standardized fixation protocols can give comparable results. By this means, the throughput of MN automated scoring can be increased to permit a rapid response to a large scale radiation emergency. 129 13.2.4. Premature chromosome condensation assay The approach here is essentially similar to that for dicentric analysis by using automated metaphase finding on Giemsa stained preparations . The images are then passed to the operator for scoring by eye. The speed of analysis is approximately three times faster than fully manual analyses. > 13.2.5. FISH based translocation assay Some considerable success has been obtained by using FISH staining of 3 or 4 pairs of chromosomes . A system consisting of a PC and a cooled CCD camera was developed. It was based on a two-step approach: the finding
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of metaphases with counterstain fluorescence, followed by the detection of translocations involving chromosomes labelled with whole chromosome paint. From the candidate list of translocations, similar false positive and false negative rates have been measured on fluorescence stained lymphocyte preparations (about 10%), as were reported for candidate dicentrics on Giemsa stained slides . A longer screening time was needed for fluorescence: 1 hour per slide, of which 25 min are necessary for autofocussing, compared with a few minutes per slide with bright field microscopy. Therefore, a larger chip size is being used with the CCD camera to increase the speed of scoring. For detection of chromosome paints, a relatively simple threshold based on the grey value histogram combined with some morphological operations seems to be sufficient to detect the chromosomes or chromosome parts labelled with the whole painting probe . The suitability of the system for scoring translocations was tested in a study to detect X ray induced translocations involving chromosome #4. A comparison was made between automatic and manual scoring, and the efficiency of the automatic assay was found to be approximately 90% of that obtained manually. By increasing the number of hybridized chromosomes in one colour, the sensitivity of the method can be improved. However, when more chromosomes are painted, procedures to separate eventual touching and/or overlapping chromosomes are essential . Piper et al. reported on the construction of a fluorescence metaphase finder with commercially available hardware and a standard Unix workstation. A cocktail of the three chromosomes #1, #2 and #4 was used and a comparison was made with manual scoring. The results showed that the amount of time required for analysis was reduced by a factor of three. Furthermore, the metaphase finder found more scorable spreads than did visual scanning. Machine assisted scoring had additional benefits
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notably that digitized images of metaphases sometimes assisted the analysis of chromosome rearrangements because cells could be revisited easily for re-examination and further analysis. This system is further modified by using a binary decision tree for classification of observed metaphases and for improving scanning accuracy . Another advantage found with digitized coloured images held in a computer is that they can be enhanced electronically, and this can sometimes permit better discrimination than can be achieved by eye, of very small translocated pieces of chromosomes. An obvious extension that is being addressed is to analyse multicolour FISH preparations by combining chromosome and centromere specific DNA libraries for automated analysis of translocations and dicentrics simultaneously. 13.3. LABORATORY INFORMATION MANAGEMENT SYSTEM (LIMS) A customized, commercially available LIMS can be an indispensable tool for addressing challenges arising from increased sample preparation/analysis throughput. In addition, a LIMS can help to maintain general laboratory records regarding personnel training, instrument calibration and chemical inventory, etc. Electronic data management via LIMS > 130 offers benefits that include the ability to link, search, and retrieve data and to rapidly report results after a radiation disaster. Several modules are available and a brief description follows: • Sample identification — Samples assigned unique bar code identifier. • Sample transport — Structured templates to input data about sample conditions during transport and on arrival (e.g. data from a temperature logger). • Test setup — Assigning to suitable processing and cytogenetic tests for any given sample (e.g. whole blood culture or lymphocytes isolation; dicentric or CBMN assay). • Sample scheduling — Prioritizing sample analysis depending on case urgency and logging cases to specific laboratory personnel. • Security — Requirement of user authentication through passwords. Users may be assigned different privileges within the subsystem. • Auditing — Records and modification are tracked. • Archiving
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— Keeps the database working efficiently, helps maintain record integrity and ensures that scientific data are securely backed up. • Reporting — Generating formatted individual case reports which can be communicated to the treating physician. • Instrument integration — Data can be automatically collected and collated directly from the component modules reducing the risk of transcription errors. This helps to improve data accuracy and consistency, which are critical during response to a mass casualty event. A schematic representation of a scalable and high-throughput automated cytogenetic laboratory is shown in Fig. 46 [301, 302]. FIG. 46. Schematic representation of a high throughput cytogenetic laboratory and automation with LIMS network. The slides produced in the central laboratory can be physically transferred to scoring stations/laboratories or captured images securely transferred electronically to the receiving laboratories for analysis (courtesy Ramakuma and Prasanna, AFRRI, USA). > Physical transfer of slides > Blood Handler Incubator > Harvester > Spreader > Auto-stainer Metaphase finder > Internet > Remote Analysing Station 2 Remote Analysing Station 1 > Slides > Metaphase spread images LIMS Server > Samples Analysed Info retrieval & Back-up on LIMS > Analysis Preparation 131 14. MASS CASUALTY EVENTS A mass casualty event is defined as one that involves injury to a sufficient number of individuals such that it exceeds the response capability of the local responders [322, 323]. When this type of event involves radiation, the result can be a large population, who may have received a range of radiation doses spanning from background levels to those large enough to cause medical consequences. These individuals need to be rapidly assessed for exposure levels to determine whether medical intervention is required [4, 322, 324–326]. Events involving radiation can result from accidents or malicious acts, both of which, if they were to happen, may cause casualties within
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the general public. Confounding factors such as conventional physical injuries could also be present and dealing with life threatening injuries takes precedence over dosimetric and other activities . Planning and preparedness is critical for an effective response to a mass casualty event. In the case of a radiation emergency the generic accepted guidelines include: a) establishment and training of local and national response teams equipped with critical equipment and supplies, b) knowledge and application of appropriate and available diagnostic approach for assessing radiation injury and dose, and c) access to reach-back reference laboratories, including expert laboratories for dose assessment by cytogenetic biological dosimetry [322, 328, 329]. A critical component in the biological dosimetry ‘concept of operations’ is the process to prioritize the selection of samples for rapid cytogenetic triage-dose assessment that requires dynamic communication between the medical responders and reference cytogenetic biological dosimetry laboratory staff. 14.1. POTENTIAL RADIATION EXPOSURE SCENARIOS > 14.1.1. Malicious Events A number of possible scenarios for malicious exposure to radiation have been identified and are listed here in three broad categories [322, 324, 326]. (a) Radiological Exposure Devices (RED) involve sealed sources distributed in an environment but not presenting a contamination threat. Individuals who come close to these sources can receive significant localized doses but numbers of highly exposed individual are anticipated to be low. (b) Radiological Dispersal Devices (RDD) use explosive or mechanical devices to distribute radiological material resulting in radioactive contamination. A relatively small area would be affected and radiation exposures could take the form of both internal and external contamination, however exposures are expected to be lower than medically significant. (c) Improvised Nuclear Devices (IND) incorporate nuclear material that can produce nuclear explosions. This can cause extensive radiation and thermal injuries with large numbers of fatalities and casualties with high doses of radiation.
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The result of such an event would be catastrophic. > 14.1.2. Accidental Events Radiation exposures could result from several scenarios including but not limited to : (a) Reactor emergencies with a breach of irradiated fuel elements during loss of coolant. These emergencies may result in high doses to workers and general public near the > 133 site and contamination leading to low doses to the general public in the vicinity (e.g. Chernobyl). (b) Criticality accidents may occur when sufficient quantities of special nuclear material are inadvertently allowed to undergo fission. This results in high levels of exposure to persons in close proximity (e.g. Tokai-mura). (c) Emergencies involving lost or stolen ‘orphan’ sources can result in several exposure scenarios depending on the activity, length of time of exposure and distribution of the source. Such emergencies can result in high doses to the whole body or partial body exposures as well as internal or external contamination (e.g. Goiânia). 14.2. HISTORICAL EXPERIENCE There have been several examples in the recent past where cytogenetic biological dosimetry has been used to assess exposures to radiation after accidental events involving multiple casualties (Table 17). TABLE 17. SELECTED EXAMPLES FOR USE OF CYTOGENETIC BIOLOGICAL DOSIMETRY IN RADIATION ACCIDENTS INVOLVING MULTIPLE CASUALTIES Year of accident Accident location Number of people involved Cytogenetic Assay Cases References Dicentrics PCC FISH CBMN 1986 Chernobyl, Ukraine >100 000 436 [342, 343, 344] 1755 97 [235, 236, 347] 140 a [259, 260, 297] 1986–1987 Lilo, Georgia 11 11 4 1995 Istanbul, Turkey 21 21 10 5 10 1997 Goiânia, Brazil 250 129 1998 Matkhoji, Georgia multiple 85 1999 Tokai-mura, Japan 43 43 3 2000 Bangkok, Thailand multiple 28 28 2005 Concepción, Chile 233 45 1 2006 Dakar, Senegal 63 33 a
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Retrospective Accidents can have different characteristics such as a sudden recognized event with many identified casualties in a short period of time (e.g. Chernobyl) or a more slowly evolving situation with delayed discovery of exposed individuals (e.g. Goiânia). An accident could 134 also involve just a few real cases but with tremendous public pressure to extend biological dosimetry to the surrounding community even though there was little to no physical evidence to justify this action (e.g. Tokai-mura). In this case, the cytogenetic biological dosimetry laboratory of the National Institute of Radiological Sciences (NIRS) was able to determine the doses to these 265 concerned individuals through surveys of their location during the event and assure them that no significant dose was received . A cohort of 43 persons at a uranium processing facility, who were confirmed to have been exposed slightly on the basis of measurements of whole-body counting of 24 Na, were also assessed for dose by chromosome aberrations analysis. Historically, cytogenetic biological dosimetry using dicentric analysis, along with routine leukocyte counting, is used as an initial assay for dose estimates following accidental exposures involving multiple casualties (see Table 17). Other cytogenetic assays (FISH, PCC and CBMN) have been used to confirm dose estimates, however, often this has been performed from months to years after the accident. 14.3. ROLE OF BIOLOGICAL DOSIMETRY 14.3.1. Radiation Exposure Assessment Methods After a mass casualty radiation event , physicians are primarily concerned with preserving life and evaluating medical signs and symptoms for early treatment decisions. Several radiation exposure assessments, evaluated by an international consensus of experts, are applicable for early-phase acute radiation [6, 25, 322, 325–327, 329]. Depending on the radiation scenario and available resources, appropriate radiation assessment methods should be implemented in a mass casualty radiation emergency. 14.3.2. Biological dosimetry concept-of-operations Generic guidelines
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for the ‘concept of operations’ for first-responders in a mass casualty radiological incident are well described by IAEA resources [322, 323, 331]. The implementation of a multiparameter biological dosimetry assessment approach in a mass casualty radiation emergency, however, can be a significant confounder without access to expert teams [322, 323]. Fig. 47 illustrates the components of the REAC/TS and AFRRI treatment strategy along with the concept of operations for use of multiparameter biological dosimetry . 135 FIG. 47. Biological dosimetry concept of operations during management of radiation emergency with trauma or illness. Biological dosimetry functions are illustrated for the individual action steps of the REAC/TS and AFRRI ‘Radiation Patient Treatment’ algorithm . Current radiation exposure assessment methods and emerging technologies can offer a potential to contribute in radiation injury and dose assessment response. Research and development are needed to establish a diagnostic triage concept to facilitate a functional biological dosimetry concept of operations in a mass casualty radiation emergency . The initial screening radiation assay must be rapid (1 assay per min or less), use a hand-held device, and ideally involve a self-use test. Secondary and tertiary radiation assay may require more expertise and take longer (>1 day) for use but have higher radiation specificity. Once identified as potentially exposed, patients may be recommended for biological dosimetry to provide confirmation of the suspected exposure and to determine a dose level. In the early-response phase of a radiation emergency, the initial purpose of cytogenetic triage is to rapidly estimate the dose for each referred patient to supplement this early clinical assessment. Although these first dose estimates may not be extremely accurate, the goal is to quickly place the patient into one of 4 dose ranges (1 Gy to 2 Gy, 2 Gy to 4 Gy, 4 Gy to 6 Gy and
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> 6 Gy) to provide timely information to the medical community that can be used for patient treatment . At this stage it is also possible to refute false positive samples due to symptoms such as vomiting from other causes. Partial-body exposures may also be identified at this stage. Once the initial urgency of the requirement for rapid triage dosimetry has passed, those patients identified as having received significant doses can be further analysed to provide more accurate dose estimates. After the emergency has passed and more accurate dosimetry is complete on those identified as exposed, further follow up will continue on those individuals who received very > 136 low or no doses, but still require reassurance. Also, follow-up epidemiological studies with other techniques such as FISH will be required. > 14.3.3. Communication with the medical community Communication between the medical community and the biological dosimetry laboratories is essential. This should be done taking due regard for medical confidentiality [3, 4]. Any information from the medical community that can assist the biodosimetrists with sample prioritization is extremely helpful. Equally essential is the communication of the biological dosimetry laboratory back to the medical community in a timely manner that will assist them with making decisions on the treatment of the patients. This requirement of continuous communication highlights the importance of robust sample tracking during response to the event. It is essential to have a unique, well established, documented sample coding system (e.g. LIMS described in Section 13.4) such that samples can be tracked from collection, through processing, analysis and reporting back to the medical community. Cytogenetic laboratories work with blinded samples while medical professionals work with names. The laboratory needs to identify individuals who will be accessing information from LIMS or similar documents to communicate to the medical professionals. These individuals
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will have to break the coding to be able to communicate to the physicians and therefore should probably not be involved in sample scoring. 14.4. EXISTING MASS CASUALTY STRATEGIES > 14.4.1. Triage scoring Rapid triage scoring can be applied to several of the cytogentic assays used for biological dosimetry. It has been determined that by scoring 50 cells (or 30 dicentrics) in the dicentric assay, dose estimates can be made with sufficient accuracy to provide useful dose estimates to the medical community. It has been shown that this method of scoring will deliver dose estimates within 1 Gy [332, 333]. Compared to full dicentric scoring of 500 or 1000 cells, this triage method increases throughput up to 20 times. To further increase scoring speed, a QuickScan method has been introduced by Flegal et al. in which only the damage in each cell is scored without the requirement of ensuring the presence of 46 centromeres, however, only cells which appear complete are scored. This method of scoring reduces the microscopy time by an additional factor of 6 . For mass casualties, PCC is particularly useful for high dose exposures. The PCC-ring method has been shown to be useful for triage dosimetry of doses above 6 Gy, measuring 300 PCC cells or 50 rings . This assay, however, has limitations for the low dose region. Triage scoring can also be applied to the CBMN assay. For standard biological dosimetry, it is recommended that 1000 binucleated cells be scored. However, it has recently been demonstrated that scoring 200 BN cells allowed the identification of doses greater than 1 Gy . The time required to score 200 BNC is approximately 15 min which is significantly faster than triage dicentric scoring and still slightly faster than QuickScan. Another advantage of this method is that the
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expertise and training required for scoring is much less than the dicentric assay so that scorers could be quickly trained in a mass casualty situation. > 14.4.2. Automation Automation has been discussed in detail in Section 13. It is clear that automation will increase throughput and free up human resources for other tasks required during mass casualty events. This can include automation of the blood processing, metaphase harvesting, metaphase finding and dicentric or micronucleus scoring. > 137 14.4.3. Networks Many nations have established reference expert cytogenetic biological dosimetry laboratories. Recently some of these laboratories have established national and regional networks to enhance their capabilities [118, 337, 338]. Others have reviewed individual national resources and capabilities with a view to forming a regional network . United Nations (UN) agencies (IAEA, WHO) that provide international cooperation in biological dosimetry have also established cytogenetic networks [340, 341] (Table 18). There are very few countries with more than one cytogenetics laboratory having the primary function of undertaking biological dosimetry. Nevertheless there may be a lot of cytogenetics expertise in other research institutes and particularly in hospitals’ clinical genetics departments. National networks (e.g. France, Korea, Japan, Canada) have implemented arrangements, including training, whereby this expertise can be mobilized promptly under the leadership of the specialist reference biological dosimetry laboratory. The networking, whether national or international, requires a coordination of infrastructure of logistics, data management, and communications. These networks also afford an excellent platform for exercises and inter-comparison studies to ensure suitable performance of individual laboratories and the cytogenetic biological dosimetry networks. Use of cytogenetic networks enhances the capabilities for use of triage and reference dose assessment by cytogenetic analysis for mass casualty radiation events. > 138 TABLE 18. SUMMARY OF EXISTING BIOLOGICAL DOSIMETRY NETWORKS Location Name Lead Participants (number or name) 2Assays in use International
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Worldwide Response and Assistance Network (RANET) IAEA Continue to change 4DCA, FISH, PCC, CBMN Worldwide BioDoseNet WHO 63 DCA, FISH, PCC, CBMN Europe Tri-Partite dependent on location of event 3UK, France, Germany DCA, FISH, PCC, CBMN Latin America Latin American Biological Dosimetry Network 1Argentina —Nuclear Regulatory Authority and Cuba — Centro de Protección e Higiene de las Radiaciones Argentina (2), Brazil, Chile, Cuba, Mexico, Peru, Uruguay 2DCA, FISH, CBMN National Canada Cytogenetic Emergency Network Health Canada 4 reference 18 satellite DCA, FISH,CBMN > France Réseau de dosimetry biologique Institut de Radioprotection et de Sûreté Nucléaire (IRSN) 2 labs from CEA and one from the MNHN DCA, FISH, PCC Japan The Chromosome Network National Institute of Radiological Sciences (NIRS) 7 DCA, PCC, FISH South Korea Korean Radiation Biodosimetry Network Korea Institute of Radiological & Medical Sciences 6 DCA, FISH, PCC,CBMN 1 With rotating leadership among participant countries every 2 years. 2 DCA is the technique applied for mutual cooperation purpose; PCC, FISH and CBMN are established in at least some of the network partners. 3 Co-leads. 4 Part of broader assistance and response network. 139 15. QUALITY PROGRAMMES AND THE ISO STANDARDS 15.1. THE RATIONALE FOR A QUALITY ASSURANCE AND QUALITY CONTROL PROGRAMME This publication has demonstrated that there are no universally adopted procedures for the cytogenetic assays employed for biological dosimetry. In broad outline laboratories follow similar methods but when fine detail is considered some variations occur in methods which potentially can influence the quality of results. Therefore, it is reasonable to expect each service laboratory to develop a quality programme that ensures the robustness, accuracy and reproducibility of its procedures. To ensure the quality of a biological dosimetry laboratory's output over extended periods of time, its production process must be solidly based on scientific principles, method validation, and product
