Cluster division is a critical issue in fluorescence microscopy-based analytical cytology when preparation protocols do not provide appropriate separation of objects. Overlooking clustered nuclei and analyzing only isolated nuclei may dramatically increase analysis time or affect the statistical validation of the results. Automatic segmentation of clustered nuclei requires the implementation of specific image segmentation tools. Most algorithms are inspired by one of the two following strategies: 1) cluster division by the detection of internuclei gradients; or 2) division by definition of domains of influence (geometrical approach). Both strategies lead to completely different implementations, and usually algorithms based on a single view strategy fail to correctly segment most clustered nuclei, or perform well just for a specific type of sample. An algorithm based on morphological watersheds has been implemented and tested on the segmentation of microscopic nuclei clusters. This algorithm provides a tool that can be used for the implementation of both gradient- and domain-based algorithms, and, more importantly, for the implementation of mixed (gradient- and shape-based) algorithms. Using this algorithm, almost 90% of the test clusters were correctly segmented in peripheral blood and bone marrow preparations. The algorithm was valid for both types of samples, using the appropriate markers and transformations. Cytometry 28:289–297, 1997.

Purpose: NKG2D ligands (NKG2DL) are expressed on various tumor types and immunosuppressive cells within tumor microenvironments, providing suitable targets for cancer therapy. Various immune cells express NKG2D receptors, including natural killer (NK) cells and CD8+ T cells. Interactions between NKG2DL and NKG2D receptors are essential for NK-cell elimination of osteosarcoma tumor-initiating cells. In this report, we used NKG2D–NKG2DL interactions to optimize an immunotherapeutic strategy against osteosarcoma. We evaluated in vitro and in vivo the safety and cytotoxic capacity against osteosarcoma cells of CD45RA− memory T cells expressing an NKG2D-4-1BB-CD3z chimeric antigen receptor (CAR). Experimental Design: CD45RA− cells from healthy donors were transduced with NKG2D CARs containing 4-1BB and CD3z signaling domains. NKG2D CAR expression was analyzed by flow cytometry. In vitro cytotoxicity of NKG2D-CAR+ CD45RA− T cells against osteosarcoma was evaluated by performing conventional 4-hour europium-TDA release assays. For the in vivo orthotopic model, 531MII YFP-luc osteosarcoma cells were used as targets in NOD-scid IL2Rgnull mice. Results: Lentiviral transduction of NKG2D-4-1BB-CD3z markedly increased NKG2D surface expression in CD45RA− cells. Genetic stability was preserved in transduced cells. In vitro, NKG2D-CAR+ memory T cells showed significantly increased cytolytic activity than untransduced cells against osteosarcoma cell lines, while preserving the integrity of healthy cells. NKG2D-CAR+ memory T cells had considerable antitumor activity in a mouse model of osteosarcoma, whereas untransduced T cells were ineffective. Conclusions: Our results demonstrate NKG2D-4-1BB-CD3z CAR–redirected memory T cells target NKG2DL-expressing osteosarcoma cells in vivo and in vitro and could be a promising immunotherapeutic approach for patients with osteosarcoma. Clin Cancer Res; 23(19); 5824–35. ©2017 AACR.

The evaluation of an automated system for Fluorescence In Situ Hybridization (FISH) spot counting in interphase nuclei is presented in this paper. Different types of experiments have been performed with samples from known populations. In all of them the goal is to detect mosaicism of chromosome X in leukocytes from mixtures in known proportions of healthy male and female blood. First the initial results from the automatic FISH analysis system were obtained and evaluated. Then the analysis was modified to reduce systematic errors, so that the results are closer to what an experienced human operator would have obtained (system calibration step). Finally, an additional control probe of chromosome Y was used to detect and discard cells where incorrect hybridization or other abnormal situations had occurred. In each step the system sensitivity was determined by the use of two statistical validation tests, so that the improvement brought about by the correction methods could be assessed. The results obtained in the study showed that, using both corrections, the system is able to detect 10% monosomies with a significance level α = 0.1%. Cytometry 31:93–99, 1998.

Background and objectives:celiac disease is associated with the HLA class II alleles: DQA1*05-DQB1*02 and DQB1*0302. The genetic risk for celiac disease may depend on the presence or absence of such alleles, their combination or number of copies. This study aimed to establish the differences in HLA genotypes between celiac patients diagnosed during childhood and adulthood, and between patients and healthy controls, and to determine the risk of disease in each genotypic category. Methods: we classified 350 celiac patients at time of diagnosis and 218 controls into 14 categories according to their HLA genotype, based on the presence or absence of risk alleles. Results: we found statistically significant differences between the genotype frequencies of celiac patients diagnosed as being children and adults. DQA1*05 (x 1 copy), DQB1*02 (x 1 copy), DQB1*0302 (x 0 copies) was the most frequent genotype in individuals diagnosed in childhood, whereas DQA1*05 (x 1 copy), DQB1*02 (x 2 copies), DQB1*0302 (x 0 copies) was the most frequent in adults. The risk for disease in each genotypic category in celiac children and adults turned out to be different. The presence of DQB1*0302 did not increase risk in children, but did in adults. Conclusion: in our celiac population, we found a different genetic pattern according to age of diagnosis. That could suggest that the pathogenic mechanism of the disease is not exactly the same in both age groups, which could somehow determine clinical presentation of the disease, its epidemiology, coexisting diseases, and complications