Image analysis of fibrosis in labial salivary glands of patients with systemic autoimmune diseases. Close correlation of lobular fibrosis to seropositive rheumatoid arthritis and increased anti-CCP and RF titres in the serum.

Lobular fibrosis in labial salivary glands of patients with systemic autoimmune disease is a rarely examined and rather neglected histological change. Its significance and disease association is poorly understood. Our aim was to explore the clinical correlations of fibrosis in labial salivary gland samples using objective methods and laboratory parameters. Labial salivary gland samples from more than 300 patients over a 3-year period were selected from the archives of the pathology department, histologically examined, digitised, image analysed and statistically evaluated to identify the presence and intensity of lobular fibrosis, its relation to age, clinical diagnoses of systemic autoimmune disease and the presence of rheumatoid factor (RF), anti-cyclic citrullinated peptide (CCP), antinuclear antibodies (ANAs), and anti-dsDNA serum markers. Significant correlation was found between lobular fibrosis and the presence of autoimmune disease (p = 0.023), mainly seropositive rheumatoid arthritis (p < 0.001). Also significant association was found between the fibrosis and the presence of serum anti-CCP (p < 0.001) and IgA/IgG/IgM-RF (p < 0.001, p < 0.001 and p = 0.008, respectively). Significant association was explored between the anti-dsDNA positivity and the negative histology groups (p = 0.033) and between the ANA positivity and the inflammation only group (p = 0.021). The results suggest that lobular fibrosis tends to associate to certain systemic autoimmune diseases, mainly seropositive rheumatoid arthritis, and seems to be rare in labial salivary gland biopsies of autoimmune diseases characterised by presence of anti-dsDNA. The close correlation of ANA positivity and the inflammation only histology was not surprising, since the majority of patients (62%) have Sjögren's syndrome, known for its inflammatory infiltrate. These findings emphasise that evaluation of lobular fibrosis and inflammation in histological samples of labial salivary gland biopsies are equally important.

Image analysis of fatty infiltration in labial salivary gland biopsies: extent and its correlation to age, obesity and diabetes.

BACKGROUND: Fatty infiltration of minor salivary gland parenchyma is relatively frequent, but not extensively examined histopathological phenomenon in biopsy samples. Its extent and relation to several suspected background diseases are not well understood. METHODS: In this study, we examined the presence and extent of fatty infiltration on digitally scanned versions of the periodic acid/Schiff-stained minor salivary gland slides of 275 patients. As a result of the image analysis, fatty infiltration was expressed in per cent of the whole selected area. The presence and extent of this change were compared to age, diabetes mellitus and body mass index in various statistical analyses. RESULTS: Significantly higher age and body mass index values were found in the fatty infiltration positive than in the negative group. We also found that not only the number of fatty infiltration positive cases was increased significantly in the gradually worsened body mass index groups, but the extent of fatty infiltration also increased as the obesity worsened. Age also showed significant correlation with the extent of fatty infiltration. DISCUSSION: All of these findings support that the age (which seemed the only independent variable) shows strong correlation with the presence of the fatty infiltration but obesity may also play important role in the development and the extent of this change. Because of its frequency in elderly, at least partly, the fatty infiltration might be responsible for the xerostomia. We also think that presence of fatty infiltration should be mentioned in the histopathological report of salivary gland biopsies.

Automated signal pattern evaluation of a bladder cancer specific multiprobe-fish assay applying a user-trainable workstation.

OBJECTIVE: Signal pattern enumeration of Urovysion Fluorescence in Situ Hybridization test is tedious and requires great experience. Our aim was to eliminate human interaction by automating the process, using an adoptable, automated image acquisition, and analysis system. METHODS: For extensive analytical analysis control, cell populations were used, while preliminary clinical study was performed on 21 patients with clinical suspicion for bladder cancer. All investigations were carried out using an automated user-trainable workstation (Metafer4-Metacyte). RESULTS: The system identified nuclei with a specificity and sensitivity of 92.7 and 96.6%, respectively, while signal detection accuracy was 81.1% on average. Both analytical and diagnostic accuracy of automated analysis was comparable to manual approach (94.8 and 71% vs. 97.9 and 76%, respectively), but classification accuracy increased with degree of polysomy, thus diagnostic sensitivity in low grade, low stage cases was poor. CONCLUSION: It is possible to automate signal enumeration of Urovysion using a user-trainable system, and achieve efficiency comparable to manual analysis. Previously introduced automated immunophenotypic targeting should further increase diagnostic sensitivity, while resulting in a comprehensively automated method. However, the problem of reduced detection accuracy in cases featured with low polysomy is likely to remain a great challenge of automated signal enumeration.

Urovysion: Considerations on modifying current evaluation scheme, including immunophenotypic targeting and locally set, statistically derived diagnostic criteria.