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verification. A complete quality programme provides the strategy for safeguarding the quality of the laboratory's product, whether it is a measurement or a service. Furthermore, these capabilities require periodic comparison with those of other certified or suitably qualified cytogenetic biological dosimetry laboratories, continued stability of the laboratory process, and periodic evaluation of the final product to confirm that it meets predefined specifications. Operating within the guidance of the documented criteria under an in-house quality assurance programme, periodic peer assessments and documented quality procedures assure stable operation between formal proficiency evaluations. The in-house quality assurance programme must provide for programme assessments, adequate operational environment, personnel qualifications, procedure manual, instrumentation, calibration, data reduction, record system and data reporting. Control over the cytogenetic process between proficiency evaluations provides another assurance of end products with reproducible quality. Adoption of a total quality management approach would assure continued improvement of operations. The proficiency tests periodically evaluate measurement consistency with other certified or suitably qualified cytogenetic biological dosimetry laboratories, (see Annex VII) and test the laboratory and its capabilities to verify their ability to produce high quality products and/or services, i.e. dose estimations. An essential element is successful completion of tests within specified limits of accuracy. In addition, this measurement process can be used to verify the quality of a laboratory's service/product output. For the specific area of biological dosimetry, two measurement proficiency testing strategies can be used: 1) samples exposed in vitro to a known radiation dose, dose rate, and quality of radiation, are sent to the service laboratory for analysis, and 2) the laboratory engages in an interlaboratory comparison study of samples sent to certified or suitability-qualified laboratories for analysis. In both cases, analyses are carried out and comparisons are made between the value obtained by the laboratory and that obtained by its testing
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laboratory. The laboratory is then notified of the percentage difference through a report. For direct testing, only the laboratory's measurement capabilities are being tested. On the other hand, when the laboratory assays its own product and also sends an aliquot to the testing laboratory for confirmational and explicit traceability measurements, both the laboratory's analytical processes and measurement capabilities are being tested. > 141 Through the combination of all these quality assurance strategies, the quality and integrity of the laboratory's measurements or services can be assured. Of these strategies, a major emphasis should be placed on strong in-house quality assurance programmes, active and thorough on-site expert evaluations, strict adherence to the documented operational criteria, and laboratory evaluation by ‘blind’ testing. This combination of checks will assure that the analytical processes will remain in control within specified precision objectives. Although periodic end-product evaluation is a requirement (e.g. between 1 and 3 years), its frequency can be minimal when the analytical processes remain under control. Quality assurance plans for service laboratories performing biological dosimetry should include the following elements: • identification and preparation of samples • validation of procedures or methods • measurement • data reduction • documentation Systematic actions should be included in the quality assurance plan to provide adequate confidence that a measurement or procedure will be performed satisfactorily. The International Organization for Standardization (ISO) seemed an appropriate environment to define and write such a set of common rules. The general principles, by which standards are developed within the ISO, are voluntary, consensus and industry-wide. In addition, each standard draft is peer reviewed, by a specialist-working group followed by participating countries via the national representatives of ISO. After publication, each standard may be used directly, or implemented into national standards. The creation of a working group on the standardization of biological
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dosimetry was proposed in 1998 and accepted by ISO in 1999 within Technical Committee 85, Nuclear Energy, at the level of Subcommittee 2, Radiation Protection. The working group includes 13 specialists from 11 countries, plus a representative of IAEA. ISO 19238 standard, published in 2004, provides Standard Criteria for Service Laboratories Performing Biological Dosimetry by Cytogenetics . 15.2. CURRENT STRUCTURE OF THE ISO 19238 DOCUMENT In its current format, the document is divided into 11 chapters and 4 informative annexes. The main features described in this document address: (a) The confidentiality of personal information with respect to: (i) The transmission of confidential data concerning the patient or the overexposure circumstances, from the doctor representing the patient (or the patient him/herself) to the laboratory. (ii) The anonymity of the blood sample and the confidentiality of the results and of the report. (iii) The delegation of confidentiality within the laboratory. > 142 (b) The potential risks incurred by the laboratory staff during the processing of a potentially infective blood sample. While this problem is not specific to biological dosimetry per se , it appeared essential to emphasize, the minimal microbiological, chemical and optical safety requirements. (c) The establishment of at least one appropriate calibration curve within the service laboratory is an essential condition for dose estimation. In particular, this curve has to be produced with the same laboratory protocols as used by that laboratory for all its dose assessments. A report must include the experimental conditions of the calibration curve fit, e.g. nature of source and source physical calibration, dose ranges and minimum detection levels. (d) While the service laboratory does not control some conditions, such as the quality of blood sample taken and its despatch, the service laboratory must upon receipt provide sound processing of the sample, a dose estimate
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and, finally, a report that is reviewed and endorsed by a qualified expert. (e) Routinely, the laboratory report should reproduce any relevant information provided by the customer since this may influence the interpretation of the findings. All observed aberrations must be listed and interpreted according to the current understanding of mechanisms for radiation-induced chromosome aberration formation. (f) Quality assurance plans for service laboratories should comprise in-house procedures to ensure long term accuracy and stability of performance plus periodic peer assessment/cross-calibrations with an external reference programme. It addresses the following broad elements: identification and preparation of samples, validation of procedures or methods, measurement and instrumentation, data interpretation, record keeping and documentation. 15.3. APPLICATION TO POPULATION TRIAGE As already discussed in Section 14 the potential for nuclear and radiological emergencies involving mass casualties from accidents or malicious acts is ever-present. After such event, individuals will be assessed clinically and categorized on the basis of any prodromal signs and symptoms of overexposure plus available information concerning their involvement in the emergency. In this early response phase of a radiation emergency, cytogenetic triage, i.e. the use of chromosome damage to evaluate approximately and rapidly radiation doses received by individuals, is also appropriate in order to supplement the early clinical categorization of casualties. However as time progresses clinicians would request more accurate estimations of doses, both in the low-dose range on risks of late stochastic effects and also for higher doses for anticipating severe tissue reactions. A secondary cytogenetic inspection should achieve a quantitatively more precise estimate of dose, and also search for any evidence of heterogeneity of exposure. However, this event can also exceed the resources of the locally involved biological dosimetry laboratory, requiring the intervention of other laboratories within the constitution of a network (see Section 14.5.3). Several biological dosimetry laboratories have independently
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and successfully performed rapid dose assessment in mass casualty emergencies or exercises. Their approach, essentially based on the > 143 dicentric assay, included preplanning, reagent stockpiling, simplified sample processing, and automation, scoring criteria, and networking with other expert laboratories. Whilst following the principles of ISO 19238 , some departures from the exact protocol are needed in order to satisfy the requirement for rapid response and delivery of dose estimations. Building upon this experience, a new ISO 21243 standard, published in 2008, defined the “Performance criteria for laboratories performing cytogenetic triage for assessment of mass casualties in radiological or nuclear emergencies. General principles and application to the dicentric assay” . The standard is written in the form of procedures to be adopted for biological dosimetry triage where the criteria required for such measurements will usually depend upon the application of the results: medical management when appropriate, radiation protection management, record keeping and medical/legal requirements. For example, selected cases would be analysed to produce more accurate evaluation of high partial-body exposure; secondly, doses would be estimated for persons exposed below the threshold for deterministic effects, by using the ISO 19238 criteria. The content of the ISO 21243 standard can be summarized as follows: > (1) Before the event each laboratory is responsible for: (a) Maintaining a stockpile of the required reagents and other laboratory consumables or must be able to immediately access them from a local, state or national stockpile or a commercial supplier. (b) Maintaining established communication links with the local/state/federal healthcare facilities. (c) Specifying and documenting the responsibilities, roles, and interrelations of all laboratory personnel whose functions affect the quality of emergency biological dosimetry response. (d) Knowing its maximum throughput capability for samples processing (time versus number). (e) Maintaining its own quality control and quality assurance programme. (f) Participating, as
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appropriate, in relevant educational, training and exercise programmes. (g) Participating in periodic interlaboratory comparison studies. > (2) During the event: (a) The reference laboratory responsible for the dose estimation calls for collaboration of network laboratories when the number of cases to be examined is above its own capacity. (b) When the decision to activate the network is made, the reference laboratory becomes the focus for communication between the network. The reference laboratory informs the partners of the circumstances of the incident, and together they establish the extent of cooperation needed. (c) Cytogenetic examination for dose estimation is performed at the request of physicians. Selection of cases to be examined is made by discussion between experts in cytogenetic dose estimation, scene managers, and physicians. > 144 (d) The reference laboratory and the network laboratories discuss the details of work sharing in biological dosimetry. (e) An informed consent in written form has to be submitted from each individual or a treating physician, as applicable, prior to blood taking. Special care has to be taken to protect privacy throughout the assignment. (f) The reference laboratory organizes the blood sampling and dispatching of specimens to the partners, or designates another suitable agency to take over. (g) The results of scoring (and sometimes dose estimation) are reviewed by more than one laboratory, and dose estimation for each person is made based on the reviewed results. (h) The associate laboratories send to the reference laboratory the raw data including the aberration distribution data. They also send the dose estimates, adjusted when necessary for dose protraction or heterogeneity, obtained from their own calibration curve most appropriate for the type of radiation involved. (i) The reference laboratory receives the results from the network partners and acts as the central point of communication/liaison with the physicians. (j) Following review
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with medical staff some patients may be selected for increased cell scoring in order to improve statistical uncertainties on dose estimates and better discrimination of inhomogeneous overexposure. Such further examination will be made according to the performance criteria described in the ISO standard 19238. According to these different configurations, the flow chart (Fig. 48) describes the interactions between the reference laboratory, the network and the medical team. > 145 FIG. 48. A flow chart describing the interactions between the reference biological dosimetry laboratory, the network and the medical team, in the context of a dose assessment of few individuals (ISO standard 19238 (blue) ) or a mass casualty (ISO standard 21243 (red) ) (this figure is reproduced from the ISO standard ISO 21243:2008 with the permission of AFNOR on behalf of ISO. Copyright remains with ISO). Reference lab decides to: Do full analyses alone Completes analyses Reports final dose to medical team Do triage alone Reports preliminary data to medical team Ref lab/medical team joint decision on full Lab completes analyses Final dose reports to medical team Activate network Network does triage-mode analyses Reference lab collates data ra p idl yPreliminary reports to medical team Reference lab completes analyses Network completes analy sReference lab collates data Final dose reports to medical team Ref lab/medical team j oint decision on full Few individuals (100) A mass casualty (>100) Biological dosimetry request Patients to medical care 146 16. SAFETY OF LABORATORY STAFF ‘Laboratory biosafety’ is the term used to describe the containment principles, technologies and practices that are implemented to prevent unintentional exposure to pathogens and toxins, or their accidental release. Global events of the recent past have highlighted the need to protect laboratories and the materials they contain from being intentionally compromised in ways that may harm people,
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livestock, agriculture or the environment. It has thus become necessary to expand biosafety through the introduction of laboratory biosecurity measures. ‘Laboratory biosecurity’ describes the protection, control and accountability for valuable biological materials within laboratories, in order to prevent their unauthorized access, loss, theft, misuse, diversion or intentional release. Considering cytogenetic laboratories, biosafety and laboratory biosecurity are comprehensively presented in the WHO Laboratory Biosafety Manual and the accompanying document Biorisk Management: Laboratory biosecurity guidance, such as: laboratory design and facilities, equipment (i.e. biological safety cabinets), safe working practices, occupational health and medical surveillance, disinfection and sterilization, waste handling, chemical exposures, fire, electrical, radiation and equipment safety. This information is fully applicable and of particular use for operating a cytogenetics laboratory [355, 356]. In addition to guidance documents, staff should conform to their national and institutional legislation or regulations regarding safe and secure working practices in laboratories. The following are some particular features concerning safety in cytogenetics laboratories that are worth highlighting. 16.1. INFECTION Universal precautions should always be applied and adopted when handling human blood, and all specimens should be regarded as being potentially infectious. Specimens should be unpacked and manipulated in appropriately used, maintained and certified biological safety cabinets. The use of sharps, e.g. hypodermic needles, should be limited to reduce risk of needle stick injuries. Contaminated sharps should always be collected in puncture-proof containers fitted with covers and treated as infectious waste. Suitable disinfectants should be available to deal with spills and to decontaminate work surfaces and equipment after specimens are processed. All biological waste and used disposable plastic ware should be sterilized, for example by autoclaving, before disposal. If infectious waste has to be removed from laboratories for decontamination and disposal, it must be transported in sealed, leak proof containers according to national and/or international regulations, as
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appropriate. If a particular vaccine or toxoid is locally licensed and available, it should be offered after an appropriate risk assessment of possible exposure and a clinical health assessment of the individual have been carried out . 16.2. OPTICAL The use of Ultraviolet (UV) light may be necessary for certain procedures. > 147 UV lights are not required in biosafety cabinets. If they are used, they must be cleaned frequently to remove any dust and dirt the germicidal effectiveness of the light. Where UV lights are in use, they must be turned off while the room is occupied, to protect eyes and skin from inadvertent exposure . When UV lamps are used in exposing slides during the FPG staining procedure, shielding and working procedures should be in place to avoid direct irradiation of the skin or eyes of laboratory staff. Fluorescence microscopes are generally engineered to be inherently safe during normal use. 16.3. CHEMICAL Certain fine chemicals and pharmaceuticals are used routinely in the procedures covered in this publication. For more information about hazardous chemicals and chemical safety see the WHO Laboratory Biosafety Manual, part VI [356, part VI] with its thorough list of chemicals, detailing their hazards and precautions to be used. When present in cultures or used in staining procedures, chemicals and pharmaceuticals are mostly used in small volumes and in dilutions that generally present no health hazard. They are, however, made up and stored in concentrated stock solutions. The main reagents of concern and their internationally agreed risk phrases (R numbers) are listed in Table 19. Storage of chemicals in laboratories should be limited to amounts necessary for daily use. Bulk stocks should be kept in specially designated rooms or buildings. Chemicals should not be stored in alphabetical order! TABLE 19. MAIN REAGENTS OF CONCERN
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FOR BIOLOGICAL DOSIMETRY LABORATORY AND THEIR INTERNATIONALLY AGREED RISK PHRASES Reagent Risk phrase (R number a )Acetic acid 10; 25 Acridine orange 36; 37; 38 Barium hydroxide 20; 22; 34 Benzylpenicillin 42; 43 Bromodeoxyuridine 20; 21; 22; 46; 61 Calyculin A 23; 24; 25; 38 Colcemid 25; 63 Cytochalasin B 26; 27; 28; 63 DAPI (4’,6-diamidino-2-phenylindole) 36; 37; 38 DMSO (Dimethyl sulphoxide) 36; 37; 38 148 Reagent Risk phrase (R number a )Formaldehyde 23; 24; 25; 34; 40; 43 Formamide 37; 38; 41; 61 Giemsa stain 20; 21; 22; 40; 41 Heparin 36; 37; 38 Hoechst stain 23; 24; 25; 36; 37; 38 Hydrochloric acid 34; 37 Hypaque 42; 43 Methanol 11; 23; 24; 25; 39 Okadaic acid 23; 24; 25; 38 Pepsin 36; 37; 38; 42 Phytohaemagglutinin 20; 21; 22; 43 Ribonuclease A 20; 21; 22; 38 Sodium hydroxide 35 Streptomycin sulphate 20; 21; 22; 61 Xylene 10; 20; 21; 38 a R 10: flammable R 11: highly flammable R 20: harmful by inhalation R 21: harmful in contact with skin R 22: harmful if swallowed R 23: toxic by inhalation R 24: toxic in contact with skin R 25: toxic if swallowed R 26: very toxic by inhalation R 27: very toxic in contact with skin R 28: very toxic if swallowed R 34: causes burns R 35: causes severe burns R 36: irritating to eyes R 37: irritating to respiratory system R 38: irritating to skin R 39: danger of very serious irreversible effects R 40: possible risk of irreversible effects R 41: risk of serious damage to eyes R 42: may cause sensitization by inhalation R 43: may cause sensitization by skin contact R 46: may cause heritable genetic damage R 61: may cause harm to the unborn child R 63: possible risk of harm to
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the unborn child 149 REFERENCES INTERNATIONAL ATOMIC ENERGY AGENCY, Biological Dosimetry: Chromosomal Aberration Analysis for Dose Assessment, Technical Reports Series No. 260, IAEA, Vienna (1986). TURAI, I., The IAEA’s co-ordinated research project on biodosimetry, 1998– 2000, Int. J. Appl. Radiat. Isot. 52 (2000) 1113–1116. INTERNATIONAL ORGANIZATION FOR STANDARDIZATION, Radiation Protection-Performance Criteria for Service Laboratories Performing Biological Dosimetry by Cytogenetics, ISO 19238, ISO, Geneva (2004). INTERNATIONAL ORGANIZATION FOR STANDARDIZATION, Radiation Protection-Performance Criteria for Laboratories Performing Cytogenetic Triage for Assessment of Mass Casualties in Radiological or Nuclear Emergencies-General Principles and Application to Dicentric Assay, ISO 21243, ISO, Geneva (2008). FENECH, M., et al., HUMN Project: detailed description of the scoring criteria for the cytokinesis-block micronucleus assay using isolated human lymphocyte cultures, Mutat. Res. 534 (2003) 65–75. ROJAS-PALMA, C., et al. (Eds) TMT Handbook, Triage, Monitoring and Treatment of People Exposed to Ionizing Radiation Following a Malevolent Act, Lobo Media AS, Norway (2009). BERTHO, J.M., et al., New biological indicators to evaluate and monitor radiation-induced damage: an accident case report, Radiat. Res. 169 (2008). 543–550 INTERNATIONAL ATOMIC ENERGY AGENCY, Cytogenetic Analysis for Radiation Dose Assessment. A Manual, Technical Reports Series No. 405, IAEA, Vienna (2001). HUBBLE, J.H., Photon mass attenuation and energy — Absorption coefficients from 1 keV to 20 MeV, Int. J. Appl. Radiat. Isot. 33 (1982) 1269– 1290. GRAY, L.H., An ionization method for the absolute measurement of gamma-ray energy, Proc. Soc. Lond., A156 (1936) 578–596. DENG, W., MORRISON, D.P., GALE, K.L., LUCAS, J.N., Biological dosimetry of beta-ray exposure from tritium using chromosome translocations in human lymphocytes analyzed by fluorescence in situ hybridization, Radiat. Res. 150 (1998) 400–405. INTERNATIONAL COMMISSION ON RADIATION UNITS AND MEASUREMENTS, Quantities and Units in Radiation Protection Dosimetry, ICRU Rep. 51 (1993). STRAUME, T.,
