The quantification of DNA damage, both in vivo and in vitro, can be very time consuming, since large amounts of samples need to be scored. Additional uncertainties may arise due to the lack of documentation or by scoring biases. Image analysis automation is a possible strategy to cope with these difficulties and to generate a new quality of reproducibility. In this communication we collected some recent results obtained with the automated scanning platform Metafer, covering applications that are being used in radiation research, biological dosimetry, DNA repair research and environmental mutagenesis studies. We can show that the automated scoring for dicentric chromosomes, for micronuclei, and for Comet assay cells produce reliable and reproducible results, which prove the usability of automated scanning in the above mentioned research fields.

The micronucleus assay (MNT) in human lymphocytes is frequently used to assess chromosomal damage as a consequence of environmental mutagen exposure, to assess the effect of mutagens or to search for reduced DNA repair capacity after a mutagenic challenge. We have established an automated scoring procedure for the cytokinesis blocked MNT based on computerized image analysis (Metasystems Metafer 4 version 2.12). To evaluate the results we used the reproducibility of counts, established a dose-response curve for gamma-irradiation and used the ability of the system to differentiate between breast cancer patients and controls as a biological reference, a difference which we had observed before by visual counting. Blood cultures were irradiated with gamma-rays (2 Gy) at the beginning and treated with cytochalasin B during the last 24 h. The slides were stained with Giemsa for visual counting and with DAPI for automated analysis. Our test sample consisted of 73 persons (27 with breast cancer and 26 female and 20 male controls). A comparison between visual counting (controls, mean MN frequency 313) and automated counting (mean MN frequency 106) in slides from the same culture revealed a large drop for the automated counts. However, the automated counts were as reproducible as the visual counts [coefficient of variation (CV) on the sample approximately 20%; CV on repeated counts of the same slides approximately 5%] and both counts were highly correlated. Furthermore, the discrimination between cases and controls improved for automated counting of slides from the same cultures [visual odds rato (OR) < or = 4.0, P = 0.009; automated OR > 16, P < 0.0001], with a strong dependence on the set of parameters used. This improvement was confirmed in a validation sample of an additional 21 controls and 20 cases (OR = 11, P = 0.0018) performed as a prospective or diagnostic test.

The proliferative life span of human cells is limited by telomere shortening, but the specific telomeres responsible for determining the onset of senescence have not been adequately determined. We here identify the shortest telomeres by the frequency of signal-free ends after in situ hybridization with telomeric probes and demonstrate that probes adjacent to the shortest ends colocalize with gammaH2AX-positive DNA damage foci in senescent cells. Normal BJ cells growth arrest at senescence before developing significant karyotypic abnormalities. We also identify all of the telomeres involved in end-associations in BJ fibroblasts whose cell-cycle arrest at the time of replicative senescence has been blocked and demonstrate that the 10% of the telomeres with the shortest ends are involved in >90% of all end-associations. The failure to find telomeric end-associations in near-senescent normal BJ metaphases, the presence of signal-free ends in 90% of near-senescent metaphases, and the colocalization of short telomeres with DNA damage foci in senescent interphase cells suggests that end-associations rather than damage signals from short telomeres per se may be the proximate cause of growth arrest. These results demonstrate that a specific group of chromosomes with the shortest telomeres rather than either all or only one or two sentinel telomeres is responsible for the induction of replicative senescence.

The detection of disseminated tumor cells (DTCs) in the hematopoetic system is important for various reasons. It is essential for tumor staging. According to the International Neuroblastoma Staging System (INSS) only the cytomorphological examination of bone marrow smears is accepted despite the fact that an infiltrate below 0.1%, can hardly be detected and even infiltrates of more than 10% are sometimes overlooked. Another important aspect is the monitoring of the disease response to cytotoxic drugs by quantifying DTCs. Moreover, bone marrow aspirates represent an ideal source to determine the genetic and biological make up of DTCs at diagnosis and during follow up. Key issues that can be tested on DTCs are: determination of the proliferation capacity, the apoptotic rate, the drug sensitivity etc. The prerequisite for such a bone-marrow diagnosis, however, is the unequivocal identification of disseminated tumor cells. Thus, in order to avoid false positive and false negative results, which are a risk in bone-marrow diagnostics, a system was developed to distinguish tumor cells from non-neoplastic cells and to facilitate the gain of insights into the biological make-up of tumor cells more easily.