Urovysion multitarget fluorescence in situ hybridization (FISH) assay is a promising tool for detection of bladder cancer, however, there is still no consensus regarding abnormal signal pattern and cut-off level, and the recommended targeting carries limitations similar to urine cytology. Aim of this study was to explore diagnostic benefits of a recently introduced method featuring target specific genotyping, as well as to investigate the feasibility of locally and statistically determined cut-off, compared with conventional evaluation scheme. Histology, cytology, and comparative FISH approaches were performed on 42 patients with high clinical suspicion for urothelial carcinoma (UC). FISH parallels were (1) Urovysion-alone (according to manufacturer's instruction); (2) Targeted-Urovysion (cytokeratin7 immunophenotyping followed by Urovysion), both of which evaluated by both conventional and statistical evaluation scheme. For statistical evaluation cut-offs and sufficient sample size were determined on controls and ratio of positive cells was recorded, whereas conventional evaluation relied on manufacturer's recommendations. The specificity of cytology, Urovysion-alone in general and targeted-Urovysion in general appeared 86%, 86%, and 100%, respectively. In the same comparison, overall sensitivity was 60%, 80%, and 93%, respectively. In superficial cases sensitivity was 48% for cytology, 72% for Urovysion-alone and 91% for targeted-Urovysion, while no prominent differences were seen in muscle invasive cases. The ratio of FISH positive cells was proportionate with both stage and grade, however, targeted genotyping could separate high grade/high stage cases more effectively. In conclusion, CK7 targeting raises diagnostic efficiency of Urovysion, and could be an ideal tool for identifying tumor cells in ambiguous cases or when other tumors are present. Statistical evaluation produces accuracy comparable with results of conventional evaluation, and with laboratories setting cut-offs individually but according harmonized protocol, it could aid method standardization. Furthermore, by providing additional quantitative information about tumor characteristics, is likely to have therapy relevant value in the future.

Increased efficiency of detecting genetically aberrant cells by UroVysion test on voided urine specimens using automated immunophenotypical preselection of uroepithelial cells.

There is a steady search for procedure which could replace or at least reduce the frequency of the invasive cystoscopy in the surveillance of heterogeneous superficial transitional cell carcinoma (TCC) of the bladder. Recently, UroVysion FISH assay has been shown to provide with better sensitivity than the urine cytology except for the lowest stage pTa and grade I-II TCCs. Data indicate that this failure of the sensitive FISH might be due to mistargeting. Therefore, our aim was to elaborate a procedure enabling FISH analysis in phenotypically preselected urothelial cells, only. Cytokeratin 7 (CK-7) chromogenic immunolabeling was applied to various mixtures of negative and positive control cells as well as voided urine specimens. Cellular targets and CK-7 positive cells were identified by morphometric and pixel intensity indices using an automated microscope workstation. UroVysion FISH pattern was analyzed only in the subsequently relocalized CK-7 positive events. Automated phenotypical preselection of urothelial cells proved to have 97.3% sensitivity, 96.1% specificity, and 99.0% accuracy, whereas combined pheno- and genotyping revealed 93.3% sensitivity and 99.8% specificity, respectively. In clinical samples, the overall 20.4% FISH positivity gained by traditional target identification contrasted with the 55.6% positivity obtained by the combined method, by which the efficiency of identifying chromosomally aberrant cells proved to be two to threefold higher even in grade I lesions. FISH analysis of phenotypically preselected urothelial cells might represent a reliable asset in surveillance of low stage-low grade TCCs.

Automated detection of residual leukemic cells by consecutive immunolabeling for CD10 and fluorescence in situ hybridization for ETV6/RUNX1 rearrangement in childhood acute lymphoblastic leukemia.

Among the various methods available for analyzing minimal residual disease, a new procedure for the cell-based approaches using consecutive phenotypic and genotypic analysis as revealed by immunofluorescent labeling and subsequent fluorescent in situ hybridization (FISH) has been developed. We are introducing a fluorescent microscopy-based technique by which not only cellular targets and immunological marker positivity, but also the FISH pattern was identified by automated scanning. For the latter one translocation-specific FISH pattern recognition was accomplished by using an automated scanning mode for the 3D determination of valid distances between FISH signals, to define the cutoff value for the shortest green-red spot distance differentiating positive cells from negative ones. The procedure was tested with CD10(+) acute lymphoblastic leukemia cell line harboring the t(12;21)(p13;q22) resulting in the ETV6/RUNX1 rearrangement (formerly TEL/AML1), as well as peripheral blood lymphocytes of healthy individuals. Using the combined, automated method, a sensitivity of 98.67% and a specificity of 99.97% were obtained. The mean false positivity + 2 standard deviations cutoff level (0.09%) allows detection of leukemic cells with high accuracy, even a bit below the tumor load dilution of 10(-3), a value reported to be critical in clinical decision making.

Correlation between BCR-ABL expression and tumor burden is restricted to the transition from minor to major cytogenetic response in interferon treated CML patients.

The interferon treatment of chronic myeloid leukaemia has been monitored by investigating the tumour burden as revealed by fluorescence in situ hybridization and the expression of BCR-ABL chimera determined by quantitative reverse transcription polymerase chain reaction. These parameters were obtained from the peripheral blood of 51 untreated and 104 follow-up patient samples. Poor correlation (r=0.31) was found between BCR-ABL expression and tumor load in all samples as well as in untreated patients, and this correlation was even less in all follow-up cases (r=0.28). Regarding chimera expression five order of magnitude difference existed in the untreated patients and this value dropped to two in those with complete cytogenetic response. Only the major and the complete cytogenetic response groups differed significantly (p<0.001) in the BCR-ABL expression from that of patients at diagnosis. Among the different cytogenetic response groups the only significant difference (p<0.01) in the BCRABL expression was obtained between the major and the minor responders. In the individual patients not only correlated changes of residual tumour mass and chimera expression, but mainly independent changes of these two parameters were observed. This indicates that the BCR-ABL expression and the tumor burden are largely independent variables.