|
{
"page_id": null,
"source": 7334,
"title": "from dpo"
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High-energy gamma rays in Hiroshima and Nagasaki: Implications for risk and W R , Health Phys. 69 (1995) 954–956. SCHMID, E., BAUCHINGER, M., LET dependence of dicentric yields in human lymphocytes induced by low doses of sparsely ionizing radiations and its implication for risk assessments, Health Phys. 74 (1998) 719–721. INTERNATIONAL ATOMIC ENERGY AGENCY, WORLD HEALTH ORGANIZATION, Generic procedures for medical response during a nuclear or radiological emergency, EPR-MEDICAL, IAEA, Vienna (2005). LEA, D.E., Actions of Radiation on Living Cells, 2nd edn, Cambridge University Press, Cambridge, UK (1956). 151 GOODHEAD, D.T., The initial physical damage produced by ionizing radiations, Int. J. Radiat. Biol. 56 (1989) 623–634. EDWARDS, A.A., LLOYD, D.C., PURROTT, R.J., “Dicentric chromosome aberration yield in human lymphocytes and radiation quality. A resume including recent results using alpha particles”, Microdosimetry (Proc. 7th Symp. Harwood Academic, Oxford, 1980), Vol. 2 (BOOZ, J., EBERT, H.G., HARTFIEL, H.D., Eds), Harwood Academic Publishers, Brussels and Luxembourg (1981) 1263–1273. INTERNATIONAL COMMISSION ON RADIOLOGICAL PROTECTION, The 2007 Recommendations of the International Commission on Radiological Protection, (Publication 103) Ann. ICRP Vol. 37, (2007). NOWELL, P.C., Phytohemagglutinin — An initiator of mitosis in cultures of normal human leukocytes, Cancer Res. 20 (1960) 462–466. CARSTAIRS, K., The human small lymphocyte — its possible pluripotential quality, Lancet (1962) 829–832. WUESTERMANN, P.R., CRONKITE, E.P., Physiological and pathophysiological aspects of the immune system contributing to abiomathematical model of lymphocytes, Stem Cells 13 Suppl. 1 (1995) S268– S275. CHENG, C. K-W., CHAN, J., CEMBROWSKI, G.S., VAN ASSENDELFT, O.W., Complete blood count reference interval diagrams derived from NHANES III: Stratification by age, sex, and race, Lab. Haematol. 10 (2004) 42–53. DAINIAK, N., WASELENKO, J.K., ARMITAGE, J.O., MacVITTEE, T.J., FARESE, A.M., The hematologist and radiation casualties, Hematology, 2003 (2003) 473–496. INTERNATIONAL ATOMIC
|
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"page_id": null,
"source": 7334,
"title": "from dpo"
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|
ENERGY AGENCY, WORLD HEALTH ORGANIZATION, Diagnosis and Treatment of Radiation Injuries, Safety Report Series No. 2, IAEA, Vienna (1998). McLEAN, A.R., MICHIE, C.A., In vivo estimates of division and death rates of human T lymphocytes, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 3707–3711. BOGEN, K.T., Reassessment of human peripheral T-lymphocyte life span deduced from cytogenetic and cytotoxic effects of radiation, Int. J. Radiat. Biol. 64 (1993) 195–204. OBE, G., BEEK, B., DUDIN, G., The human leukocyte test system: V. DNA synthesis and mitoses in PHA-stimulated 3-day cultures, Humangenetik 28 (1975) 295–302. PAULSON, J.R., LAEMMLI, U.K., The structure of histone-depleted metaphase chromosomes, Cell 12 (1977) 817–828. CREMER, C., et al., Nuclear architecture and the induction of chromosomal aberrations, Mutat. Res. 366 (1996) 97–116. SCHAFFER, L.G., TOMMERUP, N. (Eds), Recommendations of the International Standing Committee on Human Cytogenetic Nomenclature, ISCN, Karger, Basel (2005). MORTON, N.E., Parameters of the human genome, Proc. Natl. Acad. Sci. U.S.A. 88 (1991) 7474–7476. CARRANO, A.V., Induction of chromosomal aberrations in human lymphocytes by X rays and fission neutrons: Dependence on cell cycle stage, Radiat. Res. 63 (1975) 403–421. MÜLLER, H.J., Artificial transmutation of the gene, Science 66 (1927) 84–87. > 152 PAINTER, T.S., MÜLLER, H.J., Parallel cytology and genetics of induced translocations and deletions in Drosophila, J. Hered. 20 (1929) 287–298. SAX, K., Types and frequencies of chromosomal aberrations induced by X-rays, Cold Spring Harbor Symp. Quant. Biol. 9 (1941) 93–103. REVELL, S.H., “A new hypothesis for chromatid exchanges”, Radiobiology (Proc. Symp. Liège, 1954), Butterworths, London (1955) 243–253. CHADWICK, K.H., LEENHOUTS, H.P., The rejoining of DNA double-strand breaks and a model for the formation of chromosomal rearrangements, Int. J. Radiat. Biol. 33 (1978) 517–529. MOZUMDER, A., MAGEE J.L., Model of tracks of ionizing
|
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"page_id": null,
"source": 7334,
"title": "from dpo"
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|
radiations for radical reaction mechanisms, Radiat Res. 28 (1966) 203–14. GEORGAKILAS, A.G., Processing of DNA damage clusters in human cells: current status of knowledge, Mol. BioSyst., 4 (2008) 30–35. JAEA R&D REVIEW, Dependency of yield of DNA damage refractory to enzymatic repair on ionization and excitation density of radiation – Exploring the induction mechanism of clustered DNA damage, (2007), WARD, J.F., DNA damage produced by ionizing radiation in mammalian cells: Identities, mechanisms of formation and repairability, Prog. Nucleic Acids Res. Mol. Biol. 35 (1988) 95–135. RYDBERG, B., Radiation-induced DNA damage and chromatin structure, Acta Oncol. 40 (2001) 682–685. FORTINI, P., DOGLIOTTI, E., Base damage and single-strand break repair: mechanisms and functional significance of short and long-patch repair subpathways, DNA Repair 6 (2007) 398–409. CALDECOTT, K.W., Mammalian single-strand break repair: mechanisms and links with chromatin, DNA Repair 6 (2007) 443–453. IDE, H., et al., Repair of DNA-protein crosslink damage: Coordinated actions of nucleotide excision repair and homologous recombination, Nucleic Acids Symp. Ser. (Oxf.) 52 (2008) 57–58. JEGGO, P.A., LÖBRICH, M., Contribution of DNA repair and cell cycle checkpoint arrest to the maintenance of genomic stability, DNA Repair 5 (2006) 1192–1198. WYMAN, C., KANAAR, R., Homologous recombination: down to the wire, Curr. Biol. 14 (2004) R629–R631; HEFFERIN, M.L., TOMKINSON, A.E., Mechanism of DNA double-strand break repair by non-homologous end joining, DNA Repair 4 (2005) 639–648. ROTHKAMM, K., KRÜGER, I., THOMPSON, L.H., LÖBRICH, M., Pathways of DNA double-strand break repair during the mammalian cell cycle, Mol. Cell. Biol. 23 (2003) 5706–5715. SASAKI, M.S., Advances in the biophysical and molecular bases of radiation cytogenetics, Int. J. Radiat. Biol. 85 (2009) 26–47. SAVAGE, J.R.K., Annotation: Classification and relationships of induced chromosomal structural changes, J. Med. Genet. 13 (1976) 103–122. EVANS,
|
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"page_id": null,
"source": 7334,
"title": "from dpo"
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|
H.J., Chromosome aberrations induced by ionizing radiations, Int. Rev. Cytol. 13 (1962) 221–321. MESTRES, M., et al., Analysis of alpha-particle induced chromosome aberrations in human lymphocytes, using pan-centromeric and pan-telomeric probes, Int. J. Radiat. Biol. 80 (2004) 737–744. > 153 BENKHALED, L., et al., Analysis of gamma-rays induced chromosome aberrations: a fingerprint evaluation with a combination of pan-centromeric and pan-telomeric probes, Int. J. Radiat. Biol. 82 (2006) 869–875. GEBHART, E., Double minutes, cytogenetic equivalents of gene amplification in human neoplasia: a review, Clinical and Translational Oncology 7 (2005) 477–485. BLOOM, A.D., NEEL, J.V., CHOI, K.W., IDA, S., CHAGNON, N., Chromosome aberrations among the Yanomama Indians, Proc. Natl. Acad. Sci. USA 66 (1970) 920–927. AWA, A.A., NEEL, J.V., Cytogenetic “rogue” cells: what is their frequency, origin and evolutionary significance? Proc. Natl. Acad. Sci. USA 83 (1986) 1021–1025. SEVAN’KAEV, A.V., et al., Rogue cells observed in children exposed to radiation from the Chernobyl accident, Int. J. Radiat. Biol. 63 (1993) 361–367. NEEL, J.V., AWA, A.A., KODAMA, Y., NAKANO, M., MABUCHI K., Rogue lymphocytes among Ukranians not exposed to radioactive fall-out from the Chernobyl accident: the possible role of this phenomenon in oncogenesis, teratogenesis and mutagenesis, Proc. Natl. Acad. Sci. USA 89 (1992) 6973– 6977. NEEL, J.V., et al., Hypothesis: “rogue cell”-type chromosomal damage in lymphocytes is associated with infection with the JC human polyoma virus and has implications for oncopenesis, Proc. Nat. Acad. Sci. USA 93 (1996) 2690–2695. FOMINA, J., DARROUDI, F., BOEI, J. J., NATARAJAN, A.T., Discrimination between complete and incomplete chromosome exchanges in X-irradiated human lymphocytes using FISH with pan-centromeric and chromosome specific DNA probes in combination with telomeric PNA probe, Int. J. Radiat. Biol. 76 (2000) 807–813. BUCKTON, K.E., SMITH, P.G., COURT BROWN, W.M., “The estimation of lymphocyte lifespan
|
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"source": 7334,
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from studies on males treated with X-rays for ankylosing spondylitis”, Human Radiation Cytogenetics (Proc. Conf. Edinburgh, 1966) (EVANS, H.J., COURT BROWN, W.M., McLEAN, A.S., Eds), North-Holland, Amsterdam (1967) 106–114. EDWARDS, A.A., et al., Review of translocations detected by FISH for retrospective biological dosimetry applications, Radiat. Prot. Dosim. 113 (2005) 396–402. DURAN, M., BARQUINERO, J.F., CABALLIN, M.R, RIBAS, M., BARRIOS, L., Persistence of radiation-induced chromosome aberrations in a long-term cell culture, Radiat. Res. 171 (2009) 425–437. PANTELIAS, G.E., MAILLIE, H.D., A simple method for premature chromosome condensation induction in primary human and rodent cells using polyethylene glycol, Somat. Cell. Genet. 9 (1983) 533–547. PANTELIAS, G.E., MAILLIE, H.D., The use of peripheral blood mononuclear cell prematurely condensed chromosomes for biological dosimetry, Radiat. Res. 99 (1984) 140–150. KANDA, R., HAYATA, I., LLOYD, D.C., Easy biodosimetry for high-dose radiation exposure using drug-induced, prematurely condensed chromosomes, Int. J. Radiat. Biol. 75 (1999) 441–446. > 154 DURANTE, M., FURUSAWA, Y., GOTOH, E., A simple method for simultaneous interphase-metaphase chromosome analysis in biodosimetry, Int. J. Radiat. Biol. 74 (1998) 457–462. VYAS, R.C., DARROUDI, F., NATARAJAN, A.T., Radiation-induced chromosomal breakage and rejoining in interphase-metaphase chromosomes of human lymphocytes, Mutat. Res. 249 (1991) 29–35. PANTELIAS, G.E., MAILLIE, H.D., Direct analysis of radiation induced chromosome fragments and rings in unstimulated human peripheral blood lymphocytes by means of the premature chromosome condensation technique, Mutat. Res. 149 (1985) 67–72. DARROUDI, F., FOMINA, J., MEIJERS, M., NATARAJAN, A.T., Kinetics of formation of chromosome aberrations in X-irraidated human lymphocytes, using PCC and FISH, Mutat. Res. 404 (1998) 55–65. WALDREN, C.A., JOHNSON, R.T., Analysis of interphase chromosome damage by means of premature chromosome condensation after X and UV irradiation, Proc. Nat. Acad. Sci. USA 71 (1974) 1137–1141. GOTOH, E., DURANTE, M., Chromosome condensation outside
|
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"source": 7334,
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of mitosis: mechanisms and new tools, J. Cell Physiol. 209 (2006) 297–304. BLAKELY, W.F., PRASANNA, P.G.S., KOLANKO, C.J., PYLE, M.D., MOSBROOK, D.M., LOATS, A.S., RIPPEON, T.L., LOATS, H., Application of the premature chromosome condensation assay in simulated partial-body radiation exposures: evaluation of the use of an automated metaphase-finder, Stem Cells Suppl. 1 (1995) S223–S230. PRASANNA, P.G.S., ESCALADA, N.D., BLAKELY, W.F., Induction of premature chromosome condensation by a phosphatase inhibitor and a protein kinase in unstimulated human peripheral blood lymphocytes: a simple and rapid technique to study chromosome aberrations using specific whole-chromosome DNA hybridization probes for biological dosimetry, Mutat. Res. > 466 (2000) 131–141. PRASANNA, P.G.S., BLAKELY, W.F., “Premature chromosome condensation in human resting peripheral blood lymphocytes for chromosome aberration analysis using specific whole-chromosome DNA hybridization probes”, Methods in Molecular Biology, Vol. 291 Methods in Molecular Biology (KEOHAVONG, P., GRANT, S.G., Eds), Humana Press, Totowa, NJ (2004) 49–57. PATHAK, R., RAMAKUMAR, A., SUBRAMANIAN, U., and PRASANNA, P.G.S., Differential radio-sensitivities of human chromosomes 1 and 2 in interphase- and metaphase–spreads after 60Co γ-irradiation in one human donor, BMC Med. Phys. 9 (2009) 1–8. PANTELIAS, G.E., ILIAKIS, G.E., SAMBANI, C.D., POLITIS, G., Biological dosimetry of absorbed radiation by C-banding of interphase chromosomes in peripheral blood lymphocytes, Int. J. Radiat. Biol. 63 (1993) 349–354. LAMADRID, A.I., GARCIA, O., DELBOS, M., VOISIN, P., ROY L., PCC-ring induction in human lymphocytes exposed to gamma and neutron irradiation, J. Radiat. Res. 48 ( 2007) 1–6. GOTOH, E., TANNO, Y., TAKAKURA, K., Simple biodosimetry method for use in cases of high-dose radiation exposure that scores the chromosome number of Giemsa-stained drug-induced prematurely condensed chromosomes (PCC), Int. J. Radiat. Biol. 81 (2005) 33–40. > 155 COUNTRYMAN, P.I., HEDDLE, J.A., The production of micronuclei from chromosome aberrations in irradiated cultures of human
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}
|
lymphocytes, Mutat. Res. 41 (1976) 321–331. FENECH, J., MORLEY, A.A., Measurement of micronuclei in lymphocytes, Mutat. Res. 147 (1985) 29–36. FENECH, J., MORLEY, A.A., Cytokinesis-block micronucleus method in human lymphocytes: Effect of in vivo ageing and low-dose X-irradiation, Mutat. Res. 161 (1986) 193–198. VRAL, A., THIERENS, H., DE RIDDER, L., In vitro micronucleus-centromere assay to detect radiation-damage induced by low doses in human lymphocytes, Int. J. Radiat. Biol. 71 (1997) 61–68. FENECH, M., Cytokinesis-block micronucleus cytome assay, Nat. Protoc. 2 (2007) 1084–1104. WILLEMS, P., et al., Automated micronucleus (MN) scoring for population triage in case of large scale radiation events, Int. J. Radiat. Biol. 86 (2010) 2– 11. FENECH, M., The cytokinesis-block micronucleus cytome assay and its application in radiation biodosimetry, Hlth Phys. 98 (2009) 234–243. THOMAS, P., UMEKAGI, K., FENECH, M., Nucleoplasmic bridges are a sensitive measure of chromosome rearrangement in the cytokinesis-block micronucleus assay, Mutagenesis. 18 (2003) 187–94. TAMURA, H., SUGIYAMA, Y., SUGAHARA, T., Changes in the number of circulating lymphocytes with chromosomal aberrations following a single exposure of the pelvis to γ-irradiation in cancer patients, Radiat. Res. 59 (1974) 653–657. BREWEN, L.G., PRESTON, R.J., LITTLEFIELD, L.G., Radiation-induced human chromosome aberration yields following an accidental whole-body exposure to 60Co γ-rays, Radiat. Res. 49 (1972) 647–656. GRANATH, F., et al., Retrospective dose estimates in Estonian Chernobyl clean-up workers by means of FISH, Mutat. Res. 369 (1996) 7–12. WORLD HEALTH ORGANISATION, Guidance on regulations for the Transport of Infectious Substances, 009-2010 WHO/HSE/EPR/2008.10, WHO, Geneva (2008). WILKINS, R.C., et al., Interlaboratory comparison of the dicentric chromosome assay for radiation biodosimetry in mass casualty events, Radiat. Res. 169 (2008) 551–560. FABRY, L., LEMAIRE, M., Dose response relationships for radiation induced chromosome aberrations in human lymphocytes in vivo and in
|
{
"page_id": null,
"source": 7334,
"title": "from dpo"
}
|
vitro ,Strahlentherapie 162 (1986) 63–67. LLOYD, D.C., EDWARDS, A.A., PROSSER, J.S., CORP, M.J., The dose response relationship obtained at constant irradiation times for the induction of chromosome aberrations in human lymphocytes by cobalt-60 gamma rays, Radiat. Environ. Biophys. 23 (1984) 179–189. MYSKA, J.C., et al., Arrays for Use at the Cobalt Irradiation Facility, TR97-2, Armed Froces Radiobiology Research Institute (1997), EDWARDS, A.A., LLOYD, D.C., PURROT, R.J., Radiation induced chromosome aberrations and the Poisson distribution, Radiat. Environ. Biophys. 16 (1979) 89–100. > 156 RAO, C.R., CHAKRAVARTI, I.M., Some small sample tests of significance for a Poisson distribution, Biometrics 12 (1956) 264–282. SAVAGE, J.R.K., Sites of radiation induced chromosome exchanges, Curr. Top. Radiat. Res. 6 (1970) 129–194. LLOYD, D.C., EDWARDS, A.A., “Chromosome aberrations in human lymphocytes: effect of radiation quality, dose and dose rate”, Radiation-Induced Chromosome Damage in Man (ISHIHARA, T., SASAKI, M.S., Eds), Alan R. Liss, New York (1983) 23–49. BARQUINERO, J.F., et al., Establishment and validation of a dose-effect curve for gamma-rays by cytogenetic analysis, Mutat. Res. 326 (1995) 65–69. DI GIORGIO, M., et al., Chromosome aberrations induced in human lymphocytes by heavy charged particles in track segment mode, Radiat. Prot. Dosim. 108 (2004) 47–53. PAPWORTH, D.G., Curve fitting by maximum likelihood, Radiat. Bot. 15 (1975) 127–140. MERKLE, W., Statistical methods in regression and calibration analysis of chromosome aberration data, Radiat. Environ. Biophys. 21 (1983) 217–233. LLOYD, D.C., PURROTT, R.J., REEDER, E.J., The incidence of unstable chromosome aberrations in peripheral blood lymphocytes from unirradiated and occupationally exposed people, Mutat. Res. 72 (1980) 523–532. SIGURDSON, AJ., et al., International study of factors affecting human chromosome translocations, Mutat. Res. 652 (2008) 112–121. FENECH, M., The cytokinesis-block micronucleus technique: a detailed description of the method and its application to
|
{
"page_id": null,
"source": 7334,
"title": "from dpo"
}
|
genotoxicity studies in human populations, Mutat. Res. 285 ( 1993) 35–44. PETERSON, L.E., PIRLS, Poisson iteratively reweighted least squares computer program for additive, multiplicative, power, and non-linear models, J. Stat. Soft. 2 (1997) 1–28. AINSBURY, E.A., BARQUINERO J.F., Biodosimetric tools for a fast triage of people accidentally exposed to ionising radiation: statistical and computational aspects, Ann. Ist Super Sanita, 45 (2009) 307–12. DEPERAS, J., et al., CABAS: a freely available PC program for fitting calibration curves in chromosome aberration dosimetry, Radiat. Prot. Dosim. > 124 (2007) 115–123. AINSBURY, E.A., LLOYD, D.C., Dose estimation software for radiation biodosimetry, Hlth Phys. 98 (2010) 290–295. PURROTT, R.J., VULPIS, N., LLOYD, D.C., Chromosome dosimetry: The influence of culture media on the proliferation of irradiated and unirradiated human lymphocytes, Radiat. Prot. Dosim. 1 (1981) 203–208. PERRY, P., WOLFF, S., New Giemsa method for the differential staining of sister chromatids, Nature 251 (1974) 156–158. LAMBERT, B., HANSSON, K., LINDSTEN, J., STEN, M., WERELIUS, B., Bromodeoxyuridine-induced sister chromatid exchanges in human lymphocytes, Hereditas 93 (1976) 163–174. PROSSER, J.S., MOQUET, J.E., The effect of blood storage on differential chromosome staining of human lymphocytes, Experientia 39 (1983) 778–780. HAYATA, I., KAJIMA, J., OKABE, N., Distribution of metaphases in the 1st cell-cycle for automated-system in radiation-dosimetry, Radiat. Phys. Chem. > 39 (1992) 517–520. > 157 WILKINSON, D.T., et al., Canadian biodosimetry capacity for response to radiation emergencies, Radiat. Meas. 42 (2007) 1128–1132. STRICKLIN, D., et al., An initial limited biodosimetry inter-comparison exercise: FOI and DRDC Ottawa, Radiat. Meas. 42 (2007) 1133–1137. PURROTT, R.J., VULPIS, N., LLOYD, D.C., The influence of incubation temperature on the rate of human lymphocyte proliferation in vitro ,Experientia 37 (1981) 407–408. MOORHEAD, P.S., NOWELL, P.C., MELLMANN, W.J., BATTIPS, D.M., HUNGERFORD, D.A., Chromosome preparations
|
{
"page_id": null,
"source": 7334,
"title": "from dpo"
}
|
of leukocytes cultured from human peripheral blood, Exp. Cell Res. 20 (1960) 613–616. HUNGERFORD, D.A., Leukocytes cultured from small inocula of whole blood and the preparation of metaphase chromosomes by treatment with hypotonic KCl, Stain Technol. 40 (1965) 333–338. HAYATA, I., et al., Robot system for preparing lymphocyte chromosomes, J. Radiat. Res. 33 Suppl. (1992) S231–S241. McFEE, A.F., SAYER, A.M., SALOMAA, S.I., LINDHOLM, C., LITTLEFIELD, L.G., Methods for improving the yield and quality of metaphase preparations for FISH probing of human lymphocyte chromosomes, Environ. Molecul. Mutagen. 29 (1997) 98–104. SASAKI, M.S., “Radiation damage and its repair in the formation of chromosome aberrations in human lymphocytes”, Mutagen-Induced Chromosome Damage in Man (EVANS, H.J., LLOYD, D.C., Eds), Edinburgh University Press, Edinburgh 62–76 (1978). SCOTT, D., LYONS, C.Y., Homogeneous sensitivity of human peripheral blood lymphocytes to radiation-induced chromosome damage, Nature 279 (1979) 756–758. KOLIN-GERRESHEIM, J., BAUCHINGER, M., Dependence of the frequency of harlequin-stained cells on BrdU concentration in human lymphocyte cultures, Mutat. Res. 91 (1981) 251–254. HAYATA, I., Removal of stainable cytoplasmic substances from cytogenetic slide preparations, Biotechn. Histochem. 68 (1993) 150–152. LATT, S.A., Microfluorometric detection of deoxyribonucleic acid replication in human metaphase chromosomes, Proc. Nat. Acad. Sci. U.S.A. 70 (1973) 3395–3399. FERNANDES, T.S., LLOYD, D., AMARAL, A., A comparison of different cytological stains for biological dosimetry, Int. J. Radiat. Biol. 84 (2008) 703– 711. SAVAGE, J.R.K., PAPWORTH, D.G., Constructing a 2B calibration curve for retrospective dose reconstruction, Radiat. Prot. Dosim. 88 (2000) 69–76. SZ ŁUI ŃSKA, M., EDWARDS, A., LLOYD, D., Presenting statistical uncertainty on cytogenetic dose estimates, Radiat. Prot. Dosim. 123 (2007) 443–449. CROW, E.L., GARDNER, R.S., Confidence intervals for the expectation of a Poisson variable, Biometrika 46 (1959) 441–453. SCHENKER, N., GENTLEMAN, J.F., On judging the significance
|
{
"page_id": null,
"source": 7334,
"title": "from dpo"
}
|
