Lymphocytes In Tissue Sections Research Articles

Quantitative assessment of the spatial heterogeneity of tumor-infiltrating lymphocytes in breast cancer.

BackgroundTumor-infiltrating lymphocyte (TIL) count in breast cancer carries prognostic information and represents a potential predictive marker for emerging immunotherapies. However, the distribution of the lymphocyte subpopulations is not well defined. The goals of this study were to examine intratumor heterogeneity in TIL subpopulation counts in different fields of view (FOV) within each section, in different sections from the same biopsy, and between biopsies from different regions of the same cancer using quantitative immunofluorescence (QIF).MethodsWe used multiplexed QIF to quantify cytokeratin-positive epithelial cells, and CD3-positive, CD8-positive and CD20-positive lymphocytes in tissue sections from multiple biopsies obtained from different areas of 31 surgically resected primary breast carcinomas (93 samples total). Log2-transformed QIF scores or concordance and variance component analyses with linear mixed-effects models were used. Cohen’s kappa index [k] of high versus low scores, defined as above and below the median, was used to measure sample similarity between areas.ResultsWe found a strong positive correlation between CD3 and CD8 levels across all patients (Pearson correlation coefficient [CC] = 0.827). CD3 and CD8 showed a weaker but significant association with CD20 (CC = 0.446 and 0.363, respectively). For each marker, the variation between different FOVs in the same section was higher than the variation between sections or between biopsies of the same cancer. The intraclass correlation coefficients (ICC) were 0.411 for CD3, 0.324 for CD8, and 0.252 for CD20. In component analysis, 66–69 % of the variance was attributable to differences between FOVs in the same section and 30–33 % was due to differences between biopsies from different areas of the same cancer. Section to section differences were negligible. Concordance for low versus high marker status assignment in single biopsies compared to all three biopsies combined yielded k = 0.705 for CD3, k = 0.655 for CD8, and k = 0.603 for CD20.ConclusionsT and B lymphocytes show more heterogeneity across the dimensions of a single section than between different sections or regions of a given breast tumor. This observation suggests that the average lymphocyte score from a single biopsy of a tumor is reasonably representative of the whole cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0737-x) contains supplementary material, which is available to authorized users.

Atypical Lymphocytes and Cellular Cannibalism: A Phenomenon, First of its Kind to be Discovered in Chronic Periapical Lesions.

Lymphocytes are often termed to be isomorphic, having a monotonous light microscopic appearance. Morphological aspects of lymphocytes in tissue sections thereby are not routinely taken notice of as their morphology seems to vary only in case of lymphoid malignancies, hematological malignancies apart from certain viral infections. Atypical lymphocytes are the lymphocytes with unusual shape, size or overall structure. These are more commonly known as reactive lymphocytes. The unusual histomorphological feature of these cells include larger size than normal lymphocytes; in some cells the size exceeds even 30 microns. The large size is the result of antigenic stimulation of the cell. Alongwith these, the other rare feature which is recently coming under light is "Cellular Cannibalism" which is defined as a large cell enclosing a slightly smaller one within its cytoplasm. Previously, this feature was noted only in cases of malignant tumors. The objectives of this study were to determine the proportion of atypical lymphocytes in chronic periapical granulomas and cysts; to determine the proportionate cellular cannibalism in these periapical lesions. This was a descriptive, observational study conducted in the Department of Oral Medicine and Radiology and Oral Pathology and Microbiology. Haematoxylin and eosin stained 30 slides of chronic periapical granulomas and 20 slides of cysts reported in the year 2014-15 and the clinical proformas of the patients were retrieved from the files of the Department of Oral Medicine and Radiology and Oral Pathology and Microbiology. These slides were evaluated by 3 experts from the specialization of Oral Pathology and Microbiology to determine the presence of atypical lymphocytes and cellular cannibalism under high power magnification (400X). Out of the 30 slides of chronic periapical granulomas, about 12 slides (40%) revealed presence of atypical lymphocytes. In case of slides of chronic periapical cysts, however, only 4 out of the 20 slides (20%) examined histopathologically showed presence of atypical lymphocytes. An interesting feature of cellular cannibalism was noted in tissues with atypical cells. Cannibalistic cells were present in 12 out of the 30 slides of chronic periapical granulomas (40%). None of the cysts, however, revealed cannibalistic cells (0%). In the present study, we have quoted our observations on the unique cellular composition that was seen in histopathological sections of chronic periapical lesions. As also it was noted that those cases in which atypical cells & cellular cannibalism was evident the lesion clinically showed size more than 5cm. The question arises that whether presence of atypical cells from the tissue sections in these lesions denotes an aggressive clinical behavior and should be given a due consideration in deciding the treatment protocols for such cases to provide an optimum patient care.

