Mast Cells Are Abundant in Primary Cutaneous T-Cell Lymphomas: Results from a Computer-Aided Quantitative Immunohistological Study.

BACKGROUND: Mast cells (MC) are bone marrow derived haematopoetic cells playing a crucial role not only in immune response but also in the tumor microenvironment with protumorigenic and antitumorigenic functions. The role of MC in primary cutaneous T-cell lymphomas (CTCL), a heterogeneous group of non-Hodgkin lymphomas with initial presentation in the skin, is largely unknown. OBJECTIVE: To gain more accurate information about presence, number, distribution and state of activation (degranulated vs. non-degranulated) of MC in CTCL variants and clinical stages. MATERIALS AND METHODS: We established a novel computer-aided tissue analysis method on digitized skin sections. Immunohistochemistry with an anti-MC tryptase antibody was performed on 34 biopsies of different CTCL subtypes and on control skin samples. An algorithm for the automatic detection of the epidermis and of cell density based CTCL areas was developed. Cells were stratified as being within the CTCL infiltrate, in P1 (a surrounding area 0-30 µm away from CTCL), or in P2 (30-60 µm away from CTCL) area. RESULTS: We found high MC counts within CTCL infiltrates and P1 and a decreased MC number in the surrounding dermis P2. Higher MC numbers were found in MF compared to all other CTCL subgroups. Regarding different stages of MF, we found significantly higher mast cell counts in stages IA and IB than in stages IIA and IIB. Regarding MC densities, we found a higher density of MC in MF compared to all other CTCL subgroups. More MC were non-degranulated than degranulated. CONCLUSION: Here for the first time an automated method for MC analysis on tissue sections and its use in CTCL is described. Eliminating error from investigator bias, the method allows for precise cell identification and counting. Our results provide new insights on MC distribution in CTCL reappraising their role in the pathophysiology of CTCL.

Development of Blood and Lymphatic Endothelial Cells in Embryonic and Fetal Human Skin.

Blood and lymphatic vessels provide nutrients for the skin and fulfill important homeostatic functions, such as the regulation of immunologic processes. In this study, we investigated the development of blood and lymphatic endothelial cells in prenatal human skin in situ using multicolor immunofluorescence and analyzed angiogenic molecules by protein arrays of lysates and cell culture supernatants. We found that at 8 to 10 weeks of estimated gestational age, CD144(+) vessels predominantly express the venous endothelial cell marker PAL-E, whereas CD144(+)PAL-E(-) vessels compatible with arteries only appear at the end of the first trimester. Lymphatic progenitor cells at 8 weeks of estimated gestational age express CD31, CD144, Prox1, and temporary PAL-E. At that developmental stage not all lymphatic progenitor cells express podoplanin or Lyve-1, which are acquired with advancing gestational age in a stepwise fashion. Already in second-trimester human skin, the phenotype of blood and lymphatic vessels roughly resembles the one in adult skin. The expression pattern of angiogenic molecules in lysates and cell culture supernatants of prenatal skin did not reveal the expected bent to proangiogenic molecules, indicating a complex regulation of angiogenesis during ontogeny. In summary, this study provides enticing new insights into the development and phenotypic characteristics of the vascular system in human prenatal skin.

Digital microscopy assessment of angiogenesis in different breast cancer compartments.

BACKGROUND/AIM: Tumour angiogenesis defined by microvessel density (MVD) is generally accepted as a prognostic factor in breast cancer. However, due to variability of measurement systems and cutoffs, it is questionable to date whether it contributes to predictive outline. Our study aims to grade vascular heterogeneity by comparing clear-cut compartments: tumour associated stroma (TAS), tumour parenchyma, and tumour invasive front. MATERIAL AND METHODS: Computerized vessel area measurement was performed using a tissue cytometry system (TissueFAXS) on slides originated from 50 patients with breast cancer. Vessels were marked using immunohistochemistry with CD34. Regions of interest were manually defined for each tumour compartment. RESULTS: Tumour invasive front vascular endothelia area was 2.15 times higher than that in tumour parenchyma and 4.61 times higher than that in TAS (P < 0.002). Worth to mention that the lymph node negative subgroup of patients show a slight but constant increase of vessel index in all examined compartments of breast tumour. CONCLUSION: Whole slide digital examination and region of interest (ROI) analysis are a valuable tool in scoring angiogenesis markers and disclosing their prognostic capacity. Our study reveals compartments' variability of vessel density inside the tumour and highlights the propensity of invasive front to associate an active process of angiogenesis with potential implications in adjuvant therapy.

Automated REcognition of tissue-associated erythrocytes (ARETE)-a new tool in tissue cytometry.