of differences by examining the overlap between confidence intervals, Am. Stat. > 55 (2001) 182–182. AUSTIN, P.C., HUX, J.E., A brief note on overlapping confidence intervals, J. Vas. Surg. (2002) 194–195. > 158 BRAME, R.S., GROER, P.G., Bayesian methods for chromosome dosimetry following a criticality accident, Radiat. Prot. Dosim. 104 (2003) 61–63. VOISIN P., et al., Criticality accident dosimetry by chromosomal analysis, Radiat. Prot. Dosim. 110 (2004) 443–447. DOLPHIN, G.W., “Biological dosimetry with particular reference to chromosome aberration analysis. A review of methods”, Handling of Radiation Accidents (Proc. Int. Symp. Vienna, 1969), IAEA, Vienna (1969) 215–224 LLOYD, D.C., PURROTT, R.J., DOLPHIN, G.W., Chromosome aberration dosimetry using human lymphocytes in simulated partial body irradiation, Phys. Med. Biol. 18 (1973) 421–431. BARQUINERO, J.F., et al., Biological dosimetry in simulated in vitro partial irradiations, Int. J. Radiat. Biol. 71 (1997) 435–440. VOISIN, P., et al., Mathematical methods in biological dosimetry: the 1996 Iranian accident, Int. J. Radiat. Biol. 76 (2000) 1545–1554. SASAKI, M.S., MIYATA, H., Biological dosimetry in atom bomb survivors, Nature 220 (1968) 1189–1193. BUCKTON, K.E., HAMILTON, G.E., PATON, L., LANGLANDS, A.O., “Chromosome aberrations in irradiated ankylosing spondylitis patients”, Mutagen-Induced Chromosome Damage in Man, Edinburgh University Press, Edinburgh (1978) 142–150. BUCKTON, K.E., Chromosome aberrations in patients treated with X-irradiation for ankylosing spondylitis, Radiation-Induced Chromosome Damage in Man, Alan R. Liss, New York (1983) 491–511. AWA, A., Analysis of chromosome aberrations in atom bomb survivors for dose assessment: studies at the Radiation Effects Research Foundation from 1968–1993, Stem Cells 15 Suppl. 2 (1997) S163–S173. LEA, D.E., CATCHESIDE, D.G., The mechanism of the induction by radiation of chromosome aberrations in Tradescantia, J. Genet. 44 (1942) 216– 245. BAUCHINGER, M., SCHMID, E., DRESP, J., Calculation of the dose-rate dependence of the dicentric
|
{
"page_id": null,
"source": 7334,
"title": "from dpo"
}
|
yield after 60Co-irradiation of human lymphocytes, Int. J. Radiat. Biol. 35 (1979) 229–233. DUFRAIN, R.J., LITTLEFIELD, L.G., JOINER, E.E., FROME, E.L., “ In vitro human cytogenetic dose-response systems”, The Medical Basis for Radiation Accident Preparedness, Elsevier North-Holland, Amsterdam (1980) 358–374. INTERNATIONAL ATOMIC ENERGY AGENCY, The Radiological Accident in Goiânia, IAEA, Vienna (1988). INTERNATIONAL ATOMIC ENERGY AGENCY, Dosimetric and Medical Aspects of the Radiological Accident in Goiânia in 1987, IAEA-TECDOC-1009, IAEA, Vienna (1998). EUROPEAN COMMISSION, INTERNATIONAL ATOMIC ENERGY AGENCY, WORLD HEALTH ORGANIZATION, One Decade after Chernobyl — Summing up the Consequences of the Accident (Proc. Int. Conf. Vienna, 1996), IAEA, Vienna (1996). PROSSER, J.S., LLOYD, D.C., EDWARDS, A.A., STATHER, J.W., The induction of chromosome aberrations in human lymphocytes by exposure to tritiated water in vitro, Radiat. Prot. Dosim. 4 (1983) 21–26. > 159 PRESTON, R.J., BREWEN, J.G., GENGOZIAN, N., Persistence of radiation-induced chromosome aberrations in marmoset and man, Radiat. Res. 60 (1974) 516–524. VOISIN, P., LLOYD, D., EDWARDS, A., Chromosome aberrations scoring for biological dosimetry in a criticality accident, Radiat. Prot. Dosim. 70 (1997) 467–470. BASSON, J.K., HANEKOM, A.P., COETZEE, F.C., LLOYD, D.C., Health Physics Evaluation of an Accident Involving Acute Overexposure to aRadiography Source, Rep. PEL-279, Pelindaba, South Africa (1981). STEPHAN, G., HADNAGY, W., HAMMERMAIER, C., IMHOF, U., Biologically and physically recorded doses after an accidental exposure to 60 Co γ-rays, Hlth Phys. 44 (1983) 409–411. ISHIHARA, T., et al., Chromosome aberrations in persons accidentally exposed to 192 Ir gamma rays, J. Radiat. Res. 14 (1973) 328–335. INTERNATIONAL ATOMIC ENERGY AGENCY, The Radiological Accident in Istanbul, IAEA, Vienna (2000). TURAI, I., GÜNALP, B., LLOYD, D., COSSET, J.M., “Medical aspects of the radiological accident in Istanbul”, (Proc. 8th WHO/REMPAN Conf., NRPB), Oxford (2000). LLOYD, D.C., EDWARDS, A.A., PROSSER,
|
{
"page_id": null,
"source": 7334,
"title": "from dpo"
}
|
J.S., Accidental intake of tritiated water: a report of two cases, Radiat. Prot. Dosim. 15 (1986) 191–196. PINKEL, D., STRAUME, T., GRAY, J.W., Cytogenetic analysis using quantitative high-sensitivity fluorescence hybridisation, Proc. Nat. Acad. Sci. U.S.A. 83 (1986) 2934–2938. GREULICH, K.M., et al., Rapid detection of radiation-induced chromosomal aberrations in lymphocytes and hematopoietic progenitor cells by mFISH, Mutat. Res. 452 (2000) 73–81. MANTI, L., et al., Rearrangements in human chromosome 1 visualized by arm-specific probes in the progeny of blood lymphocytes exposed to iron ions, Adv. Space Res. 39 (2007) 1066–1069. CHUDOBA, I., et al., High resolution multicolor-banding: a new technique for refined FISH analysis of human chromosomes, Cytogenet. Cell Genet. 84 (1999) 156–160. HORSTMANN, M., DURANTE, M., JOHANNES, C., OBE, G., Chromosomal intrachanges induced by swift iron ions, Adv. Space Res. (2005) 35 276–279. BRENNER, D.J., et al., Biomarkers specific to densely-ionising (high LET) radiations, Radiat. Prot. Dosim. 97 (2001) 69–73. DARROUDI, F., et al., A new tool to characterize primary cervical carcinomas: to assess hall-marks and stage specificity, Cancer Lett. 287 (2010) 67–74. PRIEUR, M., et al., Acquired chromosome rearrangements in human lymphocytes: effect of ageing, Human Genet. 79 (1988) 147–150. DARROUDI, F., Use of FISH translocations analyses for retrospective biological dosimetry: how stable are stable chromosome aberrations?, Radiat. Prot. Dosim. 88 (2000) 101–109. LUCAS, J.N., et al., Rapid translocation frequency analysis in human decades after exposure to ionizing radiation, Int. J. Radiat. Biol. 62 (1992) 53–63. 160 TAWN, E.J., The non-random occurrence of exchanges involving chromosomes 7 and 14 in human lymphocytes: a prospective study of control individuals, Mutat. Res. 199 (1988) 215–220. KODAMA, Y., et al., Estimation of minimal size of translocated chromosome segments detectable by fluorescence in situ hybridisation, Int. J. Radiat. Biol. >
|
{
"page_id": null,
"source": 7334,
"title": "from dpo"
}
|
71 (1997) 35–39. SAVAGE, J.R., TUCKER, J.D., Nomenclature systems for FISH-painted chromosome aberrations, Mutat. Res. 366 (1996) 153–161. LINDHOLM, C., EDWARDS A.A., Long-term persistence of translocations in stable lymphcytes from victims of a radiological accident, Int. J. Radiat. Biol. 80 (2004) 559–566. TUCKER, J.D., et al., PAINT: A proposed nomenclature for structural aberrations detected by whole chromosome painting, Mutat. Res. 347 (1995) 21–24. SAVAGE, J.R.K., SIMPSON, P., On the scoring of FISH painted chromosome exchange aberrations, Mutat. Res. 307 (1994) 345–353. SAVAGE, J.R.K., SIMPSON, P., FISH ‘painting’ patterns resulting from complex exchanges, Mutat. Res. 312 (1994) 51–60. KNEHR, S., ZITZELSBERGER, H., BAUCHINGER, M., FISH-based analysis of radiation-induced chromosomal aberrations using different nomenclature systems, Int. J. Radiat. Biol. 73 (1998) 135–141. NATARAJAN, A.T., VYAS, R.C., DARROUDI, F., VERMEULEN, S., Frequencies of x-ray induced chromosome translocations in human peripheral lymphocytes as detected by in situ hybridization using chromosome-specific DNA libraries, Int. J. Radiat. Biol. 61 (1992) 199–203. SIMPSON, P.J., SAVAGE, J.R.K., Dose–response curves for simple and complex chromosome aberrations induced by X-rays and detected using fluorescence in situ hybridisation, Int. J. Radiat. Biol. 69 (1996) 429–436. SIMPSON, P.J., PAPWORTH, D.G., SAVAGE, J.R., X-ray-induced simple, pseudosimple and complex exchanges involving two distinctly painted chromosomes, Int. J. Radiat. Biol. 75 (1999) 11–18. CORNFORTH, M.N., Analyzing radiation-induced complex chromosome rearrangements by combinatorial painting, Radiat. Res. 155 (2001) 643–659. LUCAS, J.N., DENG, W., Views on issues in radiation biodosimetry based on chromosome translocations measured by FISH, Radiat. Prot. Dosim. 88 (2000) 77–86. KNEHR, S., ZITZELSBERGER, H., BRASELMANN, H., NAHRSTEDT, U., BAUCHINGER, M., Chromosome analysis by fluorescence in situ hybridization: further indications for a non-DNA-proportional involvement of single chromosomes in radiation-induced structural aberrations, Int. J. Radiat. Biol. 70 (1996) 385–392. BARQUINERO, J.F., KNEHR, S.,
|
{
"page_id": null,
"source": 7334,
"title": "from dpo"
}
|
BRASELMANN, H., FIGELI, M., BAUCHINGER, M., DNA-proportional distribution of radiation-induced chromosome aberrations analysed by fluorescence in situ hybridization painting of all chromosomes of a human female karyotype, Int. J. Radiat. Biol. > 74 (1998) 315–323. CIGARRAN, S., et al., Relationship between the DNA content of human chromosomes and their involvement in radiation-induced structural aberrations analysed by painting, Int. J. Radiat. Biol. 74 (1998) 449–455. > 161 WU, H., DURANTE, M., LUCAS, J.N., Relationship between radiation-induced aberrations in individual chromosomes and their DNA content: effects of interaction distance, Int. J. Radiat. Biol. 77 (2001) 781–786. CORNFORTH, M.N., et al., Chromosomes are predominantly located randomly with respect to each other in interphase human cells, J. Cell. Biol. > 159 (2002) 237–244. WHITEHOUSE, et al., Translocation yields in peripheral blood lymphocytes from control populations, Int. J. Radiat. Biol. 81 (2005) 139–145. PRESSL, S., EDWARDS, A. STEPHAN, G., The influence of age, sex and smoking habits on the background level of FISH-detected translocations, Mutat. Res. 442 (1999) 89–95. RAMSEY, M.J., et al., The effects of age and lifestyle factors on the accumulation of cytogenetic damage as measured by chromosome painting, Mutat. Res. 338 (1995) 95–106. BADR, F.M., HUSSAIN, F.H., Chromosomal aberrations in chronic male alcoholics, Alcohol. Clin. Exp. Res. 6 (1982) 122–129. BURIM, R.V., et al., Clastogenic effect of ethanol in chronic and abstinent alcoholics, Mutat. Res. 560 (2004) 187–198. BOTHWELL, A.M., WHITEHOUSE, C.A., TAWN, E.J., The application of FISH for chromosome aberration analysis in relation to radiation exposure, Radiat. Prot. Dosim. 88 (2000) 7–14. TAWN, E.J., CARTMELL, C.L., The effect of smoking on the frequencies of asymmetrical and symmetrical chromosome exchanges in human lymphocytes, Mutat. Res. 224 (1989) 151–156. VAN DIEMEN, P.C.M., MAASDAM, D., VERMEULEN, S., DARROUDI, F., NATARAJAN, A.T., Influence of
|
{
"page_id": null,
"source": 7334,
"title": "from dpo"
}
|
smoking habits on the frequencies of structural and numerical chromosomal aberrations in human peripheral blood lymphocytes using the fluorescence in situ hybridization (FISH) technique, Mutagenesis 10 (1995) 487–495. TUCKER, J.D., et al., Multi-endpoint biological monitoring of phosphine workers, Mutat. Res. 536 (2003) 7–14. MAENG, S.H., et al., Chromosome aberration and lipid peroxidation in chromium-exposed workers, Biomarkers 9 (2004) 418–434. BESKID, O., et al., Chromosomal aberrations by fluorescence in situ hybridization (FISH) — Biomarker of exposure to carcinogenic PAHs, Mutat. Res. 620 (2007) 62–70. PLUTH, J.M., RAMSEY, M.J., TUCKER, J.D., Role of maternal exposures and newborn genotypes on newborn chromosome aberration frequencies, Mutat. Res. > 465 (2000) 101–111. STEENLAND, K., et al., Thyroid hormones and cytogenetic outcomes in backpack sprayers using ethylenebis (dithiocarbamate) (EBDC) fungicides in Mexico, Environ. Hlth Perspect. 105 (1997) 1126–1130. KIM Y. K., et al., Chromosomal aberrations in workers exposed to low levels of benzene: association with genetic polymorphisms, Pharmacogenetics 14 (2004) 453–463. SMITH, M. T., et al., Increased translocations and aneusomy in chromosomes 8 and 21 among workers exposed to benzene, Cancer Res. 58 (1998) 2176– 2181. > 162 ZHANG, L., et al., Aberrations in chromosomes associated with lymphoma and therapy-related leukemia in benzene-exposed workers, Environ. Mol. Mutagen. 48 (2007) 467–474. BESKID, O., DUSEK, Z., SOLANKSY, I., SRAM, R.J., The effects of exposure to different clastogens on the pattern of chromosomal aberrations detected by FISH whole chromosome painting in occupationally exposed individuals, Mutat. Res. 594 (2006) 20–29. SRAM R.J., et al., Environmental exposure to carcinogenic polycyclic aromatic hydrocarbons-The interpretation of cytogenetic analysis by FISH, Toxicol. Lett. 172 (2007) 12–20. DOHERTY, A.T., et al., Increased chromosome translocations and aneuploidy in peripheral blood lymphocytes of patients having revision arthroplasty of the hip, J. Bone Joint Surg. 83 (2001)
|
{
"page_id": null,
"source": 7334,
"title": "from dpo"
}
|
1075–1081. DULOUT, F.N., et al., Chromosomal aberrations in peripheral blood lymphocytes from native Andean women and children from northwestern Argentina exposed to arsenic in drinking water, Mutat. Res. 370 (1996) 151–158. NATARAJAN, A. T., et al., 137 Cs-induced chromosome aberrations analyzed by fluorescence in situ hybridization: eight years follow up of the Goiânia radiation accident victims, Mutat. Res. 400 (1998) 299–312. SAKAMOTO-HOJO, E.T., NATARAJAN, A.T., CURADO, M.P., Chromosome translocations in lymphocytes from individuals exposed to 137 Cs 7.5 years after the accident in Goiânia (Brazil), Radiat. Prot. Dosim. 86 (1999) 25–32. BAUCHINGER, M., et al., Collaborative exercise on the use of FISH chromosome painting for retrospective biodosimetry of Mayak nuclear-industrial personnel, Int. J. Radiat. Biol. 77 (2001) 259–267. SALASSIDIS, K., et al., Chromosome painting in highly irradiated Chernobyl victims: a follow-up study to evaluate the stability of symmetrical translocations and the influence of clonal aberrations for retrospective dose estimation, Int. J. Radiat. Biol. 68 (1995) 257–262. LINDHOLM, C., TEKKEL, M., VEIDEBAUM, T., ILUS, T. SALOMAA, S., Persistence of translocations after accidental exposure to ionizing radiation, Int. J. Radiat. Biol. 74 (1998) 565–571. LINDHOLM, C., et al., Intercomparison of translocation and dicentric frequencies between laboratories in a follow-up of the radiological accident in Estonia, Int. J. Radiat. Biol. 78 (2002) 883–890. DURAN, A., et al., Suitability of FISH painting techniques for the detection of partial-body irradiations for biological dosimetry, Radiat. Res. 157 (2002) 461–468. GUERRERO-CARBAJAL, Y.C., MOQUET, J.E., EDWARDS, A.A., LLOYD, D.C., The persistence of FISH translocations for retrospective biological dosimetry after simulated whole or partial body irradiation, Radiat. Prot. Dosim. 76 (1998) 159–168. SEVAN’KAEV, A.V., et al., A cytogenetic follow-up of some highly irradiated victims of the Chernobyl accident, Radiat. Prot. Dosim. 113 (2005) 152–161. LINDHOLM, C., SALOMAA, S.,
|
{
"page_id": null,
"source": 7334,
"title": "from dpo"
}
|
Dose assessment of past accidental or chronic exposure using FISH chromosome painting, Radiat. Prot. Dosim. 88 (2000) 21–25. 163 MULLER, I., et al., Time-course of radiation-induced chromosomal aberrations in tumor patients after radiotherapy, Int. J. Radiat. Oncol. Biol. Phys. 63 (2005) 1214–1220. XUNCLA, M., et al., Cytogenetic damage induced by radiotherapy. Evaluation of protection by amifostine and analysis of chromosome aberrations persistence, Int. J. Radiat. Biol. 84 (2008) 243–251. SCHMIDBERGER, H., et al., Reciprocal translocations in patients with testicular seminoma before and after radiotherapy, Int. J. Radiat. Oncol. Biol. Phys. 50 (2001) 857–864. GEBHART, E., NEUBAUER, S., SCHMITT, G., BIRKENHAKE, S. and DUNST, J., Use of a three-colour chromosome in situ suppression technique for the detection of past radiation exposure, Radiat. Res. 145 (1996) 47–52. PALA, F.S., MOQUET, J.E., EDWARDS, A.A., LLOYD, D.C., In vitro transmission of chromosomal aberrations through mitosis in human lymphocytes, Mutat. Res. 474 (2001) 139–146. GARDNER, S.N., TUCKER, J. D., The cellular lethality of radiationinduced radiation-induced chromosome translocations in human lymphocytes, Radiat. Res. 157 (2002) 539–552. HANDE, M.P., NATARAJAN, A.T., Induction and persistence of cytogenetic damage in mouse splenocytes following whole-body X irradiation analysed by fluorescence in situ hybridization. IV. Dose response, Int. J. Radiat. Biol. 74 (1998) 441–448. XIAO, Y., DARROUDI, F., GRIGOROVA, M., NATARAJAN, A.T., Induction and persistence of chromosomal exchanges in mouse bone marrow cells following whole-body exposure to X-rays, Int. J. Radiat. Biol. 75 (1999) 1119–1128. ROMM, H., STEPHAN, G., Dose dependency of FISH-detected translocations in stable and unstable cells after Cs gamma irradiation of human lymphocytes in vitro , Cytogenet. Genome Res. 104 (2004) 162–167. RODRIGUEZ, P., et al., Analysis of translocations in stable cells and their implications in retrospective biological dosimetry, Radiat. Res. 162 (2004) 31-38. BAUCHINGER, M., SCHMID,
|
{
"page_id": null,
"source": 7334,
"title": "from dpo"
}
|
E., ZITZELBERGER, H., BRASELMANN, H., AHRSTEDT, U., Radiation-induced chromosomal aberrations analyzed by two colour fluorescence in situ hybridization with composite whole chromosome-specific DNA probes and a pancentromeric DNA probe, Int. J. Radiat. Biol. 64 (1993) 179–84. FERNANDEZ, J. L., et al., X-ray biological dosimetry performed by selective painting of human chromosomes 1 and 2, Int. J. Radiat. Biol. 67 (1995) 295-302. FINNON, P., LLOYD, D.C., EDWARDS, A.A., Fluorescence in situ hybridization detection of chromosomal aberrations in human lymphocytes: applicability to biological dosimetry, Int. J. Radiat. Biol. (1995) 68 429–435. HSIEH, W.A., et al., Alpha coefficient of dose-response for chromosome translocations measured by FISH in human lymphocytes exposed to chronic 60 Co gamma rays at body temperature, Int. J. Radiat. Biol. 75 (1999) 435–439. DARROUDI, F., NATARAJAN, A.T., Application of FISH chromosome painting assay for dose reconstruction: State of the art and current views, Radiat. Prot. Dosim. 88 (2000) 51–58. 164 SALISSIDIS, K., SCHMID, E., PETER, R.U., BRASELMANN, H., BAUCHINGER, M., Dicentric and translocation analysis for retrospective dose estimation in humans exposed to ionising radiation during the Chernobyl power plant accident, Mutat. Res. 311 (1994) 39–48. LLOYD, D.C., et al., “Retrospective dosimetry by chromosomal analysis”, The Radiological Consequences of the Chernobyl Accident (Proc. Mtg Minsk, 1996), Rep. Eur 16544 EN, (1996) 965–973. SNIGIRYOVA, G., BRASELMANN, H., SALASSIDIS, K., SHEVCHENKO, V., BAUCHINGER, M., Retrospective biodosimetry of Chernobyl clean-up workers using chromosome painting and conventional chromosome analysis, Int. J. Radiat. Biol. 71 (1997) 119–127. DARROUDI, F., NATARAJAN, A.T., “Biological dosimetric studies in the Chernobyl radiation accident, on populations living in the contaminated areas (Gomel regions) and in Estonian clean-up workers, using FISH technique”, The Radiological Consequences of the Chernobyl Accident (Proc. Mtg Minsk, 1996) Eur 16544 EN, (1996) 1067–1072. SALISSIDIS, K., et al., Analysis
|
{
"page_id": null,
"source": 7334,
"title": "from dpo"
}
|
of symmetrical translocations for retrospective biodosimetry in radiation workers of the Mayak nuclear-industrial complex (Southern Urals) using FISH-chromosome painting, Int. J. Radiat. Biol. 74 (1999) 431–439. BAUCHINGER, M., et al., FISH-based analysis of stable translocations in a Techa river population, Int. J. Radiat. Biol. 73 (1998) 605–612. TUCKER, J.D., et al., Biological dosimetry of radiation workers at the Sellafield nuclear facility, Radiat. Res. 148 (1997) 216–226. NAKAMURA, N., MIYAZAWA, C., SAWADA, S., AKIYAMA, M., AWA, A.A., A close correlation between spin resonance (ESR) dosimetry from tooth enamel and cytogenetic dosimetry from lymphocytes of Hiroshima atomic-bomb survivors, Int. J. Radiat. Biol. 73 (1998) 619–627. LLOYD, D.C., MOQUET, J.E., ORAM, S., EDWARDS, A.A., LUCAS, J.N., Accidental intake of tritiated water: A cytogenetic follow-up case on translocation stability and dose reconstruction, Int. J. Radiat. Biol. 73 (1998) 543–547. RAMALHO, A.T., NASCIMENTO, A.C.H., NATARAJAN, A.T., Dose assessments by cytogenetic analysis in the Goiânia (Brazil) radiation accident, Radiat. Prot. Dosim. 25 (1988) 97–100. STRAUME, T., et al., Novel biodosimetry methods applied to victims of the Goiânia accident, Hlth Phys. 60 (1991) 71–76. STEPHAN, G., PRESSL, S., Chromosome aberrations in human lymphocytes analysed by fluorescence in situ hybridization after in vitro irradiation, and in radiation workers, 11 years after an accidental radiation exposure, Int. J. Radiat. Biol. 71 (1997) 293–299. LINDHOLM, C., et al., Biodosimetry after accidental radiation exposure by conventional chromosome analysis and FISH, Int. J. Radiat. Biol. 70 (1996) 647– 656. INTERNATIONAL ATOMIC ENERGY AGENCY, The Radiological Accident in Lilo, IAEA, Vienna (2000). ROY, L., et al., Study of the tools available in biological dosimetry to estimate the dose in cases of accidental complex overexposure to ionizing radiation: the Lilo accident, Int. J. Radiat. Biol. 82 (2006) 39–48. DARROUDI, F., et al., Detection
|
{
"page_id": null,
"source": 7334,
"title": "from dpo"
}
|
of total and partial body irradiation in a monkey model: a comparative study of chromosomal aberration, micronuclei and > 165 premature chromosome condensation assays, Int. J. Radiat. Biol. 74 (1998) 207-215. PRASANNA, P.G.S., KOLANKO, C.J., GERSTENBERG, H.M., BLAKELY, W.F., Premature chromosome condensation assay for biodosimetry: Studies with fission neutrons, Hlth Phys. 72 (1997) 594–600. TERZOUDI, G.I., PANTELIAS G.E., Cytogenetic methods for biodosimetry and risk individualization after exposure to ionizing radiation, Radiat. Prot. Dosim . > 122 (2006) 513–520. DARROUDI, F., NATARAJAN, A.T., “Premature chromosome condensation, a novel method for biological dosimetry”, High Levels of Natural Radiation (Proc. 10th Int. Congr. Vienna, 1993), IAEA, Vienna (1993) 479–485. KANDA, R., MINAMIHISAMATSU, M., HAYATA, I., Dynamic analysis of chromosome aberrations in three victims of the Tokai-mura criticality accident, Int. J. Radiat. Biol. 78 (2002) 857–862. HAYATA, I., KANDA, R., MINAMIHISAMATSU, M., FURUKAWA, A., SASAKI, M.S., Cytogenetical dose estimation for 3 severely exposed cases in the JCO criticality accident in Tokai-mura, J. Radiat. Res. 42 Suppl. (2001) S149– S155. ISHIGURE, N., ENDO, A., YAMAGUCHI, Y., KAWACHI, K., Calculation of the absorbed dose for the overexposed patients at the JCO criticality accident in Tokai-mura, J. Rad. Res. 42 Suppl. (2000) S137–S148. BAUCHINGER, M., BRASELMANN, H., “Use of micronuclei in biological dosimetry of absorbed radiation dose”, Chromosome Aberrations — Basic and Applied Aspects, Springer-Verlag, Berlin (1989) 202–211. KOTELES, G.J., The human lymphocyte micronucleus assay. A review on its applicabilities in occupational and environmental medicine, Centr. Europ. J. Occup. Environ. Med. 2 (1996) 12–30. FENECH, M., The in vitro micronucleus technique, Mutat. Res. 455 (2000) 81-95. FENECH, M., PEREPETSKAYA, G., MIKHALEVICH, L., A more comprehensive application of the micronucleus technique for biomonitoring of genetic damage rates in human populations — Experiences from the Chernobyl catastrophe, Environ. Mol.