The effects of FasL on inflammation and tumor survival are dependent on its expression levels

The apoptosis-inducing Fas ligand (FasL) is expressed in a variety of human cancers and has been implicated in tumor immune evasion. Paradoxically, ectopic expression of FasL in experimental tumors triggers a neutrophil-mediated inflammatory response and tumor rejection. To resolve these conflicting findings, we have established B16 melanoma and P29 Lewis lung carcinoma lines expressing different levels of FasL and examined their tumorigenicity in vivo. While tumors with a high level of FasL were rapidly rejected as previously reported, those expressing a low level of FasL were not rejected but grew faster than did FasL-negative parental cells. The growth enhancement of FasL(low) tumors was not observed in T-cell-deficient nude mice, suggesting that FasL expressed in tumors at low levels counteracted against T-cell-dependent antitumor responses. In support of this notion, FasL(low) tumors were found to grow faster than parental cells in mice that had acquired tumor-specific immunity. Furthermore, histological examinations revealed apoptosis of lymphocytes in tissue sections of FasL(low) tumors. These results collectively suggest that FasL on tumors is a double-edged sword: at high levels it triggers tumor rejection whereas at low levels it facilitates tumor growth possibly by suppressing antitumor immune responses.

“Virtual flow cytometry” of immunostained lymphocytes on microscopic tissue slides:iHCFlow™ tissue cytometry

A method and approach is developed for fully automated measurements of immunostained lymphocytes in tissue sections by means of digital color microscopy and patent pending advanced cell analysis. The validation data for population statistic measurements of immunostained lymphocytes in tissue sections using tissue cytometry (TC) is presented. The report is the first to describe the conversion of immunohistochemistry (IHC) data to a flow cytometry-like two parameter dot-plot display, hence the technique is also a virtual flow cytometry. We believe this approach is a paradigm shift, as well as novel, and called the system iHCFlow TC. Seven issues related to technical obstacles to virtual flow cytometry (FC) are identified. Segmentation of a 512 x 474 RGB image and tabular display of statistical results table took 12-15 s using proprietary developed algorithms. We used a panel of seven antibodies for validation on 14 cases of mantle cell lymphoma giving percentage positive, total lymphocytes, and staining density. A total of 2,027 image frames with 810,800 cell objects (COBs) were evaluated. Antibodies to CD3, CD4, CD8, Bcl-1, Ki-67, CD20, CD5 were subjected to virtual FC on tissue. The results of TC were compared with manual counts of expert observers and with the results of flow cytometric immunophenotyping of the same specimen. The correlation coefficient and 95% confidence interval by linear regression analysis yielded a high concordance between manual human results (M), FC results, and TC results per antibody, (r = 0.9365 M vs. TC, r= 0.9537 FC vs. TC). The technical issues were resolved and the solutions and results were evaluated and presented. These results suggest the new technology of TC by iHCFlow could be a clinically valid surrogate for both M and FC analysis when only tissue IHC is available for diagnosis and prognosis. The application for cancer diagnosis, monitoring, and prognosis is for objective, rapid, automated counting of immunostained cells in tissues with percentage results. We report a new paradigm in TC that converts IHC staining of lymphocytes to automated results and a flow cytometry-like report. The dot plot histogram display is familiar, intuitive, informative, and provides the pathologists with an automated tool to rapidly characterize the staining and size distribution of the immunoreactive as well as the negative cell population in the tissue. This systems tool is a major improvement over existing ones and satisfies fully the criteria to perform Cytomics (Ecker and Tarnok, Cytometry A 2005;65:1; Ecker and Steiner, Cytometry A 2004;59:182-190; Ecker et al., Cytometry A 2004;59:172-181).

“Virtual Flow Cytometry” of Immunostained Lymphocytes on Microscopic Tissue Slides.