Automated microscopic image analysis of immunofluorescence-stained targets on tissue sections is challenged by autofluorescent elements such as erythrocytes, which might interfere with target segmentation and quantification. Therefore, we developed an automated system (Automated REcognition of Tissue-associated Erythrocytes; ARETE) for in silico exclusion of erythrocytes. To detect erythrocytes in transmission images, a cascade of boosted decision trees of Haar-like features was trained on 8,640/4,000 areas (15 × 15 pixels) with/without erythrocytes from images of placental sections (4 µm). Ground truth data were generated on 28 transmission images. At least two human experts labelled the area covered by erythrocytes. For validation, output masks of human experts and ARETE were compared pixel-wise against a mask obtained from majority voting of human experts. F1 score, specificity, and Cohen's κ coefficients were calculated. To study the influence of erythrocyte-derived autofluorescence, we investigated the expression levels of a protein (receptor for advanced glycated end products; RAGE) in placenta and number of Ki-67-positive/cytokeratin 8-positive epithelial cells in colon sections. ARETE exhibited high sensitivity (99.87%) and specificity (99.81%) on a training-subset and processed transmission images (1,392 × 1,024 pixels) within 4 sec. ARETE and human expert's F1-scores were 0.55 versus 0.76, specificities 0.85 versus 0.92 and Cohen's κ coefficients 0.41 versus 0.68. A ranking of Cohen's κ coefficient by the scale of Fleiss certified "good agreement" between ARETE and the human experts. Applying ARETE, we demonstrated 4-14% false-positive RAGE-expression in placenta, and 18% falsely detected proliferative epithelial cells in colon, caused by erythrocyte-autofluorescence. ARETE is a fast system for in silico reduction of erythrocytes, which improves automated image analysis in research and diagnostic pathology.

3D parallel coordinate systems--a new data visualization method in the context of microscopy-based multicolor tissue cytometry.

BACKGROUND: Presentation of multiple interactions is of vital importance in the new field of cytomics. Quantitative analysis of multi- and polychromatic stained cells in tissue will serve as a basis for medical diagnosis and prediction of disease in forthcoming years. A major problem associated with huge interdependent data sets is visualization. Therefore, alternative and easy-to-handle strategies for data visualization as well as data meta-evaluation (population analysis, cross-correlation, co-expression analysis) were developed. METHODS: To facilitate human comprehension of complex data, 3D parallel coordinate systems have been developed and used in automated microscopy-based multicolor tissue cytometry (MMTC). Frozen sections of human skin were stained using the combination anti-CD45-PE, anti-CD14-APC, and SytoxGreen as well as the appropriate single and double negative controls. Stained sections were analyzed using automated confocal laser microscopy and semiquantitative MMTC-analysis with TissueQuest 2.0. The 3D parallel coordinate plots are generated from semiquantitative immunofluorescent data of single cells. The 2D and 3D parallel coordinate plots were produced by further processing using the Matlab environment (Mathworks, USA). RESULTS: Current techniques in data visualization primarily utilize scattergrams, where two parameters are plotted against each other on linear or logarithmic scales. However, data evaluation on cartesian x/y-scattergrams is, in general, only of limited value in multiparameter analysis. Dot plots suffer from serious problems, and in particular, do not meet the requirements of polychromatic high-context tissue cytometry of millions of cells. The 3D parallel coordinate plot replaces the vast amount of scattergrams that are usually needed for the cross-correlation analysis. As a result, the scientist is able to perform the data meta-evaluation by using one single plot. On the basis of 2D parallel coordinate systems, a density isosurface is created for representing the event population in an intuitive way. CONCLUSIONS: The proposed method opens new possibilities to represent and explore multidimensional data in the perspective of cytomics and other life sciences, e.g., DNA chip array technology. Current protocols in immunofluorescence permit simultaneous staining of up to 17 markers. Showing the cross-correlation between these markers requires 136 scattergrams, which is a prohibitively high number. The improved data visualization method allows the observation of such complex patterns in only one 3D plot and could take advantage of the latest developments in 3D imaging.

An improved method for discrimination of cell populations in tissue sections using microscopy-based multicolor tissue cytometry.

BACKGROUND: In tissue context, researchers and pathologists lack a generally applicable standard for quantitative determination of cytological parameters. Increasing knowledge of disease-specific markers calls for an appropriate in situ tissue cytometry. METHODS: Microscopy-based multicolor tissue cytometry (MMTC) permits multicolor analysis of single cells within tissue context. RESULTS: Tissue specimens stained for CD45/CD3/CD4/CD8 were analyzed. Specificity as well as reproducibility of MMTC is demonstrated and a novel MMTC-based function to improve visual discrimination of subpopulations is introduced. CONCLUSIONS: Our data demonstrate that MMTC constitutes an important step toward automated and quantitative fluorometry of solid tissues and cell monolayers.