|
{
"page_id": null,
"source": 7334,
"title": "from dpo"
}
|
Mutagen. 30 (1997) 112–118. MIKHALEVICH, L.S., et al., Radiation effects in lymphocytes of children living in a Chernobyl contaminated region of Belarus, Int. J. Radiat. Biol. 76 (2000) 1377–1385. FUCIC, A., et al., Genomic damage in children accidentally exposed to ionizing radiation: A review of the literature, Mutat. Res. 658 (2008) 111–123. KIRSCH-VOLDERS, M., FENECH, M., Inclusion of micronuclei in non-divided mononuclear lymphocytes and necrosis/apoptosis may provide a more comprehensive cytokinesis block micronucleus assay for biomonitoring purposes, Mutagenesis 16 (2001) 51–58. ROSEFORT, C., FAUTH, E., ZANKL, H., Micronuclei induced by aneugens and clastogens in mononucleate and binucleate cells using the cytokinesis block assay, Mutagenesis 19 (2004) 277–284. NORPPA, H., RENZI, L., LINDHOLM, C., Detection of whole chromosomes in micronuclei of cytokinesis-block human lymphocytes by antikinetochore staining and in situ hybridization, Mutagenesis 8 (1993) 519–525. > 166 HUBER, R., et al., Detection of centromeres in vinblastine- and radiation-induced micronuclei of human lymphocytes using FISH with an alpha satellite pancentromeric DNA probe, Environ. Mol. Mutagen. 27 (1996) 105–109. THIERENS, H., et al., Scoring of different cytogenetic endpoints after in vitro low dose γ-exposure: interlaboratory comparison for biomonitoring of radiological workers, Int. J. Radiat. Biol. 75 (1999) 23–34. PALA, F.S., et al., The effects of micronuclei with whole chromosomes on biological dose estimation, Turk. J. Biol. 32 (2008) 283–290. MUELLER, W.U., RODE, A., The micronucleus assay in human lymphocytes after high radiation doses (5-15 Gy), Mutat. Res. 502 (2002) 47–51. ABRAMSSON-ZETTERBERG, L., ZETTERBERG, G., BERGQVIST, M., GRAWE, J., Human cytogenetic biomonitoring using flow-cytometric analysis of micronuclei in transferring-positive immature peripheral blood reticulocytes, Environ. Mol. Mutagen. 36 (2000) 22–31. STOPPER, H., et al., Pilot study for comparison of reticulocyte-micronuclei with lymphocyte-micronuclei in human biomonitoring, Toxicol. Lett. 156 (2005) 351-360. GRAWE, J., Evaluation
|
{
"page_id": null,
"source": 7334,
"title": "from dpo"
}
|
of the reticulocyte micronucleus assay in patients treated with radioiodine for thyroid cancer, Mutat. Res. 583 (2005) 12–25. VRAL, A., Micronuclei induced by fast neutrons versus 60 Co γ-rays in human peripheral blood lymphocytes, Int. J. Radiat. Biol. 65 (1994) 321–328. VERHAEGEN, F., VRAL, A., Sensitivity of micronucleus induction in human lymphocytes to low LET radiation qualities: RBE and correlation of RBE and LET, Radiat. Res. 139 (1994) 208–213. WUTTKE, K., MULLER, W.U., STREFFER, C., The sensitivity of the in vitro cytokinesis-block micronucleus assay in lymphocytes for different and combined radiation qualities, Strahlentherapie Onkol. 174 (1998) 262–268. GERSEY, B., et al., Micronuclei induction in human fibroblasts exposed in vitro to Los Alamos high-energy neutrons, Adv. Space Res. 40 (2007) 1754–1757. FENECH, M., AITKEN, C., RINALDI, J., Folate, vitamin B12, homocysteine status and DNA damage in young Australian adults, Carcinogenesis 19 (1998) 1163–1171. FENECH, M., HOLLAND, N., CHANG, W.P., ZEIGER, E., BONASSI, S., The HUMN Project - An international collaborative study on the use of the micronucleus technique for measuring DNA damage in humans, Mutat. Res. 428 (1999) 271–283. THIERENS, H., VRAL, A., DE RIDDER, L., A cytogenetic study of radiological workers : effect of age, smoking and radiation burden on the micronucleus frequency, Mutat. Res. 360 (1996) 75–82. THIERENS, H., et al., Cytogenetic monitoring of hospital workers occupationally exposed to ionizing radiation using the micronucleus-centromere assay, Mutagenesis 15 (2000) 245–249. THIERENS, H., et al., A cytogenetic study of nuclear power plant workers using the micronucleus-centromere assay, Mutat. Res. 445 ( 1999) 105–111. CATALAN, J., et al., Age-dependent inclusion of sex chromosomes in lymphocyte micronuclei of man, Am. J. Hum. Genet. 63 (1998) 1464–1472. CARERE, A., ANTOCCIA, A., CIMINI, D., et al., Analysis of chromosome loss and non-disjunction in cytokinesis-block
|
{
"page_id": null,
"source": 7334,
"title": "from dpo"
}
|
lymphocytes of 24 male subjects, Mutagenesis 14 (1999) 491–496. 167 THIERENS, H., VRAL, A., DERIDDER, L., Biological dosimetry using the micronucleus assay for lymphocytes: interindividual differences in dose-response, Hlth Phys. 61 (1991) 623–630. EASTMOND, D.A., TUCKER, J.D., Identification of aneuploidy-inducing agents using cytokinesis-block human lymphocytes and an antikinetochore antibody, Environ. Mol. Mutagen. 13 (1989) 34–43. LEE, T.K., et al., Micronuclei in lymphocytes of prostate cancer patients undergoing radiation therapy, Mutat. Res. 469 (2000) 63–70. THIERENS, H., VRAL, A., VAN EIJKEREN, M., SPELEMAN, F., DE RIDDER, L., Micronucleus induction in peripheral blood lymphocytes of patients under radiotherapy treatment for cervical cancer or Hodgkin's disease, Int. J. Radiat. Biol., 67 (1995) 529–539. SILVA-BARBOSA, I., PEREIRA-MAGNATA, S., AMARAL, A., SOTERO, G., MELO, H.C., Dose assessment by quantification of chromosome aberrations and micronuclei in peripheral blood lymphocytes from patients exposed to gamma radiation, Genet. Mol. Biol. 28 (2005) 452–457. LE ROUX, J., SLABBERT, J., SMIT, B., BLEKKENHORST, G., Assessment of the micronucleus assay as a biological dosimeter using cytokinesis-block lymphocytes from cancer patients receiving fractionated partial-body radiotherapy, Strahlenther. Onkol. 174 (1998) 75–81. IARMARCOVAI, G., et al., Micronuclei frequency in peripheral blood lymphocytes of cancer patients: a meta-analysis, Mutat. Res. 659 (2008) 274–283. MONSIEURS, M., et al., Patient dosimetry after 131I lipiodol therapy, Eur. J. Nucl. Med. Mol. Imag. 30 (2003) 554–561. LIVINGSTON, G.K., FOSTER, A.E., ELSON, H.R., Effect of in vivo exposure to iodine-131 on the frequency and persistence of micronuclei in human lymphocytes, J. Toxicol. Environ. Hlth 40 ( 1993) 367–375. FENECH, M., et al., Micronuclei in cytokinesis-block lymphocytes of cancer patients following fractionated partial-body radiotherapy, Int. J. Radiat. Biol. 57 (1990) 373–383. DIAS, F.L., et al., Cytogenetic analysis in lymphocytes from workers occupationally exposed to low levels of ionizing radiation, Environ.
|
{
"page_id": null,
"source": 7334,
"title": "from dpo"
}
|
Toxicol. Pharmacol. 23 (2007) 228–233. SARI-MINODIER, I., et al., Cytogenetic monitoring by use of the micronucleus assay among hospital workers exposed to low doses of ionizing radiation, Mutat. Res. 629 (2007) 111–121. THIERENS, H., et al., Micronucleus assay reveals no radiation effects among nuclear power plant workers, Hlth Phys. 83 (2002) 178–182. VAGLENOV, A., et al., Cytogenetic monitoring of workers from a nuclear power plant, Centr. Eur. J. Occupation. Environ. Med. 3 (1997) 40–47. LIVINGSTON, G.K., et al., Radiobiological evaluation of immigrants from the vicinity of Chernobyl, Int. J. Radiat. Biol. 72 (1997) 703–713. TANAKA, K., et al., Unstable-type chromosome aberrations in lymphocytes from individuals living near Semipalatinsk Test Site, J. Radiat. Res. 47 (2006) Suppl. A159–A164. THIERENS, H., et al., Cytogenetic biodosimetry of an accidental exposure of a radiological worker using multiple assays, Radiat. Prot. Dosim . 113 (2005) 408–414. MARTIN, P.R., BERDYCHEVSKI, R.E., SUBRAMANIAN, U., BLAKELY, W.F., PRASANNA, P.G.S., Sample tracking in an automated cytogenetic > 168 biodosimetry laboratory for radiation mass casualties, Radiat. Meas. 42 (2007) 1119–1124. FRIEDMAN, L.I., SEVERNS, M.L., Application of robotics in blood banking, Vox Sang 51 Suppl. 1 (1986) S57–S62. HANSON, K.L., CARTWRIGHT, C.P., Evaluation of an automated liquid-handling system (Tecan Genesis RSP 100) in the Abbott LCx assay for Chlamydia trachomatis, J. Clin. Microbiol. 39 (2001) 1975–1977. DENG, W., TSAO, S.W., LUCAS, J.N., LEUNG, C.S., CHANG, A.L., A new method for improving metaphase chromosome spreading, Cytometry 51 (2003) 46–51. WEBER, J., SCHEID, W., TRAUT, H., Time saving in biological dosimetry by using the automatic metaphase finder, Metafer2. Mutat. Res. 272 (1992) 31–34. LLOYD, D.C., “Advances in cytogenetic dosimetry”, The Medical Basis of Radiation Accident Preparedness II: Clinical Experience and Follow Ups since 1979, (RICKS, R. E., FRY, S. S. E.,
|
{
"page_id": null,
"source": 7334,
"title": "from dpo"
}
|
Eds), Elsevier, New York, NY (1990) 479-487. FINNON, P., LLOYD, D.C., EDWARDS, A.A., An assessment of the metaphase finding capability of the Cytoscan 110, Mutation Res, 164 (1986) 101–108. LLOYD, D.C., “Automated aberration scoring: the requirements of an end-user”, Automation of Cytogenetics (LUNDSTEEN, C., PIPER, J., Eds), Springer-Verlag, Heidelberg (1989). 9–17. LORCH, T., WITTLER, C., STEPHAN, G., BILLE, J., “An automated chromosome aberration scoring system”, Automation of Cytogenetics (LUNDSTEEN, C., PIPER, J., Eds), Springer- Verlag, Heidelberg (1989) 19–30. FINNON, P., LLOYD, D.C., EDWARDS, A.A., “Progress in automatic dicentric hunting”, Chromosome Alterations, Origin and Significance (OBE, G., NATARAJAN, A.T., Eds), Springer-Verlag, Heidelberg (1994) 192–202. STEPHAN, G., “Automatische Analyse dizentrischer Chromosomen”, Methodische Fragen beim Human Population Monitoring in der Zytogenetik (ARNDT, D., OBE, G., Eds), MMV Verlag, München (1996). VAURIJOUX, A., et al., Strategy for Population Triage Based on Dicentric Analysis, Radiat. Res. 171 (2009) 541–548. CASTELAIN, P., et al., Automated detection of cytochalasin-B blocked binucleated lymphocytes for scoring micronuclei, Mutagenesis 8 (1993) 285–293. VERHAEGEN, F., et al., Scoring of radiation-induced micronuclei in cytokinesis-block human lymphocytes by automated image analysis, Cytometry 17 (1994) 119–127. SCHUNK, C., et al., New developments in automated cytogenetic imaging: unattended scoring of dicentric chromosomes, micronuclei, single cell gel electrophoresis, and fluorescence signals, Cytogenet. Genome Res. 104 (2004) 383–389. VARGA, D., et al., An automated scoring procedure for the micronucleus test by image analysis, Mutagenesis 19 (2004) 391–397. DECORDIER, I., et al., Automated image analysis of cytokinesis-block micronuclei: an adapted protocol and a validated scoring procedure for biomonitoring, Mutagenesis 24 (2009) 85–93. KORTHOF, K., CAROTHERS, A.D., Test of performance of four semi-automatic metaphase-finding and karyotyping systems, Clin. Gen. 40 (1991) 441-451. > 169 VROLIK, J., SLOOS, W.C., DARROUDI, F., NATARAJAN, A.T., TANKE, H.J., A system
|
{
"page_id": null,
"source": 7334,
"title": "from dpo"
}
|
for fluorescence metaphase finding and scoring of chromosomal translocations visualized by in situ hybridisation, Int. J. Radiat. Biol. 66 (1994) 287–295. WU, Q., SNELLINGS, J., AMORY, L., SUETENS, P., OOSTERIJNCK, A., “Model-based contour analysis in a chromosome segmentation system”, Automation of Cytogenetics, Springer, Heidelberg (1989) 217–229. PIPER, J., et al., Automated fluorescence metaphase finder speeds translocation scoring in FISH painted chromosomes, Cytometry 16 (1994) 7–16. MASCIO, L.N., et al., Advances in the automated detection of metaphase chromosomes labelled with fluorescence dyes, Cytometry 33 (1998) 10–18. INTERNATIONAL ATOMIC ENERGY AGENCY, Generic procedures for medical response during a nuclear or radiological emergency, EPR-MEDICAL IAEA, Vienna (2005). INTERNATIONAL ATOMIC ENERGY AGENCY, Manual for first responders to a radiological emergency, EPR-FIRST RESPONDERS, IAEA, Vienna (2006). NATIONAL COUNCIL OF RADIATION PROTECTION AND MEASUREMENTS , Key elements of preparing emergency responders for nuclear and radiological terrorism, Commentary No. 19, Bethesda, MD (2005). WASELENKO, J.K., et al., Medical management of the acute radiation syndrome: Recommendations of the Strategic National Stockpile Working Group, Ann. Intern. Med. 140 (2004) 1037–1051. ALEXANDER, G.A., et al., BiodosEPR-2006 Meeting: Acute dosimetry consensus committee recommendations on biodosimetry applications in events involving uses of radiation by terrorists and radiation accidents, Radiat. Meas. 42 (2007) 972–996. FLYNN, D.F., GOANS, R.E., Nuclear terrorism: triage and medical management of radiation and combined-injury casualties, Surg. Clin. N. Am. 86 (2006) 601-636. BLAKELY, W.F., WALTER, C.A., PRASANNA, P.G.S., Early-response biological dosimetry—recommended countermeasure enhancements for mass casualty radiological incidents and terrorism, Hlth Phys. 89 (2005) 494–504. BLAKELY, W.F., Early Biodosimetry Response: Recommendations for Mass-Casualty Radiation Accidents and Terrorism (Refresher Course for the 12th International Congress of the International Radiation Protection Association, Buenos Aires, 19–24 October 2008), MURATA, H., AKASHI, M., The report of the criticality accident in
|
{
"page_id": null,
"source": 7334,
"title": "from dpo"
}
|
a uranium conversion test plant in Tokai-mura, NIRS-M-154, National Institute of Radiation Sciences, Japan (2002). US DEPARTMENT OF HEALTH AND HUMAN SERVICES, Radiation Event Medical Management, LLOYD, D.C., EDWARDS, A.A., MOQUET, J.E., GUERRERO-CARBAJAL, Y.C., The role of cytogenetics in early triage of radiation casualties, Appl. Radiat. Isot. 52 (2000) 1107–1112. VOISIN, P., et al., The cytogenetic dosimetry of recent accidental overexposure, Cell. Mol. Biol. (Noisy-Le-Grand) 47 (2001) 557–564. FLEGAL, F.N., DEVANTIER, Y., MCNAMEE, J.P., WILKINS, R.C., QuickScan dicentric chromosome analysis for radiation biodosimetry, Hlth Phys. > 98 (2010) 276–281. > 170 LINDHOLM,C., et al., Premature chromosome condensation (PCC) assay for dose assessment in mass casualty accidents, Radiat. Res. 173 (2010) 71–78. McNAMEE, J.P., FLEGAL, F.N., BOULAY GREENE, H., MARRO, L., WILKINS R.C., Validation of the Cytokinesis-Block Micronucleus (CBMN) assay for use as a triage biological dosimetry tool, Radiat. Prot. Dosim. 135 (2009) 232–242. YOSHIDA, M.A., et al., The Chromosome Network for biodosimetry in Japan, Radiat. Meas. 42 (2007) 1125–1127. MILLER, S. M., et al., Canadian Cytogenetic Emergency Network (CEN) for biological dosimetry following radiological/nuclear accidents, Int. J. Radiat. Biol. 83 (2007) 471–477. WOJCIK, A., LLOYD, D., ROMM, H., ROY, L., Biological dosimetry for triage of casualties in a large-scale radiological emergency: Capacity of the EU member states, Radiat. Prot. Dosim. 138 (2010) 397–401. INTERNATIONAL ATOMIC ENERGY AGENCY, RANET Assistance Action Plan – Arrangements for Providing International Assistance and Sample of Assistance Action Plan, IAEA, Vienna, Austria, 2006. BLAKELY, W.F., et al., WHO 1st consultation on the development of a global biodosimetry laboratories network for radiation emergencies (BioDoseNet), Radiat. Res. 171 (2009) 127–39. SEVAN'KAEV, A.V., Results of cytogenetic studies of the consequences of the Chernobyl accident, Radiats. Biol. Radioecol. 40 (2000) 589–595. MAZNIK, N.A., VINNIKOV, V.A., LLOYD,
|
{
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"source": 7334,
"title": "from dpo"
}
|
D.C., EDWARDS, A.A., Chromosomal dosimetry for some groups of evacuees from Prypiat and Ukrainian liquidators, Radiat. Prot. Dosim. 74 (1997) 5–11. SHEVCHENKO, V.A., SNIGIRYOVA, G.P., “Cytogenetic effects of the action of ionizing radiations on human population”, Research Activities about the Radiological Consequences of the Chernobyl NPS Accident and Social Activities to Assist the Sufferers by the Accident (IMANAKA,T., Ed.), Research Reactor Institute, Kyoto University (1998) 203–215. SEVAN'KAEV, A.V., et al., A survey of chromosomal aberrations in lymphocytes of Chernobyl liquidators, Radiat. Prot. Dosim. 58 (1995) 85–91. MAZNIK, N.A., VINNIKOV, V.A., The retrospective cytogenetic dosimetry using the results of conventional chromosomal analysis in Chernobyl clean-up workers, Radiat. Biol. Radioecol. 45 (2005) 700–708. SEVAN'KAEV, A.V., et al., Novel data set for retrospective biodosimetry using both conventional and FISH chromosome analysis after high accidental overexposure, Appl. Radiat. Isot. 52 (2000) 1149–1152. EDWARDS, A., et al., Biological estimates of dose to inhabitants of Belarus and Ukraine following the Chernobyl accident, Radiat. Prot. Dosim. 111 (2004) 211–219. KOKSAL, G., PALA, F.S., DALCI, D.O., In vitro dose-response curve for chromosome aberrations induced in human lymphocytes by 60 Co gamma-radiation, Mutat. Res. 329 (1995) 57–61. RAMALHO, A.T., NASCIMENTO, A.C., The fate of chromosomal aberrations in 137 Cs-exposed individuals in the Goiânia radiation accident, Health Phys. 60 (1991) 67–70. SASAKI, M.S., HAYATA, I., KAMADA, N., KODAMA, Y., KODAMA, S., Chromosome aberration analysis in persons exposed to low-level radiation from 171 the JCO criticality accident in Tokai-mura, J. Radiat. Res. 42 Suppl. (2001) S107-S116. JINARATANA, V., The Radiological Accident in Thailand, Parthenon Publishing, (2002) 283–301. INTERNATIONAL ATOMIC ENERGY AGENCY, The Radiological Accident in Nueva Aldea, IAEA, Vienna (2009). BERTHO, J.M., ROY, L., A rapid multiparametric method for victim triage in cases of accidental protracted irradiation or delayed analysis,
|
{
"page_id": null,
"source": 7334,
"title": "from dpo"
}
|
Br. J. Rad. 82 (2009) 764–770. WORLD HEALTH ORGANIZATION, Biorisk Management: Laboratory Biosecurity Guidance, WHO, Geneva (2006). WORLD HEALTH ORGANIZATION, Laboratory Biosafety Manual, 3rd edn, WHO, Geneva (2004). INTERNATIONAL ATOMIC ENERGY AGENCY, Safety Glossary, Terminology Used in Nuclear Safety and Radiation Protection, IAEA, Vienna (2007). > 172 Annex I DICENTRIC ASSAY The variety of materials and methods for making and processing lymphocyte cultures which are commonly used by laboratories around the world have been described and discussed, and probably no two laboratories adopt precisely the same technique. This Annex provides a detailed, step by step description of a reliable method which could be of assistance for some laboratories. I–1. LYMPHOCYTE CULTURE I–1.1. Materials (1) Heparinized whole blood. (2) Phytohaemagglutinin (PHA), commercially available. If supplied freeze dried, it should be reconstituted with sterile analytical grade water. (3) Eagle’s minimum essential culture medium (MEM), commercially available: ready to use, x10 concentration or powdered. Working concentrations should be made up with sterile analytical grade water. L-glutamine may need to be added according to the manufacturer’s instructions. The pH will need to be adjusted with sterile sodium bicarbonate. (i) Antibiotics may need to be added to the medium made from concentrate. Add 1 mL of a stock solution of antibiotics in saline to 100 mL of medium. The stock solution should contain 100 IU/mL of benzylpenicillin and 100 μg/mL streptomycin sulphate and can be stored frozen. (4) Bromodeoxyuridine (BrdU). Add 1 mL of a stock solution to 100 mL of medium. The stock solution is 6.4 mg of BrdU dissolved in 10 mL of medium and membrane filtered. This will give a final concentration in the culture of 15 μM. The stock can be stored for one month in the dark at 4°C or for several months at -20°C. (5) Heat
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inactivated (56 oC for 0.5 hour) foetal calf serum, commercially available and stored frozen. (6) Colcemid: stock solution of 10 μg/mL in sterile physiological saline. It can be stored at 4°C for 6 months. (7) Sterile culture vessels. There are various options, e.g. glass bacteriology bottles or disposable plastic containers. The volume should be 15 to 20 mL. (8) Cultures should be set up in a class 2 microbiological safety cabinet, under subdued lighting. Liquids can be transferred between vessels using sterile disposable syringes or pipettes. If blood needs to be passed through a hypodermic needle, this should be done slowly by using a wide bore (19 gauge) needle to minimize shearing forces on the cells. I–1.2. Method (1) Place 0.3 mL of heparinized blood into a culture vessel. (2) Add 4.0 mL of culture medium to which antibiotics and BrdU have already been added. (3) Add 0.1 mL of reconstituted PHA. (4) Add 0.5 mL of foetal calf serum. (5) Seal the lid securely. 173 (6) Mix the contents of the vessel by gentle shaking. (7) Incubate at 37°C ± 0.5°C in the dark for 45 hours. (8) Add 50 μL of Colcemid stock solution to the culture and shake gently. (9) Return to the incubator for three more hours. I–2. FIXATION AND SLIDE PREPARATION I–2.1. Method (1) Tip the contents of the culture vessel into a centrifuge tube. (2) Spin at 200 g for 10 min (to convert g into rev/min, use g = r ω2 /981, where r = radius in cm and ω = (2 π x rev/min)/60). (3) Remove the supernatant by suction and resuspend the cell button in 5 to 10 mL of 0.075M potassium chloride solution. (4) Leave to stand at room temperature for 15 to 20 min. (5) Spin again at 200
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g for 10 min. (6) Remove supernatant and resuspend the cells in 5 to 10 mL of freshly prepared 3:1 methanol/acetic acid fixative. The fixative must be added slowly, but at a constant rate with vigorous agitation, ideally using a vortex mixer, to prevent the cell button from becoming a solid clump. A further aid to preventing clumping is to use a latex rubber bulb on a Pasteur pipette to gently mix the cell button prior to adding the fixative. (7) Spin again. (8) Remove supernatant and resuspend in 5 to 10 mL of fixative. (9) Spin again. (10) Remove supernatant and resuspend in 5 to 10 mL of fixative. (11) Spin again. (12) Remove all but 0.25 mL of the supernatant and resuspend the cell button in the remaining fluid. (13) Draw up the cell suspension into a Pasteur pipette. (14) Take a clean, grease free slide that has previously been stored in a freezer. Melt the frost on the slide with your breath. (15) Allow one or two drops of the cell suspension to drip onto the slide from a height of at least 10 cm. (16) Prepare at least two such slides from each culture. (17) Place the slides to dry in gentle heat over a hotplate. I–3. STAINING I–3.1. Materials (1) Hoechst 33258 stain. A 1000 x concentrated stock solution of 50 μg/mL in pH 6.8 phosphate buffer can be stored at 4°C in the dark. 174 (2) Giemsa stain. (3) Phosphate buffer (pH 6.8) made up from commercially available tablets. (4) 2 x SSC (sodium chloride and trisodiurn citrate): 17.53 g sodium chloride, 8.82 g sodium citrate, distilled water to make 1.0 L. (5) Xylene and DPX mountant. (6) An ultraviolet lamp (>310 nm) or a fluorescent strip lamp. I–3.2. Methods A few (up to
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five) days at room temperature should elapse between preparation of the slides and commencement of FPG staining, while the conventional Giemsa stain can be used as soon as the slides are dry. Alternatively, slides can be dried at 37 oC and stained with FPG the following day. Fluorescence plus Giemsa (FPG) (1) Place approximately 10 drops of Hoechst stain (diluted from the stock solution to 0.5 μg/mL) onto the slide and cover with a coverslip. (2) Place the slide on a sheet of aluminium foil and beneath an ultraviolet lamp for 0.5 hour. (3) Carefully remove the coverslip. (4) Wash well with pH 6.8 buffer. (5) Place in 2 x SSC at 60°C for 20 to 30 min. (6) Wash in distilled water. (7) Place slides in Giemsa stain — a 5 to 10% solution in pH 6.8 buffer for 3 min. (8) Rinse briefly in buffer. (9) Rinse briefly in distilled water. (10) Air dry. (11) Clear and mount under a coverslip. Conventional Giemsa (1) Place slide in 2% Giemsa stain in pH 6.8 buffer for 5 min. (2) Wash in buffer. (3) Rinse briefly in distilled water. (4) Air dry. (5) Clear and mount under a coverslip. 175 Annex II FISH BASED TRANSLOCATION ASSAY The procedure given here uses both directly and indirectly labelled (commercially available) probes and describes painting three pairs of chromosomes in different colours, all centromeres in a fourth colour and counterstaining the remaining chromosomes. Manufacturers do supply protocols which could be read in conjunction with the method below. II–1.1. Pre-treatment Wash slides with PBS for 5 min at room temperature. Dehydrate slides in an ethanol series (70, 90 and 100%) 2 to 5 min each step, at room temperature, and air dry. II–1.2. RNase and pepsin treatment Mix 445 μL water, 50 μL 20