<p>The BRCA2 gene is mutated in familial breast and ovarian cancer, and its product is implicated in DNA repair and transcriptional regulation. Here we identify a protein, EMSY, which binds BRCA2 within a region (exon 3) deleted in cancer. EMSY is capable of silencing the activation potential of BRCA2 exon 3, associates with chromatin regulators HP1beta and BS69, and localizes to sites of repair following DNA damage. EMSY maps to chromosome 11q13.5, a region known to be involved in breast and ovarian cancer. We show that the EMSY gene is amplified almost exclusively in sporadic breast cancer (13%) and higher-grade ovarian cancer (17%). In addition, EMSY amplification is associated with worse survival, particularly in node-negative breast cancer, suggesting that it may be of prognostic value. The remarkable clinical overlap between sporadic EMSY amplification and familial BRCA2 deletion implicates a BRCA2 pathway in sporadic breast and ovarian cancer.</p>

<p>The sensitive detection of bone marrow involvement is crucial for tumor staging at diagnosis and for monitoring of the therapeutic response in the patient's follow-up. In neuroblastoma, only conventional cytomorphological techniques are presently accepted for the detection of bone marrow involvement, yet since the therapeutic consequences of the bone marrow findings may be far-reaching, the need for highly reliable detection methods has become evident. For this purpose, we developed an automatic immunofluorescence plus FISH (AIPF) device which allows the exact quantification of disseminated tumor cells and the genetic verification in critical cases. In this study, the power of the immunofluorescence technique is compared with conventional cytomorphology. 198 samples from 23 neuroblastoma patients (stages 4 and 4s) at diagnosis and during follow-up were investigated. At diagnosis, 45.6% of the samples (26 of 57) which were positive by AIPF investigation were negative by cytomorphology. During follow-up, 74.2% (49 of 66) of AIPF-positive samples showed no cytological signs of tumor cell involvement. False negative morphological results were found in up to 10% of tumor cell content. A tumor cell infiltrate below 0.1% was virtually not detectable by conventional cytomorphology. Using the sensitive immunofluorescence technique, the analysis of only two instead of four puncture sites did not lead to false negative results. Thus, the immunofluorescence technique offers an excellent tool for reliable detection and quantification of disseminated tumor cells at diagnosis and during the course of the disease.</p>

La technique de référence en dosimétrie biologique est basée sur le dénombrement des aberrations chromosomiques de type dicentrique induit par les rayonnements ionisants. Cet article présente divers systèmesd'analyse d'images utilisés en dosimétrie biologique pour aider la détection de ces aberrations. Les systèmes présentés sont le CYTOGEN de la société IMSTAR, le CYTOSCAN (APPLIED IMAGING) et le METAFER (METASYSTEM). Tous ne présentent pas les mêmes fonctionnalités et chacun peut être utilisé de façon plus ou moins automatique. Certaines fonctionnalités communes de ces systèmes sont comparées. L'aide apportée par les systèmes porte sur 3 points : (1) localisation automatique des métaphases sur les lames, dans ce cas on a un gain de temps d'un facteur 2 à 4 par rapport au comptage manuel ; (2) un outil d'aide au comptage qui apporte un confort de lecture et une meilleure fiabilité des résultats ; (3) la détection automatique des dicentriques est particulièrement utile en cas de tri de population. En effet, dans ce cas il faut estimer très rapidement la dose reçue par un nombre important de personnes. Par contre, l'estimation de dose n'a pas besoin d'être aussi précise que dans le cas de l'expertise individuelle. Des erreurs dans la détection des dicentriques est alors tolérée et une détection automatique des dicentriques est envisageable. Le gain de temps est très appréciable puisqu'il est possible de compter 300 cellules en une demie-heure (METAFER) contre 25 avec la seule aide du chercheur de métaphases. Cependant la qualité de la détection doit encore être améliorée puisque 50 % des dicentriques ne sont pas détectées. Le marquage des centromères par technique FISH devrait permettre d'améliorer la sensibilité de la technique. Les premiers résultats sont encourageant puisque 90 % des centromères sont correctements détectés mais d'autres expériences doivent êtres réalisées pour évaluer le gain de temps.