A method and approach were developed for fully automated measurements of immunostained lymphocytes in tissue sections by means of digital color microscopy and patent pending advanced cell analysis. The validation data for population statistic measurements of immunostained lymphocytes in tissue sections using tissue cytometry is presented. Segmentation of a 512 x 474 RGB image and display of statistical results table took 12 to 15 seconds using in-house developed algorithms. We used a panel of 7 antibodies for validation on 14 cases of mantle cell lymphoma giving percentage positive, total lymphocytes, density staining. An average of 2027 image frames with 810,800 cell objects were evaluated. Antibodies to CD3, CD4, CD8, Bcl-1, Ki-67, CD20, CD5 were subjected to virtual flow cytometry on tissue. The results of tissue cytometry were compared with manual counts of expert observers and with the results of flow cytometry immunophenotyping of the same specimen. The correlation coefficient and 95 % confidence interval by linear regression analysis yielded a high concordance between manual human results (M), flow cytometry results(FC), and tissue cytometry (TC) results per antibody, (r =0.9365 manual vs TC, r =0.9537 FC vs TC). We noted a lower CD8 tumor infiltrating lymphocytes in blastic form than in non-blastic form of mantle cell and a higher Ki67 in the latter subtype than in the former. These results suggest the technology of tissue cytometry could be a clinically valid surrogate for both manual and flow cytometry analysis when only tissue immunohistochemistry is available for diagnosis and prognosis. The application for cancer diagnosis, monitoring, and prognosis is for objective counting of immunostained cells in tissues with percentage results. These results could be also be used in standardization of cut off percentage used in diagnosis; for exemplary tool for leukemic blasts(CD34) >20% for acute leukemia, for determining automated 30 % cut off for positivity for prognostic markers in diffuse large B-cell lymphoma for bcl-2, bcl-6, mum-1, and CD10.

A neural classifier enabling high-throughput topological analysis of lymphocytes in tissue sections.

A neural cell detection system (NCDS) for the automatic quantitation of fluorescent lymphocytes in tissue sections is presented in this paper. The system acquires visual knowledge from a set of training cell-image patches selected by a user. The trained system evaluates an image in 2 min calculating: the number, the positions, and the phenotypes of the fluorescent cells. For validation, the NCDS learning performance was tested by cross validation on digitized images of tissue sections obtained from inherently different types of tissue: diagnostic tissue sections across the human tonsil and across an inflammatory lymphocyte infiltrate of the human skeletal muscle. The NCDS detection results were compared with detection results from biomedical experts and were visually evaluated by our most experienced biomedical expert. Although the micrographs were noisy and the fluorescent cells varied in shape and size, the NCDS detected a minimum of 95% of the cells. In contrast, the cellular counts based on visual cell recognition of the experts were inconsistent and largely unreproducible for approximately 80% of the lymphocytes present in a visual field. The data indicate that the NCDS is rapid and delivers highly reproducible results and, therefore, enables high-throughput topological screening of lymphocytes in many types of tissue, e.g., as obtained by routine diagnostic biopsy procedures. High-throughput screening with the NCDS provides the platform for the quantitative analysis of the interrelationship between tissue environment, cellular phenotype, and cellular topology.

Automatic Recognition of Muscle‐Invasive T‐Lymphocytes Expressing Dipeptidyl‐Peptidase IV (CD26) and Analysis of the Associated Cell Surface Phenotypes

A neural cell detection system (NCDS) for the automatic quantitation of fluorescent lymphocytes in tissue sections was used to analyze CD26 expression in muscle‐invasive T‐cells. CD26 is a cell surface dipeptidyl‐peptidase IV (DPP IV) involved in co‐stimulatory activation of T‐cells and also in adhesive events. The NCDS system acquires visual knowledge from a set of training cell image patches selected by a user. The trained system evaluates an image in 2 min calculating (i) the number, (ii) the positions and (iii) the phenotypes of the fluorescent cells. In the present study we have used the NCDS to identity DPP IV (CD26) expressing invasive lymphocytes in sarcoid myopathy and to analyze the associated cell surface phenotypes. We find highly unusual phenotypes characterized by differential combination of seven cell surface receptors usually involved in co‐stimulatory events in T‐lymphocytes. The data support a differential adhesive rather than a co‐stimulatory role of CD26 in muscle‐invasive cells. The adaptability of the NCDS algorithm to diverse types of cells should enable us to approach any invasion process, including invasion of malignant cells.

Expression of the gangliosides GD3 and GD2 on lymphocytes in tissue sections of melanoma

Previous studies in vitro have shown that monoclonal antibodies (MAbs) against gangliosides GD3 and GD2 potentiate lymphocyte responses to a variety of stimuli. The purpose of the present study was to determine by immunohistological techniques whether GD3 and GD2 was expressed on lymphoid cells in vivo around melanoma cells. Studies on metastases in lymph nodes indicated that the lymphoid infiltrate around the margins of the metastases was predominantly CD4+ T cells, which were shown by dual labelling techniques to express mainly GD2 and to a lesser extent GD3. CD4+GD3+ T cells were detected more frequently in cortical regions of the lymph nodes. CD8+ T cells were less numerous than CD4+ T cells and expressed both GD3 and GD2. Expression of GD2 was also prominent on CD4+ T cells, B lymphocytes and dendritic reticular cells in germinal centres, whereas GD3 was mainly expressed on T cells in the margins of the follicles. In contrast to the predominance of CD4+ T cells in lymph nodes, CD4+ and CD8+ T cells were in approximately equal proportions about primary melanoma and metastases in skin. GD2 was largely undetectable on lymphocytes at these sites. In contrast, GD3 was detected on both CD8+ and CD4+ lymphocytes but not on B lymphocytes. The absence of GD2 on CD4+ T cells in skin suggested the latter were a different subpopulation to those in lymph nodes. There appeared to be no clear correlation, however, with subsets of CD4 T cells defined by the 2H4 and Leu 8 MAbs.(ABSTRACT TRUNCATED AT 250 WORDS)