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x SSC and 5 μL RNase A (10 μg / μL) (the mixture can be prepared in advance and should be kept at -20 oC). Pipette 100 μL of RNase A per slide, overlay with a coverslip. Incubate in a moist chamber for 60 min at 37°C. Wash three times with 2 x SSC (5 min each at room temperature). During the first wash remove coverslip. Afterwards, wash with PBS for 5 min at room temperature. For pepsin treatment (0.005% in 10 mM HCl), prepare in advance a mixture consisting of 50 μL pepsin (10%), 99 mL water and 1 mL 1N HCl. This mixture can be kept at -20°C before use. Prewarm the mixture in a water bath at 37°C and put 100 μL onto each slide for 1 to 2 min. Wash with PBS for 5 min at room temperature. Wash with 50 mM MgCl 2 -PBS (5mL MgCl 2 and 95 mL PBS) for 5 min at room tempera-ture. Wash with 1% formaldehyde in MgCl 2 –PBS for 10 min at room temperature. Rinse in PBS for 5 min at room temperature. Air dry in an ethanol series (70, 90 and 100%) 2 to 5 min each at room temperature. II–1.3. FISH protocol for chromosome paint probes in combination with a pan-centromeric probe Warm chromosome paint probes to 42°C and shake well before use. A sufficient amount of every chromosome paint should be placed in an Eppendorf tube with hybridization buffer; shake well and spin down. (a) Denaturation Chromosome paints can be denatured by incubation at 65°C for 10 min in a water bath. Then put on ice for 2 to 3 min, and transfer to a water bath (37°C) and incubate for 60 min. When using the chromosome paints in combination with a pan-centromeric probe (CP),
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start warming the CP and hybridization buffer at 37°C 30 min before probe competition. Denature the CP by incubating at 85°C for 10 min in a water bath, then immediately put on ice for 2 to 3 min. For triple colour FISH with a pan-centromeric probe, the final volume of 18 to 20 μL of hybridization mixture per slide should be used (i.e. for 3 μL of each of the three concentrated paint probes add 1.6 μL of its appropriate buffer and add 2 to 3 μL of concentrated CP). For example, when three chromosomes #1, 4 and 8 are being painted: Chromosome #1 (biotin), #4 biotin/FITC, #8 FITC and CP FITC, they will generate red, yellow, green and green colour signal, respectively. 177 (b) Pre-hybridization Pre-hybridization of slides should be started approximately 30 min before the end of probe competition. Put 100 μL of 70% formamide in 2 x SSC and 50 mM PBS per slide and overlay with a coverslip (350 μL deionized 100% formamide (store at –20°C), 50 μL 0.5 M PBS (store at -20°C) and 50 μL of 20 x SSC). The formamide should be deionized shortly before its use. Denature slides at 70°C for 2.5 min on a hot plate. Air dry the slides in an ethanol series (stored at -20°C) of 70% for 5 min, 90 and 100% for 2 to 5 min each at room temperature. Allow the slides to air dry. (c) Hybridization Mix well all chromosome paints and CP in one Eppendorf tube. Spin down for a few seconds, and put 20 μL of the mixture onto each slide, overlay with a coverslip, seal with rubber glue and air dry. Slides should then be incubated overnight in a moist chamber at 42°C. This can be extended to two days. Detection: (1)
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Prepare a wash solution (WS) of 4 x SSC containing 0.05% Tween 20. (2) Dilute blocking protein (BP) to 15% (v/v) in WS. (3) Use the diluted BP for diluting antibodies as follows: 3.1.1 First layer B3 (1:500), Texas Red Avidin. 3.1.2 Second layer B4 (1:250) biotinylated goat anti-avidin. 3.1.3 F1 (1:200) rabbit anti-FITC. 3.1.4 Third layer B3 (1:500) F2-FITC, goat anti-rabbit IgG. 3.1.5 F2 (1:100). (4) Incubate in the dark for 10 min at room temperature, microcentrifuge at 11,000 g for 10 min, and use the supernatant. (5) Prewarm the following solutions to 42 oC: i) The wash solution. ii) Some 2 x SSC. iii) 50% formamide in 2 x SSC. iv) 0.1% x SSC. (6) Carefully remove coverslips in a jar of warmed 2 x SSC. (7) Wash the slides in the warmed solutions as follows: i) The wash solution. ii) Some 2 x SSC. iii) 50% formamide in 2 x SSC. (8) Put 100 μL of the diluted blocking protein onto each side and overlay with a coverslip, incubate in a moist chamber for 15 to 20 min at 37°C. (9) Wash slides with 0.05% Tween 20 in 4 x SSC for 2 to 5 min at 42°C. 178 (10) Put 100 μL of the first layer of antibody onto each slide and overlay with a coverslip. Incubate in a moist chamber for 20 to 30 min at 37°C. (11) Wash slides in 0.05% Tween 20 in 4 x SSC, three times, 5 min each at 42°C. (12) Put 100 μL of the second layer of antibodies onto each slide and overlay with a coverslip. Incubate in a moist chamber for 20 to 30 min at 37°C. (13) Wash slides with 0.05% Tween 20 in 4 x SSC, three times, 5 min each at 42°C. (14) Put
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100 μL of the third layer of antibodies onto each slide and overlay with a coverslip, incubate in a moist chamber for 20 to 30 min at 37°C. (15) Wash slides with 0.05% Tween 20 in 4 x SSC, three times, 5 min each at 42°C. (16) Repeat steps 11 to 14 once. (17) Dehydrate slides in an ethanol series of 70, 90 and 100%, 2 to 5 min each at room temperature. (18) Allow the slides to air dry. (19) Counterstain with DAPI (0.15 μg/mL in Vectashield mountant), 25 μL per slide under a coverslip. If all the painting signals are insufficiently bright one may interpose after step 14 another round of the second and third layers. Alternatively if just one of the colours is feint then one may repeat the steps B3 / wash / B4 for Texas Red or F1 / wash / F2 for FITC. > 179 Annex III PREMATURE CHROMOSOME CONDENSATION III-1. PCC BY MITOTIC FUSION Human peripheral blood mononuclear cells are fused with mitotic Chinese hamster ovary (CHO) cells in the presence of polyethylene glycol (PEG). As a result of the cell fusion in only one hour, the mononuclear blood cells undergo chromatin condensation which is rapidly followed by dissolution of their nuclear membrane and further condensation of chromatin into 46 (2n = 46) single chromatid chromosomes. > III–1.1. Isolation of human peripheral blood lymphocytes For the separation of mononuclear cells from anti-coagulated whole blood, a LeucoPREP or, a Ficoll-Hypaque cell separation tube can be used. > A. LeucoPREP The LeucoPREP product is a tube system containing a separation medium that, like Ficoll-Hypaque, takes advantage of the lower density of mononuclear cells and platelets to separate these from the remaining components of anticoagulated whole blood. The separation occurs when blood is placed in the
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tube over the gel layer and the tube is subjected to a specified centrifuge force for a given duration. Subsequent washings and centrifugations reduce the quantity of platelets present. The resulting preparations of viable mononuclear cells can be used for PCC . (1) Store LeucoPREP tubes (10 mL) upright at room temperature (18–25°C). (2) Collect blood by venipuncture into a heparinized tube. (3) Heparin anticoagulated blood should be separated within two hours of blood sampling. (4) Add undiluted blood (8 to 10 mL) to each LeucoPREP tube, then centrifuge for 15 min at 400–600 g at room temperature. (5) After centrifugation, mononuclear cells and platelets will be in a fluffy, white layer just under the plasma layer. Aspirate plasma as much as possible without aspirating cells. Collect cell layer with a Pasteur pipette and transfer to a 10 mL conical centrifuge tube with cap. (6) Resuspend cell pellet by gently vortexing. Add F10 medium (10 mL), mix cells by inverting tubes 3 to 4 times, then centrifuge for 10 min at 100 g.(7) Repeat step 5 once again. > B. Ficoll-Hypaque gradient system Ficoll-Paque is an aqueous solution of density 1.077 ± 0.001 g/mL containing 5.7 g Ficoll 400 and 9 g sodium diatrizoate calcium disodium ethylenediaminetetracetic acid (EDTA) in every 100 mL. (1) Collect blood by venipuncture into heparinized tube. (2) Dilute blood samples with an equal volume of balanced salt solution. (3) Put about 5 mL of diluted blood (drop by drop) on top of Ficoll-Hypaque (3 mL) without intermixing. > 181 (4) Centrifuge the tubes for 30 min at 400 g at 8–10°C. (5) Collect lymphocytes (middle layer) and wash three times (centrifuge at 100 g for 10 min) with 5 mL F-10 culture medium plus 5% foetal calf serum. The isolated lymphocytes may be used immediately
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for performing PCC experiments or frozen for future use. III–1.2. Freezing the isolated lymphocytes After the second wash with F-10 and centrifugation, resuspend the cell pellet by gently vortexing and make a cell suspension in 1:1, F-10 + 40% foetal calf serum (FCS): F-10 + 40% FCS + 20% DMSO. Make cell suspensions in a manner so that each ampoule (1.5 mL) contains about 8 x 10 6 isolated lymphocytes. For freezing the best method is to use a machine that can gradually decrease the temperature. Finally store frozen ampoules at -110°C or in liquid nitrogen. III–1.3. Thawing the isolated lymphocytes Take the lymphocyte ampoules out of the freezer and put them directly into a water bath (37°C). When they are slightly melted, transfer the whole suspension into a centrifuge tube (10 mL). Add 10 mL cold (4°C) RPMI + 40% FCS onto the lymphocyte suspensions, slowly drop by drop (in about 30 min), then centrifuge for 10 min at 100 g. Resuspend the cell pellet in 5 mL RPMI + 5% FCS. These mononuclear lymphocytes can be used for PCC experiments. III–1.4. Collection and preparation of mitotic Chinese hamster ovary cells Chinese hamster ovary (CHO) cells are grown in roller bottles or flasks (750 mL) in complete medium (F-10 + 15% new-born calf serum and antibiotics (penicillin 100 IU/mL and streptomycin 100 μg/mL)). Colcemid (0.1 μg/mL) is added to the exponentially growing cells, and mitotic cells are harvested by a standard selective detachment (shake-off) procedure 4 to 5 hours later. CHO cells can also be grown for more than two cell cycles (~32 hours) in complete medium supplemented with BrdU, (final concentration of 5 μM). Mitotic CHO cells obtained will all be differentially stained and look pale in colour following FPG staining. Therefore, lymphocyte PCC will be better differentiated
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among CHO mitotic cells. (1) Freezing mitotic CHO cells Mitotic CHO cells can either be prepared and used immediately for fusion or taken from stock frozen in complete medium supplemented with 8% DMSO. Put them in small aliquots (2.5 x 10 6/ampoule in 1.5 mL) and store them at -110°C. (2) Thawing mitotic CHO cells Take the ampoules of CHO mitotic cells out of the freezer and put them into a water bath at 37°C, then transfer the cell suspension into a centrifuge tube and add 10 mL medium. Centrifuge for 10 min at 100 g. Discard the supernatant, add medium (5 mL) and keep them on ice until use. III–1.5. Preparation of polyethylene glycol (PEG) solution Put 400 mg of PEG (M.W. 1450, Sigma, 40% w/v) into a small (10 mL) round bottom cen-trifuge tube and add 600 μL Hank’s balanced salt solution (HBSS) or phosphate buffered saline (PBS) or F-10 medium, and leave the tubes in the water bath at 37°C for 15 min. PEG can also be melted first in an oven and then mixed with HBSS or PBS or F-10 medium. 182 III–1.6. Cell fusion (1) Interphase lymphocytes and mitotic CHO cells are washed once with HBSS or F-10 (5 mL) separately. Centrifuge for 5 min at 100 g then discard supernatant. In a round bottom culture tube, mix interphase cells with mitotic CHO cells (5:1) in 10 mL F-10 medium and centrifuge for 5 min at 100 g (higher speeds may cause the pellet to compact too much). (2) Pour off the supernatant and keep the tube inverted. Blot the residual drops of medium by placing the tubes upside down in a test tube rack on a paper towel. (3) If air bubbles are formed on top of the pellet in the tube, they should
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be removed with a Pasteur pipette. (4) Using a micropipette (200 μL), take 0.15 mL PEG and put it directly into the cell pellet, place in a test tube rack for 1.5 min. Shake the tube very gently, only three times (30 s interval). At this point, the cell pellet should appear detached from the bottom of the tube, forming big clumps in the PEG solution. (5) Add 1.5–2 mL F-10 or PBS very slowly over 3 min (0.5 mL per min). Mix the cell suspension gently by tapping the tube. (6) Centrifuge the tube for 5 min at 100 g. (7) Pour off supernatant completely and add 0.5 mL of culture medium (F-10 plus 15% foetal calf serum). Finally add 50 μL of Colcemid (final concentration 1 μg/mL), gently tapping the tube to form small clumps. Incubate the test tube at 37°C for 1 hour. By this time cell fusion and induction of PCC are completed. > III–1.7. Fixation protocol (1) Add 7–8 mL prewarmed hypotonic (KCl, 5.6 g/L) to each tube and incubate for 10 min at 37°C. (2) Centrifuge the tube for 5 min at 100 g.(3) Discard supernatant until 0.5 mL above pellet. Cells are fixed in 5 mL methanol: acetic acid (3:1). (4) Centrifuge the tube for 5 min at 100 g.(5) Repeat steps 3 and 4 two more times. (6) Following the last centrifugation, discard supernatant and leave about 0.3 mL fixative solution on top of the pellet. Then break the pellet gently and add about 0.5–1 mL fixative by lightly tapping the tube. > III–1.8. Slide preparation Drop cells with a drawn-out Pasteur pipette onto precleaned slides. By observing Newton rings gently blow under an infrared lamp. > III-1.9. Staining protocols When mitotic CHO cells are not prelabelled with BrdU, slides can be
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stained with a 3% aqueous Giemsa solution (Gurr Improved R66) for 5 min. When mitotic CHO cells are prelabelled with BrdU, slides can be stained according to the FPG technique. (Section 9.3.) Finally, rinse slides in distilled water, allow them to dry and then > 183 mount under a 24 x 60 mm coverslip. However, note the caveat in Section 11.2.1.6 that often this is not the preferred staining method and simple Giemsa staining should be sufficient. For C-banding of PCCs (for dicentric analysis), freshly prepared slides should be treated with 1N HCl for 5 min, followed by washing in 0.2N HCl for 5 min. Slides are then dried with a paper towel and treated with Ba(OH) 2 solution (5%) for 3 min at room temperature. They are then washed in 0.2N HCl for 5 min. Afterwards incubate slides in 2 x SSC at 60°C for 30 min. Wash with Gurr’s buffer (pH = 6.8) and stain with 6% Giemsa for 30 min. Finally, rinse in tap water, allow them to dry and mount under a coverslip. Note that this is slightly different from the method in section 9.3.3, but both methods work. For the detection of translocations, whole chromosome specific probes together with a pan-centromeric probe can be used following the same protocol as for metaphases (see Annex II and Fig. 37). Then it is possible to detect dicentrics and translocations simultaneously. III-2. PCC BY CHEMICAL INDUCTION III-2.1. Using isolated lymphocytes (1) Place 3 mL of heparinized whole blood in a LeukoPREP or Ficol-Hypaque tube. (2) Centrifuge at 700 g for 15 min at room temperature. (3) Transfer isolated lymphocytes into a 15 mL test tube containing 5ml medium supplemented with 20% fetal calf serum for washing. (4) Centrifuge at 200–400 g for 10 min at 4°C. (5) Resuspend
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the lymphocytes in 6 mL of culture medium supplemented with 20% fetal calf serum and PHA. (6) Incubate at 37°C for 47 hours (an optional step is to add Colcemid, 40 ng/mL, at 24 hours into the culture time). (7) Add calyculin A at a final concentration of 50nM into the culture and incubate at 37°C for 1 hour. (8) Prepare a warm (37 oC) hypotonic solution, 0.075M KCl. (9) Centrifuge the cells at 200–400 g for 5–10 min and remove supernatant. (10) Add 2 mL of 0.075M KCl to the cell pellet and incubate at 37°C for 20 min. (11) Add 30 μL of methanol /acetic acid (3:1) and tap the tube. (12) Centrifuge at 200–400 g for 5–10 min at room temperature. (13) Add 1.8 mL of methanol:acetic acid after removing the supernatant and transfer into a 2 mL tube. (14) Store the tube at -20°C until slide preparation. III-2.2. Using whole blood (1) Place 0.75 mL of heparinized whole blood in a 15 mL test tube. (2) Bring to a total volume of 10 mL by adding culture medium supplemented with 20% fetal calf serum and PHA. (3) Incubate at 37°C for 47 hours. (an optional step is to add Colcemid, 40 ng/mL, at 24h into the culture time). 184 (4) Add calyculin A at a final concentration of 30nM into the culture and incubate at 37°C for 1 hour. (5) Centrifuge at 200–400 g for 5–10 min at room temperature. (6) Add 5 mL of 0.075M KCl after removing the supernatant and incubate at 37°C for 25 min. (7) Add 30 μL of methanol:acetic acid and tap the tube. (8) Centrifuge at 200–400 g for 5–10 min at room temperature. (9) Add 2 mL of 3:1 methanol:acetic acid. (10) Repeat steps 8 and 9 until the
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cell pellet is clear. (11) Transfer the cell suspension into a 2 mL tube. (12) Store the tube at -20°C until slide preparation. > 185 Annex IV CYTOKINESIS-BLOCK MICRONUCLEUS ASSAY A simple standard protocol that works well is given below. There are other methods involving more procedural steps and employing cultures of isolated lymphocytes but for routine biological dosimetry purposes whole blood cultures are adequate. IV-1. STANDARD CYTOKINESIS-BLOCK MICRONUCLEUS PROTOCOL (1) The blood sample is collected using lithium heparin anticoagulant. (2) Typically 0.5 mL of whole blood is added to 4.5 mL of culture medium (RPMI-1640) supplemented with 10 to 15% heat inactivated fetal calf serum, L-glutamine and antibiotics. 100 μL of phytohaemagglutinin (e.g. PHA-M, Sigma, 25 mg/25 mL H 2 O) is added to the culture to give a final concentration of 20 μg/mL. (3) The blood is cultured in tissue culture flasks at 37°C, 5% CO 2 in a humidified atmosphere. (4) 20 μL cytochalasin-B (Cyt-B) is added to the culture, at 24 hours post PHA stimulation, to give a final concentration of 6 μg/mL. This is the optimum concentration for accumulating BN cells in whole blood cultures. As Cyt-B is difficult to dissolve in aqueous solution a Cyt-B stock solution should be prepared in dimethylsulphoxide (5 mg Cyt-B in 3.3 mL DMSO) and aliquoted and stored until required at -20°C. (5) The culture is terminated between 68–72 hours post PHA stimulation. The chosen harvest time should maximize the number of BN cells and minimize the number of mononucleated and multinucleated cells. (6) The cells are centrifuged gently at 180 g for 10 min and the supernatant culture medium is removed. (7) The cells are hypotonically treated with 7 mL of cold (4°C) 0.075M KCl to lyse red blood cells, and centrifuged immediately at 180 g for
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10 min. (8) The supernatant is removed and replaced with 5 mL freshly made fixative consisting of methanol: acetic acid (10:1) diluted 1:1 with Ringer’s solution (4.5 g NaCl, 0.21g KCl, 0.12 g CaCl 2 in 500 mL H 2 O). The fixative should be added whilst agitating the cells to prevent clumps forming. The cells are then centrifuged again at 180 g for 10 min. (9) The cells are washed with two to three further changes of freshly prepared fixative consisting of methanol:acetic acid (10:1), this time without Ringer’s solution, until the cell suspension is clear . (10) After removing the supernatant to 1 cm or less above the cell pellet (depending on pellet size), the cells are resuspended gently, and the suspension is dropped onto clean glass slides and allowed to air dry. (11) For light microscope analysis cells can be stained in 2–6% Giemsa (e.g. Giemsa’s Azur-Eosin-Methylene blue solution, Merck) in HEPES buffer (0.03M ; pH 6.5) during 10–20 min in the dark, followed by a quick rinse in distilled H 2 O and air dried. For fluorescence microscopy cells can be stained, alternatively, in acridine orange (10 μg/mL in phosphate buffered saline pH 6.9) for 2–3 sec. 187 IV-2. MICRONUCLEUS-CENTROMERE STAINING PROTOCOL For analysing centromeres in MN a commercial pan-centromeric FISH probe can be used. A pan-centromeric probe can also be made by PCR amplification (forward primer: 5’-GAA GCT TAA CTC ACA GAG TTG AA-3´ reverse primer: 5´-GCT GCA GAT CAC AAA GAA GTT TC-3´) . Below, the in situ hybridisation protocol for the commercial probe is given: (1) Slides are prepared according to the standard CBMN protocol given above (up to step 10). (2) Dehydrate cells by passing the slides through 70–90–100% ethanol series, 2 min each step and air dry. (3) Denature slides: i)
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denature the chromatin on the slide in 70% formamide in 2xSSC for 2min at 70°C; ii) immerse slides in ice cold 70% ethanol and dehydrate through 70–90–100% ethanol series for 5 min each while shaking. (4) Denature probe just before use: i) warm probe to 37 ˚C for 5 min; ii) denature the probe at 85 ˚C for 10 min (10 μL/slide); iii) vortex and spin down quickly; iv) immediately chill on ice and keep in the dark. (5) Hybridisation: i) apply 10 μL of probe to the slide, put on a coverslip and seal with rubber cement; ii) hybridize overnight at 37 ˚C in the dark in a humidified chamber. (6) Post hybridization wash: i) remove the rubber cement and briefly dip slides into 50% formamide and shake off the coverslip; ii) wash slides in 2X SSC for 5 min at 37 ˚C; iii) wash slides 2 times in 50% formamide at 37 ˚C for 5 min each; iv) wash slides in 2X SSC for 5 min at 37 ˚C; v) wash in Tween washing solution (0.05% in 2xSSC) for 5 min at 37 ˚C; vi) add a drop of DAPI/antifade mountant on a coverslip and put on the slide. (7) Slides can be stored at room temperature in the dark or scored immediately under a fluorescent microscope. IV-3 THE ISOLATED LYMPHOCYTE CYTOKINESIS-BLOCK MICRONUCLEUS CYTOME (CBMN Cyt) ASSAY The detailed protocol for the CBMN Cyt assay has been published recently . For a comprehensive photographic gallery of the various cell types scored in the CBMN Cyt assay see Fenech et al. . > 188 IV-4 CALCULATING ERROR ON NDI FOR THE CBMN ASSAY The formulae for calculating the NDI and variance on the NDI (for micronucleus, MN, assay) that are given in Section 12.4.3 is as follows: N MMMMNDI
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4321 432( +++= (IV-1) # ∑ ∑ ∑ = = +=+= 4141412 `) ``cov( `2`) var( `)var( i i ij jijiii MMMMMMNDI (IV-2) In Table IV-1 a worked example for calculating the NDI and variance are presented. TABLE IV-1. DISTRIBUTION OF MICRONUCLEI Number of cells with 1, 2, 3 or 4 micronuclei N 1 2 3 4 NDI 500 169 ± 111.878 239 ± 124.758 48 ± 43.392 44 ± 40.128 1.934 In Table IV-1, the numbers of cells with 1, 2, 3 or 4 micronuclei from a total of 500 cells is presented. The NDI is calculated according to Eq. (IV-1) above: NDI = (169 + 2 x 239 + 3 x 48 + 4 x 44) / 500 = 1.934 The values of variance on each value are calculated using the binomial equation (Eq. IV-3): )) /(1)( /()var( 1 NMNMNM ii −= (IV-3) So for M 1 :var(M 1 ) = 500 (169 / 500) (1 – (169 / 500)) = 111.878 NB: It should be noted that all the figures given here are calculated in Microsoft Excel with each value correct to a large number of decimal places. However the values presented in the text are rounded to the third decimal place for convenience, and thus using a calculator with the presented values will not yield exactly the same results. To calculate var(NDI), one must first calculate the sum of the square of each value of M i `times its variance: # ∑ = > 412 `) var( `i ii MM (IV-4) However, because the covariance can only correctly be calculated from the total number of cells with M = 1 to 4, one must replace M in the equation with the following values of M’: TABLE IV-2. CALCULATED VALUES OF M i ` AND VAR(M i