The detection and quantification of disseminated tumor cells (DTC) present in the bone marrow (BM), peripheral blood (PB) and apheresis products (AP) are becoming increasingly significant in the treatment of cancer patients. Three different applications are implemented in the clinical practice of pediatric and adult solid tumor patients: (1) the identification of tumor cells in the BM and PB at diagnosis; (2) the response of occult tumor cells to high-dose chemotherapy; and (3) the presence of tumor cells in the autograft. In solid tumors the clinical significance of DTCs at diagnosis or during the course of the disease, usually termed minimal residual disease (MRD) testing, is still under debate. These indistinct results are mainly due to methodical reasons. Therefore, a fully automated system (RCDetect/metafer) combining the detection of 'tumor-specific' immunological features together with 'tumor-typical' DNA aberrations has been developed allowing the unambiguous visualization of tumor cells in a hematopoietic surrounding.

BACKGROUND: Rare tumor cells circulating in the hematopoietic system can escape identification. On the other hand, the nature of these cells, positive for an immunologiCal tumor marker, cannot be determined without any genetic information. PROCEDURE: To overcome these problems a novel computer assisted scanning system for automatic cell search, analysis, and sequential repositioning was developed. This system allows an exact quantitative analysis of rare tumor cells in the bone marrow and peripheral blood by sequential immunological and molecular cytogenetic characterization. RESULTS AND CONCLUSIONS: In that virtually all tumor cells in a mixing experiment could be recovered unambiguously, we can conclude that the sensitivity of this approach is set by the number of cells available for analysis. Sequential FISH analyses of immunologically positive cells improve both the specificity and the sensitivity of the microscopic minimal residual disease detection.

BACKGROUND: Rare tumor cells circulating in the hematopoietic system can escape identification. On the other hand, the nature of these cells, positive for an immunologiCal tumor marker, cannot be determined without any genetic information. PROCEDURE: To overcome these problems a novel computer assisted scanning system for automatic cell search, analysis, and sequential repositioning was developed. This system allows an exact quantitative analysis of rare tumor cells in the bone marrow and peripheral blood by sequential immunological and molecular cytogenetic characterization. RESULTS AND CONCLUSIONS: In that virtually all tumor cells in a mixing experiment could be recovered unambiguously, we can conclude that the sensitivity of this approach is set by the number of cells available for analysis. Sequential FISH analyses of immunologically positive cells improve both the specificity and the sensitivity of the microscopic minimal residual disease detection.

The efficiency of the automated metaphase finding system METAFER2 is assessed in a routine mutagenicity assay using an aneuploid rat liver cell line treated with various promutagens. Data sets generated by automated and manual selection of metaphases are compared. It is demonstrated that METAFER2 routinely allows an efficient automatic identification of metaphases not only in lymphocyte preparations, but also in preparations from mammalian cell lines with varying chromosome numbers. Although larger slide areas are required for automated compared to manual metaphase scanning, the automatic system is faster by a factor of about 5. The interactive visual elimination of metaphases of insufficient quality is an easy and fast procedure. METAFER2 allows an unbiased selection of metaphases irrespective of their appearance as homogeneously stained first or harlequin-stained second division cells. Random selection of metaphases is neither influenced by various structural chromosome changes nor by increased frequencies of sister-chromatid exchanges.

The amount of time-saving by using the Metafer2 metaphase finder for routine analysis of radiation-induced chromosome aberrations (biological dosimetry) was determined. Metaphases were prepared by standard methods from cultures of human peripheral blood lymphocytes and stained either with Giemsa or with the FPG method. The metaphase finder was used for detecting metaphases on the microscope slides and for automatically processing the evaluation data. In our laboratory, standardized analysis of 1000 metaphases requires at least 3 working days for cell culturing and slide preparation and 51.5 working hours for cytogenetic analysis. When using the metaphase finder the time required for cytogenetic analysis is reduced to 17.3 working hours (time-saving factor: 51.5/17.3 h = 3.0). In our prolonged method, including more than one scoring of each slide and karyotyping of metaphases with chromosome aberrations, the analysis times for 1000 cells are 132 and 70 working hours, respectively (time saving factor: 132/70 h = 1.9).