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`) Values of M i ` for Eq. (IV-2) N 1 2 3 4967 169 ± 139.464 478 ± 241.719 144 ± 122.556 176 ± 143.967 The values of M have been recalculated so that M 1 ` = 1 x 169; M 2 ` = 2 x 239; M 3 ` = 3 x 48 and M 4 ` = 4 x 44. The value of n is the sum of these components, which is calculated as follows: n = (169 + 2 x 239 + 3 x 48 + 4 x 44) = 967 189 The values of variance are recalculated according to Eq. (IV-3), but using the new values of M i’ and n, for example: var(M 2 `) = 967 x (478 / 967) x (1 – (478 / 500)) = 241.719 The values of M i and var(M i ) from Table IV-2 can then be used to calculate the first part of the var(NDI), as given in Eq. (IV-4): # ∑ = > 412 `) var( `i ii MM = M 12 x var(M 1 ) + M 12 x var(M 1 ) + M 12 x var(M 1 ) + M 12 x var(M 1 )= (169 2 x 139.464) + (478 2 x 241.719) + (144 2 x 122.556) + (176 2 x 143.967) = 66 212 947.630 Next, according to Eq. (IV-1), one must find the covariance of each data set. This is calculated according to the formula: > jiji pnp MM −=`) `, cov( (IV-5) In this equation, p i and p j are the probability of observing each number of micronuclei in the binucleated cells, so for M 1 to M 4 , the probability is calculated as follows: p1 = 169 / 967 = 0.175 p2 = (2
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x 239) / 967 = 0.494 p3 = (3 x 48) / 967 = 0.149 p4 = (4 x 44) / 967 = 0.182 So the covariance of M 1 ` and M 2 ` is calculated according to Eq. (IV-5): cov(M 1 `,M 2 `) = - 967 x 0.175 x 0.494 = - 83.539 The values of covariance must then be similarly calculated for every set of M i `, M j `: cov(M 1 `,M 3 `) = - 967 x 0.175 x 0.149 = - 25.166 cov(M 1 `,M 4 `) = - 967 x 0.175 x 0.182 = - 30.759 cov(M 2 ,M 3 ) = - 967 x 0.494 x 0.149 = - 71.181 cov(M 2 ,M 4 ) = - 967 x 0.494 x 0.182 = - 86.999 cov(M 3 ,M 4 ) = - 967 x 0.149 x 0.182 = - 26.209 Following this, the individual components of the second half of Eq. (IV-2) must then be calculated. For example, for i = 1 and j = 2: M1 `M 2 ` cov(M 1 `,M 2 `) = 169 x 478 x (- 83.539) = -6748429.704 Similarly, so that the sums can be made from i = 1 to 4 and j = i+1 to 4: M1 `M 3 ` cov(M 1 `,M 3 `) = 169 x 144 x (- 25.166) = -612 451.806 M 1 `M 4 ` cov(M 1 `,M 4 `) = 169 x 176 x (- 30.759) = -914 897.142 M 2 `M 3 ` cov(M 2 `,M 3 `) = 478 x 144 x (- 71.181) = -4 899 528.670 M 2 `M 4 ` cov(M 2 `,M 4 `) = 478 x 176 x (- 86.999) = -7 319 049.001 M 3 `M 4 `
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cov(M 3 `,M 4 `) = 144 x 176 x (- 26.209) = -664 238.196 Once all the individual components have been calculated, these can be summed as per the second half of Eq. (IV-2), to give a total of -21 158 594.519. According to Eq. (IV-2), the variance on the NDI is thus: var(NDI) = 66 212 947.630 + 2 x (-21 158 594.519 )= 23 895 758.592 To convert this to a normalized value of standard error, for presentation with the value of NDI, the following equation is used: 190 2/3)var( )var( )( nNDI nnNDI NDI SE == (IV-6) Using the values calculated above, this gives a standard error of: SE(NDI) = (23 895 758.592) (1/2) / 967 (3/2) = 0.163 Thus the calculated value of NDI using the data presented in Table IV-1 is 1.934 ± 0.163. 191 Annex V CRITERIA FOR DETERMINING MITOTIC INDEX The procedure for determining the mitotic index for the dicentric assay is to: > • Exclude nuclei from polymorphonuclear cells, unstimulated cells (small nuclei), dead or dying cells and micronuclei. > • Count the number of nuclei from mitotic cells and stimulated cells (blast cells with large nuclei) and use Eq. (V-1) to calculate the mitotic index in stimulated cells. Given the range in sizes of nuclei from stimulated cells, an arbitrary cut-off has to be established between small stimulated nuclei and unstimulated nuclei. ‘Metaphase spreads’ would include prophases and anaphases. blasts metaphases metaphases dex Mitodic In +×= #100 )(# (V-1) In Fig. V-1, the mitotic index would be (3/(3+12)) x 100= 20%, although typically 500 cells are counted for a full mitotic index analysis. FIG. V-1. A low magnification view of a typical lymphocyte culture slide. White circles are nuclei counted as blasts, red circles are nuclei which are not counted,
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boxes are metaphase spreads. 193 Annex VI STATISTICAL ANALYSIS Examples of calculations using statistical procedures for the analysis and interpretation of cytogenetic biological dosimetry data have been given earlier in this publication, notably in Sections 8 and 9. There is a wide selection of statistics text books available, some aimed specifically towards biological and biomedical applications. Therefore this publication does not set out to cover statistics in great detail. However in this Annex a brief introduction is given to the statistical tests and distributions most frequently encountered in the field of cytogenetic biological dosimetry. Part 3 of this Annex provides a software routine for dose response curve fitting. VI-1. BASIC STATISTICAL METHODS FOR CYTOGENETICS VI-1.1. Standard error and standard deviation The standard deviation (SD) of a set of data is simply a measure of the average dispersion (distance) of the numbers from their mean value. It gives an indication of how widely spread the values in the data set are. The standard error in the mean (SEM) is a measure of how far the sample mean is likely to deviate from the true population mean. It is equivalent to the estimated standard deviation of the error in the method. The SEM quantifies how accurately the true mean of the population is known. The SEM gets smaller as sample size increases, because the mean of a large sample is much more likely to be closer to the true population mean than the mean of a small sample. VI-1.2. p values The p value represents the probability of obtaining a result at least as extreme as a given data point, assuming that the data point was the result of chance alone. For example, given a null hypothesis that two population means are identical, a p value of 0.03 would represent a 3%
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chance of observing a difference as large as the measured difference if the null hypothesis was true. Random sampling from identical populations would lead to a difference smaller than measured in 97% of experiments and larger than measured in 3% of experiments. For statistical tests, if p > the significance level (often 0.05), the data do not depart significantly from the expected model and thus the null hypothesis cannot be rejected. It is important to note that, in the above situation, it is impossible to conclude that the null hypothesis is true, just that either ‘The null hypothesis can be rejected’ (p < 0.05) or ‘The null hypothesis is not significantly not true’ (p ≥ 0.05). For multiple comparisons, the p value must be amended as follows: For a number of independent null hypotheses N, the probability of obtaining one or more p values less than the threshold, t = 0.05, by chance is 100(1.00 − 0.95 N). The threshold required to ensure that the overall risk of incorrectly rejecting a true null hypothesis is ≤ 0.05 is 1.00 − 0.95 (1/N) . VI-1.3. The chi-squared test The chi-squared test (the residual deviance or residual sum of squared deviations (Pearson χ2 ))is used to evaluate statistically significant differences between proportions of normally distributed results. The p value for χ2 (with the associated number of degrees of freedom) gives the probability that differences between the results are due to chance. It is usual to set the significance level at 95%, which means that for a normally distributed set of data, we would only expect this degree of variation 5% of the time. 195 The chi-squared test for homogeneity allows comparison of a number of measurements, testing the null hypothesis that the relative frequencies of observed events follow the chi-squared distribution. In
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cytogenetics, the chi-squared test for homogeneity is used to test for differences between a number of sets of data, for instance observed numbers of dicentrics in scored cells, to determine the number of distinct populations within the data set. In general, the chi statistic is only reliable for sample sizes greater than ~ 5. For smaller sample sizes, the Yates correction can be applied in order to reduce the error introduced by approximation of the data to the chi-squared distribution. Effectively, the correction reduces the chi-squared statistic and thus increases the associated p-value. However the applicability of the correction varies and the correction factor may be too great, thus caution is advised in its application. In the special case of comparison of two samples, the data are expected to be distributed binomially. In this case, χ2, is calculated using the normal approximation to the binomial distribution, this is the chi-squared test for one degree of freedom. A binomial version of the chi-squared test can be used to compare a single set of observed and expected counts, for example the number of dicentrics in a control, unexposed, blood sample to the number in an exposed sample. VI-1.4. The t-test The t-test is a statistical hypothesis test for which the null hypothesis is true if the test statistic, t, is t-distributed. The test is valid for small samples, for which the population cannot be specified as normally distributed because the population standard deviation is uncertain. The t-test takes the effect of chance into account, by incorporating information about the number of samples. In cytogenetics, the t-test is usually used to test for the significance of a difference between the two Poisson counts, comparing the means to determine whether the two sets of data have come from the same population. Again, the p
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value is used to identify whether differences between samples are significant and it is usual to set the significance level at 95% or 0.05. There are a number of different forms of the t-test, which are valid in different situations. The paired t-test is used for samples with direct dependence. An example of this would be numbers of dicentrics scored by two different scorers on the same set of slides. For the paired t-test, the size of the two samples, e.g. the numbers of cells scored, must always be equal. The unpaired t-test is for independent sets of data, for example numbers of dicentrics scored by two different scorers on two different sets of slides. In this case the sample sizes may be the same or different. T-tests may be one or two sided. A one sided test is used to determine whether one sample is significantly larger than a second sample. A two sided test is used to determine whether differences between data sets are significant in either direction, i.e. sample one is larger or smaller than sample two. VI-1.5. The F-test The F-distribution is a continuous probability distribution which is equivalent to the ratio of two chi-squared distributions. An F-test, based on this distribution, can thus be used to compare data to see whether they come from the same distribution. The F-test or z-test can be used to test for significance of the coefficients produced by curve fitting by maximum likelihood. In the case where there is evidence of lack of fit (e.g. from the χ2 test), the t-test should be used to test for significance of the coefficients. In contrast to the t-test, which is used to compare means, the F-test compares variances of data sets. The most common use of the F-test is in analysis of
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variance testing. 196 VI-1.6. ANOVA Analysis of Variance refers to a collection of several methods which are used to test equality of means. ANOVA uses the F-distribution to test for differences among three or more independent, normally distributed groups, with homogeneity of variances, or between repeated measurements. ANOVA evaluates the importance of one or more factors by comparing the response variable means at the different factor levels. The p value for each factor describes the probability that the high variance among the groups compared to the variation within groups, is by chance. The p value can be thought of as the probability that random sampling would result in means as far apart (or more so) as observed in the experiment. In cytogenetics, ANOVA may be used in any circumstance when comparison of three or more groups, or two or more factors, is required. This may be, for instance, to test for the combined effects of radiation dose level and dose fractionation or of radiation and chemical exposure. There are a large number of different forms of the test but most commercially available data manipulation packages have ANOVA capabilities and further guidance can be found in statistical texts. Although in principal, ANOVA is a parametric method of analysis, which can usually only be applied to Normal data, the type of data most frequently encountered in cytogenetics (i.e. Poisson distributed), approximates the Normal distribution in a manner sufficient to ensure that ANOVA can be applied. Alternatively, a large number of non-parametric analyses are available, as discussed below. VI-1.7. Non-parametric tests In cases where the condition of Normality cannot be met, non-parametric tests can be applied. The Wilcoxon test is a non-parametric test that is analogous to the paired t-test. It can be used to compare one or two sets of data. The
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test is a signed rank test, and as such requires that data are measured at repeated intervals. The test statistic looks for equality of population medians. For independent samples, the Mann Whitney test can be used. This is the non-parametric version of the t-test which can be used to test whether two sets of unpaired data come from the same distribution. For comparisons of multiple data sets, the Kruskal Wallis test is an extension of the Mann Whitney test which is analogous to ANOVA. VI-2. STATISTICAL DISTRIBUTIONS There are several forms and classes of distributions that can be used to model the probability of occurrence of events. The type of distribution chosen is very important for accurate data analysis, and several models have been proposed and implemented in assessment of cytogenetic data. A selection of the most commonly used models, and their applicability to radiation cytogenetics, are discussed below. VI-2.1. The Poisson distribution The Poisson is a discrete probability distribution which expresses the probability of occurrence of rare random events. The Poisson distribution is by far the most widely recognized and commonly used type of distribution for cytogenetic data analysis. Chromosome aberration data are usually fairly small in number, and Edwards et al. showed that it is much more realistic to assume that chromosome aberrations follow the Poisson distribution than the Normal distribution . Merkle showed that Poisson-based goodness of fit tests, including the χ2 , variance and u-test that are all discussed in this publication, were shown to be applicable for cytogenetic data, particularly in the case of large sample sizes . For curve fitting, regression analysis has been shown to be applicable for Poisson data. The resulting forms of maximum 197 likelihood and/or weighted least squares fitting are now almost universally used for creating dose based
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calibration curves for chromosome aberrations, such as dicentrics or micronuclei. > VI-2.2. Binomial distribution. The binomial distribution is a discrete probability distribution which describes the probability of the number of successful outcomes from a sequence of independent experiments, each with one of two possible outcomes. In each case, if outcome 1 has an associated level of probability of p, outcome 2 will have probability 1 – p. In cytogenetics, a good example of a set of data that can be modelled with this distribution is counting numbers of damaged cells, where the two ‘binomial’ outcomes are a cell is either damaged, or intact. Indeed, the binomial distribution is often used to calculate standard errors associated with yields of damaged cells. > VI-2.3. The mixed Poisson model Sasaki presented a method of analysis for chromosome aberration data, in an attempt to deal with the problems of inappropriate estimation of average dose which result from inhomogeneity. The cell population consists of a mix of sub populations, each exposed to a different dose, causing a different amount of damage. The distribution of chromosome damage in cells can therefore be expressed in terms of a mixed Poisson distribution, and ‘unfolding’ of this creates a dose distribution profile. The model was demonstrated to provide adequate fits for the linear-quadratic dose response for simulated and real data. > VI-2.4. The negative binomial distribution Like the Poisson distribution, the negative binomial distribution is a discrete probability distribution, however the negative binomial has an additional parameter which can be used to represent overdispersion. As the overdispersion parameter tends to 0, the negative binomial tends to Poisson . The negative binomial distribution has been used by several authors in place of the Poisson, for example in a 2008 study of frequency of translocations in airline pilots . >
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VI-2.5. The Neyman type-A distribution The Neyman distribution was first proposed in 1939 by Neyman, who introduced this new class of distribution to be used to test the difference between means of two samples with different variances. This is in contrast to other standard test such as the z-test and t-test, for example, which are based on normally distributed data with known and unknown population standard deviations respectively, and for which variance must be similar if not identical. The Neyman type-A distribution tends towards the generalized Poisson distribution with increasing sample size . In 2008, Morand et al. published a technical note describing the NETA computer program, which can be used to calculate the 95% confidence limits of Neyman type A distributed events . Morand and colleagues found that the confidence limits calculated using the Neyman distribution were smaller than those calculated using the traditional Poisson-based method for low sample sizes (numbers of cells) . > VI-2.6. Other distributions The Beta distribution defines a family of continuous probability distributions, which are defined on the interval 0–1 by two shape parameters, usually referred to as α and β. The Dirichlet distributions are an extension of the Beta distribution for multiple (>2 parameters). Stiratelli et al. compared the Poisson and Binomal distributions for chemically induced chromosome damage with the beta-binomial, negative-binomial and correlated-binomial distributions. In contrast to the Poisson and simple Binomial distributions, these models do not rely on independence of cellular response. The authors found that all the Beta distribution based > 198 models showed improved fits with respect to the Poisson and Binomial models (as tested by the χ2 test). The Beta-binomial model provided the best fit with respect to the author’s data set . The log-normal distribution was formally described by Aitchison and Shen in 1980. Logistic transformation
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of a d-dimensional normal distribution produces a log-normal distribution over the d-dimensional simplex. This distribution can be applied in statistical diagnosis where classification of the basic cases is subject to uncertainty, such as chromosomal aberration data. The authors give examples of usage, for instance in the direct statistical description and analysis of compositional and probabilistic data and also as a substitute for the Dirichlet conjugate prior class in the analysis of multinomial and contingency table data . VI-3. A ROUTINE FOR FITTING DOSE RESPONSE CURVES Curve fitting software has been described in Section 8.3. In this Annex a worked example is presented using one of the software options, the R-based tool, applied to the 60 Co data shown in Table 4. Whereas CABAS and Dose Estimate are available as ready-to-use packages, the R procedure needs a routine to be written by a mathematician. The required routine has been composed (by H. Braselmann) and is presented here in Box 1 in full because it has not been published elsewhere. The routine has four parts. The first is used to input the observed data i.e., doses, numbers of aberrations, number of cells scored and the distribution index (disp). For this index there are two options; either to use a constant value for every dose point or to ascribe a separate value to each dose. In the worked example a constant value of 1.0 is used. The alternative, which is also shown, would be to use the individual σ2/y values shown in Table 4. (Note that all lines in the routine starting with a ‘#’ symbol are for information only and will not run). The next part is for entering optimal settings; i) the sigma correlation coefficient, for which it is recommended to use the value 1 or else to estimate this coefficient;
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ii) the required weight and; iii) the function that one wishes to fit. For this, enter either ‘l’ for a linear fit or ‘lq’ for linear quadratic. The remaining two parts of the routine should only be modified by developers of the script. If one wishes to fit data to the linear dose response function, the data that would be entered using the 3 He data in Table 4 are shown beneath Box 1. Thereafter the routine is identical to that shown in Box 1. To run the routine one should download the R programme from the internet site (see Section 8.3). Using a PDF version of this publication, copy and paste directly into a word processor software the routine shown in Box 1. Replace the 60 Co worked example input data with your own data and select your desired options such a ‘l’ or ‘lq’. With the R programme on your screen, paste in the routine after the symbol >. The output is shown in Box 2 where x0, x1 and x2 are, respectively, the C, α and β coefficients as shown in Eq. (2) together with their standard errors. The z value is a test of the significance of each coefficient with its probability (Pr). Also shown are the variance and covariance values for each coefficient. It may be noted that the values of the coefficients are identical to those shown in Table 5 and the variance / covariance values with those shown in Section 9.7.3. The R output also presents the data points and the fitted curve as a graph (Fig. VI-1). 199 BOX 1. THE CURVE FITTING ROUTINE APPLIED AS A WORKED EXAMPLE TO 60 CO DATA ## latest changes: H. Braselmann, 2010, April 9 th ## Helmholtz Zentrum München, Department of Radiation Cytogenetics, Germany ##
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contact details: braselm@helmholtz-muenchen.de ## user part: data # cobalt-60 gamma (86) dose<-c(0,0.1,0.25,0.5,0.75,1,1.5,2,3,4,5) ab<-c(8,14,22,55,100,109,100,103,108,103,107) cells<-c(5000,5002,2008,2002,1832,1168,562,332,193,103,59) disp<- 1.0 #disp<- c(1.0,1.0,1.08,0.97,1.03,1.0,1.06,1.14,0.83,0.88,1.15) ## user part: option settings sigma<- 1 # regression sigma 1 or #sigma<- NULL # NULL (regression sigma estimated) wt<- 1/disp # weight setting, required! model<- "lq" #model<- "l" # "l" for linear or "lq" for linear quadratic # a background value (c) is fitted in both options ############################################################################ ## execution part: changes recommended only for developpers of the script ## ############################################################################ if (length(disp)==1) disp<- rep(disp,length(dose)) kurvendaten<-data.frame(dose,ab,cells,disp) print(kurvendaten) x0<-cells x1<-cells*dose x2<-cells*dose*dose modelldaten<-list(x0,x1,x2,ab) if (length(wt)==1) wt<- rep(wt,length(dose)) if (model=="lq" & sigma==1) result<-glm(ab ~ -1 + x0+x1+x2,family=poisson(link = "identity"), weights=wt, data=modelldaten) if (model=="lq" & is.null(sigma)) result<-glm(ab ~ -1 + x0+x1+x2,family=quasipoisson(link = "identity"), weights=wt, data=modelldaten) if (model=="l" & sigma==1) result<-glm(ab ~ -1 + x0+x1,family=poisson(link = "identity"), weights=wt, data=modelldaten) if (model=="l" & is.null(sigma)) result<-glm(ab ~ -1 + x0+x1,family=quasipoisson(link = "identity"), weights=wt, data=modelldaten) smry<-summary(result,correlation=TRUE) #smry$coefficients #smry$correlation corma<-smry$correlation bstat<-smry$coefficients seb<-bstat[,2] vakoma<-corma*outer(seb,seb) vakoma<-vcov(result) ####################### ## output of results ## ####################### cat("\n") cat("Result of curve fit 'result'\n") cat("----------------------------\n") print(result) cat("\n") cat("assumed sigma\n") print(sigma) cat("\n") cat("Coefficients 'bstat'\n") print(bstat) cat("\n") cat("variance-covariance matrix 'vakoma'\n") print(vakoma) cat("\n") cat("correlation matrix 'corma'\n") print(corma) 200 par(lwd=2) plot(dose, ab/cells) if (model=="lq") curve(bstat[1,1]+bstat[2,1]*x+bstat[3,1]*x*x,0,max(dose), add=TRUE) if (model=="l") curve(bstat[1,1]+bstat[2,1]*x,0,max(dose), add=TRUE) The input data for fitting 3 He data to the linear model. # 20 MeV helium α- particles (87) dose<-c(0,0.051,0.104,0.511,1.01,1.536,2.05,2.526,3.029) ab<-c(3,19,27,199,108,96,120,148,108) cells<-c(2000,900,1029,1136,304,142,137,144,98) disp<- 1.19 sigma<- NULL wt<- 1/disp model<- "l" BOX 2. THE OUTPUT FOR THE FIT TO THE 60 CO DATA 201 FIG. VI-1. The output for the 60 Co data presented as a graph showing the observed data points and the fitted linear quadratic curve. 202 Annex VII AN EXAMPLE OF AN INTERLABORATORY COMPARISON EXERCISE FOR QUALITY ASSURANCE This Annex provides an example of an international interlaboratory comparison performed among 14 biological dosimetry laboratories. The exercise comprised the analysis
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of slides of metaphase preparations from blood that had been irradiated in vitro to 0.75 and 2.5 Gy with 60 Co γ-rays. Participating laboratories were required to report the frequency of dicentrics that they obtained and the estimated dose after the analysis of 50, 100 cells (triage mode) and after conventional scoring of 500 cells or stopping sooner if 100 dicentrics was reached. For this laboratory interlaboratory comparison, the performance of each laboratory and reproducibility of the exercise was evaluated using robust methods (algorithms A and S) described in ISO standards ISO 5725-5 and ISO 13528:2005 [15, 16]. This annex shows as a worked example just a subset of the interlaboratory comparison results, those obtained after analysing 500 cells at 0.75 Gy. Full details are described in . To determine the laboratory performance the z-test was used: # ( ) # ( ) 22* > xref i usxxz +−= (VII-1) For each laboratory the z-test considers its reported values for the frequency of dicentrics observed or the estimated dose derived by referring the dicentric frequency to its own pre-existing dose response curve ( xi). For the frequency analysis, xref was a consensus value (robust average, x*, obtained by the algorithm A), and for the dose estimation analysis it was the physical dose administrated. The z-test also takes into account the robust standard deviation ( s *)obtained by the algorithm A and the standard uncertainty of the consensus or reference value (ux). When frequencies were evaluated ux was calculated as follows: psu ref > * 25 .1= (VII-2) where p is the number of participating laboratories. Regarding dose estimation, the uncertainty, designated u x, on the physical measurements of the actual doses delivered to the blood samples was considered to be negligible according to the criteria shown in Eq. (VII-3). Regarding
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dose estimation u x was the uncertainty on the physical dose delivered. For each analysis, ux was considered negligible according to the following criteria: # ( ) 1uss96 0 2x2 ≤+≤ **. (VII-3) To evaluate the laboratory performance, the following criteria were applied: | z | ≤ 2 satisfactory 2 -2,00 -1,50 -1,00 -0,50 0,00 0,50 1,00 1,50 L5 L1 L6 L13 L10 L9 L3 L12 L14 L8 L7 L2 L4 L11 # Laboratory z-score L5 L1 L6 L13 L12 L14 L3 L9 L10 L8 L4 L2 L7 L11 0,00 0,20 0,40 0,60 0,80 1,00 1,20 1,40 012345678910 11
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12 13 14 15 # Laboratory Dose [Gy] 204 for each participating laboratory that in the present exercise was 2. After the analysis of 500 cells at 0.75 Gy the SR values were 0.013 for the frequency and 0.116 for the dose. To compare the reproducibility of both measurements, frequency and dose, the coefficient of variation (CV) was defined. The CV indicates the global dispersion of the results and it is calculated as the ratio SR /x* . In the present example the obtained coefficients were 24.4% and 15.6% for frequency and dose respectively. These results indicated a better reproducibility when estimated doses were considered rather than the dicentric frequencies. Future interlaboratory comparisons among the same laboratories will determine if the reproducibility can be improved. If it cannot be improved, then the obtained value will be accepted as the variability associated with the random errors of the method. 205 REFERENCES TO THE ANNEXES FENECH, M., Cytokinesis-block micronucleus cytome assay, Nat. Protoc. 2 (2007) 1084–1104. WEIER, H.G., et al., Two colour hybridization with high complexity chromosome-specific probes and a degenerate alpha satellite probe DNA allows unambiguous discrimination between symmetrical and asymmetrical translocations, Chromosoma 100 (1991) 371–376. FENECH, M., et al., HUMN project: detailed description of the scoring criteria 4686 for the cytokinesis-block micronucleus assay using isolated human lymphocyte 4687 cultures, Mutat. Res. 534 (2003) 65–75. EDWARDS, A.A., LLOYD, D.C., PURROT, R.J., Radiation induced chromosome aberrations and the Poisson distribution, Radiat. Environ. Biophys. 16 (1979) 89–100. MERKLE, W., Poisson goodness-of-fit tests for radiation-induced chromosome aberrations, Int. J. Radiat. Biol. 40 (1981) 685–692. FROME, E.L., DUFRAIN, R.J., Maximum likelihood estimation for cytogenetic dose-response curves, Biometrics 42 (1986) 73–84. PAPWORTH, D.G., SAVAGE, J.R.K., “Curve fitting by maximum likelihood”, Radiation-Induced Chromosomal Aberrations in Tradescantia: Dose Response Curves, Radiat.
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Bot. 15 (1975) 87–140. SASAKI, M.S., Chromosomal biodosimetry by unfolding a mixed Poisson distribution: a generalized model, Int. J. Radiat. Biol. 79 (2003) 83–97. BRAME, R. S., and GROER, P. G., Bayesian methods for chromosome dosimetry following a criticality accident. Radiat. Prot. Dosim. 104 (2003) 61–63. YONG, L.C., et al., Increased frequency of chromosome translocations in airline pilots with long-term flying experience, Occ. Environ. Med. 66 (2008) 56–62. NEYMAN, J., On a new class of “contagious” distribution, applicable in entomology and bacteriology, Am. Math. Stat. 10 (1939) 35–55. MORAND, J., et al., Confidence limits for Neyman type A-distributed events, Radiat. Prot. Dosim. 128 (2008) 437–443. STIRATELLI, R.G., MCCARTHY, K.L., SCRIBNER, H.E., Parametric approaches to the analysis of in vivo cytogenetics studies, Environ. Mutagen. 7 Suppl. 4 (1985) S43– S54. AITCHISON, J., SHEN, S.M., Logistic-normal distributions: some properties and uses, Biometrika 67 (1980) 261–272. INTERNATIONAL ORGANIZATION FOR STANDARDIZATION, “Alternative methods for the determination of the precision of a standard measurement method”, International Standard. Accuracy (Trueness and Precision) of Measurement Methods and Results, ISO 5725-5, ISO, Geneva (1998). INTERNATIONAL ORGANIZATION FOR STANDARDIZATION, International Standard. Statistical Methods for Use in Proficiency Testing by Interlaboratory Comparisons, ISO 13528, ISO, Geneva (2005). DI GIORGIO, M., BARQUINERO, J.F., VALLERGA, M.B., RADL, A., TAJA, M.R., SEOANE, A., DE LUCA, J., STUCK OLIVEIRA, M., VALDIVIA, P., GARCIA LIMA, O., LAMADRID, A., GONZALEZ MESA, J., ROMERO AGUILERA, I., MANDINA CARDOSO, T., GUERRERO CARBAJAL, Y.C., ARCEO MALDONADO, C., ESPINOZA, M.E., MARTINEZ-LOPEZ, W., MENDEZ-ACUÑA, L., DI TOMASO, M., ROY, L., LINDHOLM, C., ROMM, H., GÜÇLÜ, I., LLOYD, D.C., Biological dosimetry intercomparison exercise: an evaluation through triage and routine mode results by robust methods. Radiation Research (2011, in press). > 207 LIST OF ABBREVIATIONS ace acentric fragment AFRRI Armed Forces Radiobiology Research
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Institute (USA) ANOVA analysis of variance ARS acute radiation syndrome AS abasic sites ATP adenosine triphosphate BD base damage BER base excision repair BN binucleated BrdU bromodeoxyuridine BSS Basic Safety Standards CABAS chromosomal aberration calculation software CBMN cytokinesis-block micronucleus assay CBMN Cyt cytokinesis-block micronucleus cytome assay CCD charge-coupled device CHO Chinese hamster ovary CP centromeric probe CRP Co-ordinated Research Programme Cyt-B cytochalasin-B DAPI 4′,6 ′-diamidino-2-phenylindole DCA dicentric chromosome assay df degrees of freedom dic dicentric chromosome DMSO dimethylsulphoxide DNA deoxyribonucleic acid DPC DNA-protein cross-links > 209 DSB double strand break EDTA ethylene diamine tetra acetic acid ESR electron spin resonance FISH fluorescence in situ hybridization FPG fluorescence plus Giemsa HBSS Hank’s balanced salt solution HIV human immunodeficiency virus HPBL human peripheral blood lymphocytes HRR homologous recombination repair HUMN human micronucleus IAEA International Atomic Energy Agency ICRP International Commission on Radiological Protection ICRU International Commission on Radiation Units and Measurements IND improvised nuclear devices IRSN Institut de Radioprotection et de Sûreté Nucléaire (France) ISO International Organization for Standardisation IU international unit LCL lower confidence limit LET linear energy transfer LIMS laboratory information management system M1, M2, ... first, second, … in vitro division metaphase MDS multiple damage sites MEM minimum essential medium mFISH multicolour fluorescence in situ hybridization MN micronucleus (micronuclei) MNCM -ve/+ve micronucleus centromere negative/positive cell 210 NBUD nuclear bud NDI nuclear division index NHEJ non-homologous end-joining NER nucleotide excision repair NIRS National Institute of Radiological Sciences (Japan) NPB nucleoplasmic bridge NPP nuclear power plant OA okadaic acid PAINT Protocol for Aberration Identification and Nomenclature Terminology PBS phosphate buffered saline PCC premature chromosome condensation PCR polymerase chain reaction PEG polyethylene glycol PHA phytohaemagglutinin QA quality assurance QC quality control RBE relative biological effectiveness RDD radiological dispersal device REAC/TS Radiation Emergency Assistance Center/Training Site (USA) RED radiological exposure device RICA rapid
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interphase chromosome assay RNase ribonuclease SD standard deviation SE standard error SEM standard error of the mean SI International System of Units > 211 SSB single strand break SSBR single strand break repair SSC saline sodium citrate TLD thermoluminescence dosimeter UCL upper confidence limit UN United Nations UV Ultraviolet WHO World Health Organization > 212 DEFINITIONS 3 absorbed dose (D)*. The fundamental dosimetric quantity D, defined as: dm dD ε= where: dε is the mean energy imparted by ionizing radiation to matter in a volume element, and dm is the mass of matter in the volume element. • The energy can be averaged over any defined volume, the average dose being equal to the total energy imparted in the volume divided by the mass in the volume. • Absorbed dose is defined at a point; for the average dose in a tissue or organ, see organ dose. • Unit: gray (Gy), equal to 1 J/kg (formerly, the rad was used). accident*. Any unintended event, including operating errors, equipment failures and other mishaps, the consequences or potential consequences of which are not negligible from the point of view of protection or safety. criticality accident. An accident involving criticality. • Typically, in a facility in which fissile material is used. acentric (ace). Terminal or interstitial chromosome fragment of varying size lacking a centromere. An acentric formed independently from a dicentric, tricentric, or centric ring aberration is usually referred to as an excess acentric. alpha radiation. Particle radiation emitted in the nuclear disintegration of certain radionuclides. Alpha particles consist of two neutrons and two protons and are identical with the nucleus of the helium atom. They are readily absorbed by a few centimetres of air and therefore the main hazards come from internally incorporated alpha emitting nuclides. aneugen. An indirect mutagen able to
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affect cell division and the mitotic spindle apparatus resulting in the loss or gain of whole chromosomes, thus inducing an aneuploidy. ankylosing spondylitis. Chronic, inflammatory arthritis which affects the spine and the sacroilium in the pelvis. Many decades ago, large field external beam radiation was used to treat the inflammation of the spines in these patients. anticoagulant. A drug which prevents the clotting (coagulation) of blood. background frequency/level/value. Incidence (or number) of chromosome aberrations or micronuclei recorded in the general population. 3 Definitions apply for the purposes of the present publication. Definitions marked with an asterisk are taken from Ref. . 213 becquerel (Bq)*. The SI unit of activity, equal to one transformation per second. • Supersedes the non-SI unit curie (Ci). 1 Bq = 27 pCi (2.7 × 10–11 Ci) approximately. 1 Ci = 3.7 × 1010 Bq. > beta radiation. Particle radiation comprising electrons with positive or negative charge emitted in the nuclear disintegration of certain radionuclides. The penetration of beta particles is a few centimetres to metres in air and a few millimetres to centimetres in soft tissue or plastic. > bias. Deviation of results or inferences from the truth or processes leading to such deviation. > binucleated. Having two nuclei. Binucleated cells occur at the end of the nuclear division cycle and can be accumulated using a cytokinesis-block inhibitor such as cytochalasin-B. Binucleated cells are scored for the presence of micronuclei and nucleoplasmic bridges in the cytokinesis-block micronucleus assay. > biological dosimetry/biodosimetry. The use of biomarkers to verify exposure to radiation and to estimate absorbed dose. > biological effects. Range of possible consequences on living material, organisms, tissues, or cells, depending on type and degree of cellular damage that may result from exposure to an external agent, such as ionizing radiation. > biomarker. An indicator of
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normal biological or pathogenic processes. Within the scope of biological dosimetry, they are used to distinguish radiation-induced biological damage from that produced by other agents. > Bragg-Gray cavity theory. Relates the ionization produced within a gas-filled cavity inside a medium to the energy absorbed in that surrounding medium. In the context of chromosome aberration formation, applying the theory means that the size of the cell nucleus is so small that the total energy absorbed is only due to electrons passing through the nucleus. Therefore secondary particles can be ignored. > 5-Bromodeoxyuridine (BrdU ). An analogue of thymidine in which the methyl group at the 6´ position in thymine is replaced by bromine. BrdU is used in biological dosimetry for differentially labelling newly synthesized DNA to identify cells having passed through mitosis more than once. > buffy coat. The layer of an anticoagulated blood sample after centrifugation that contains most of the white blood cells. > calibration curve. In biological dosimetry, a graphical or mathematical description of the dose effect relation derived by the in vitro irradiation of blood samples to known doses. The curve is used to determine, by interpolation, the absorbed radiation dose to a potentially exposed individual. > C-banding. See ‘chromosome banding’. > centromere. Primary constriction region of a chromosome that is visualized during mitosis and joins together the chromatid pair. > 214 chain-of-custody. The complete record of a blood sample by tracking its handling and storage from point of specimen collection/receipt to final disposition of the specimen. > charged particle equilibrium. Occurs when the number of each type of charged particles leaving a given volume is equal to those entering it. > chromosome banding. A technique for the differential staining of chromosomes, most commonly using Giemsa stain. Depending on the method, a selective staining of certain chromosomal
|
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regions such as centromeres (C-banding) or characteristic patterns along the arms (G-banding) are visualized. The specific pattern of dark and light stripes (bands), unique to each chromosome pair, is used to identify them and evaluate their structure. > clastogen. A physical or chemical agent that breaks DNA in chromosomes, leading to rearrangements such as the aberrations observed in metaphase. > clustered DNA lesions. More than two sites of DNA damage generated by ionizing radiation within 20 bps of the same molecule. > colchicine/Colcemid. Alkaloid compounds that inhibit spindle formation during cell division. They are used to collect a large number of metaphase cells by preventing them from progressing to anaphase. Colcemid is a synthetic analogue of the natural, plant-derived, colchicine. > complex rearrangement. An aberration involving three or more breaks in two or more chromosomes and is characteristically induced after exposure to densely-ionizing radiation or high doses of sparsely ionizing radiation. > confidence interval. An interval estimate for a variable of interest, e.g. a rate, constructed according to a chosen distribution (e.g. the Poisson) so that this range has a specified probability of including the true value of the variable. Thus, a confidence interval (described by the upper and lower confidence limit) is used to indicate the reliability of an estimate. How likely the interval is to contain the parameter is determined by the confidence level or confidence coefficient. Increasing the desired confidence level will widen the confidence interval. > confound. To ‘disturb’ the correlation between an influencing variable (e.g. exposure to ionizing radiation) and effect (e.g. induced aberrations) investigated in a study by another variable (confounder, e.g. age, smoking). If confounders are not taken into consideration a correlation that does not exist in reality can be pretended or a real correlation can be blurred. > contaminated poisson method. A
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mathematical analysis of centric ring and dicentric chromosome frequencies which permits dose assessment in cases of suspected partial body exposures. The method permits dose assessment by considering the distribution of dicentrics among all the scored cells and gives additional information about the irradiated volume of the body. See also Qdr method. > contamination*. Radioactive substances on surfaces, or within solids, liquids or gases (including the human body), where their presence is unintended or undesirable, or the process giving rise to their presence in such places. > 215 control group. A group of cells, animals, or test persons being exposed to the best possible identical conditions as the exposed individuals, with the exception that the effect to be investigated is not administered. > covariance. A measure of the correlation of the variance between two (or more) dependent sets of data, in other words, how the data vary together. It can be positive or negative, indicating a positive or negative linear relationship between the data sets. If the data are independent, the covariance is zero. > covariance of curve parameters . The parameters (C, alpha, beta) do not deviate independently from its ideally true values (see standard deviation, variance), but do so together up to a certain amount of correlation because they are simultaneously calculated from the same data set. Thus it reduces the error term in combined calculations like Eq. (7) as when calculated with variances alone. Corresponding correlations could also be calculated from the variances and covariances (covariance divided by square root of the product of the two variances, i.e. covariance divided by the product of the standard deviations). > curve fitting. Identifying an equation which describes the best fit to a series of data points, possibly with a number of other constraints, including weighting the fit by the reliability
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of each data point (assessed by the standard error on the point) and/or constraining the fit to a measured baseline value. > cytochalasin B. A natural compound, of fungal origin, with the unique property of inhibiting cytokinesis in mammalian and human cells used in the cytokinesis-block micronucleus assay. > cytogenetics. A branch of genetics that deals with the study of chromosomes > cytokinesis-block micronucleus cytome assay (CBMN Cyt). The CBMN Cyt assay is a more advanced version of the CBMN assay in which a wider range of biomarkers of chromosome damage (micronuclei, nucleoplasmic bridges, nuclear buds in binucleated cells) as well as cell death (necrotic and apoptotic cells) and cytostasis (nuclear division index based on ratios of mononucleated, binucleated and multinucleated cells) are measured. The micronucleus and nucleoplasmic bridge biomarkers are the biomarkers in this system that are best validated for biological dosimetry of ionizing radiation exposure. > densely-ionizing/high-LET radiation. Radiation which deposits its energy in closely spaced interactions along its track (e.g. alpha particles, neutrons). This spatial distribution is reflected in the relative biological effectiveness. See also ‘linear energy transfer, LET’. > detection limit. The dose represented by the lowest frequency of a given biodosimetric marker that can be discriminated above the background frequency with a certain level of confidence, normally 95%. > deterministic effect. A health effect of radiation for which generally a threshold level of dose exists above which the severity of the effect is greater for a higher dose. Such an effect is described as a ‘severe deterministic effect’ if it is fatal or life threatening or results in a permanent injury that reduces quality of life. > dicentric (dic). Aberrant chromosome bearing two centromeres derived from the misrepair of two broken chromosomes. > 216 diploid. The species specific number of chromosomes in a somatic cell;
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