An improved method for the detection of human T lymphocytes in tissue sections

The technique of haemadsorption to tissue sections for the detection of T lymphocytes is described in detail. The modification using the closed chamber technique is convenient, sensitive and reproducible. In principle, it parallels the rosette technique using blood lymphocytes in suspensions and AET-treated sheep erythrocytes. Storage of tissue at —25°C did not reduce the activity of the E-receptors in the tissue. The E-receptors were blocked by incubation of the tissue sections with an anti-T lymphocyte serum.

Ultrastructural Identification of T Lymphocytes in Tissue Sections of Mycosis Fungoides

An immunoelectronomicroscopic method has been employed to demonstrate in situ the T lymphocyte nature of the dermal and epidermal infiltrates to mycosis fungoides. A specific antiserum to the human T lymphocyte surface antigen (HTLA) was used in an indirect reaction with a peroxidase labeled anti-immunoglobulin. After histochemically revealing the peroxidase activity, T cells were easily identified by the deposition of electron dense material on the cytoplasmic membrane. Counterstaining with uranyl acetate and lead citrate enabled us morphologically to observe the maturity of infiltrating T lymphocytes and to identify Sézary cells and other cells such as eosinophils in the infiltrate. Our results confirm the T cell nature of the dermal infiltrate in mycosis fungoides and show that the epidermal infiltrate of cells forming Pautrier micro-abscesses are predominantly T lymphocytes.

Studies on human lymphocytes in frozen tissue section: Viability and spontaneous rosette formation with sheep erythrocytes

Sixty to 70% viability of lymphocytes in frozen tissue section was obtained by using dimethylsulphoxide-containing medium as a cryoprotective agent and by conditioning time and temperature for freezing, making sections on a cryostat and thawing. The viable and dead cells were differentiated by trypan blue exclusion test on section. Lymphocytes in tissue sections showed spontaneous rosette formation with sheep erythrocytes (E rosette) with specific localizations for each lymphoid organ.

Automated classification of cells in electron microscopic images of lymphoreticular tissue.

In classifying cells in tissue sections, one must consider the fact that only random projections of cells and of subcellular structures are available in the two-dimensional image. Therefore, measurement values that solely reflect the size of such projections cannot be taken on their own as a basis for cell classification. More complex morphologic features such as shape, texture and distribution pattern of cells and their components should be analyzed. Using cell nuclei as an example, the relationship between such features and geometric measurement values is evaluated. It can be shown that a well balanced combination of geometric parameters provides a suitable basis for reproducing the visual preclassification of lymphocytes in tissue sections. Moreover, using a cluster algorithm, which allows different levels of similarity to be defined, a hierarchical sequence of subclusters turns out, indicating the heterogeneity of the visually determined cell classes. Whether or not these subclusters can be correlated to functionally defined subpopulations of lymphocytes remains a matter for further investigation.

Acid Esterase in Human Lymphoid Cells and Leukaemic Blasts: a Marker for T Lymphocytes

A non-specific acid alpha-naphthyl-acetate esterase activity was investigated in human lymphoid cell populations from tonsils, blood, thymus and in different leukaemias. Four patterns have emerged. (i) The vast majority of T lymphocytes (peripheral blood, tonsils) showed a localized, intense reaction product ("T-like"). (ii) A thymocyte subpopulation expressed faint, localized enzyme activity ("Thy-like) but the majority of thymocytes showed no esterase activity. Some cells from acute lymphoblastic leukaemias with thymocyte surface characteristics had a "Thy-like" appearance. (iii) Most B lymphocytes and cells from chronic lymphocytic leukaemias were esterase negative. No activity was seen in mitogen activated peripheral blood T and B lymphoblasts from peripheral blood and in leukaemic blasts from the common form of childhood acute "lymphoblastic" leukaemia. (iv) Myeloid cells, including peripheral blood monocytes, and blast cells from acute myeloblastic and chronic granulocytic leukaemias showed an intense, diffuse reaction product (M-like). It is concluded that the modified esterase technique is a convenient marker for small mature human T lymphocytes in tissue sections and smears. It may also be helpful in the differential diagnosis of acute lymphoblastic leukaemia with T characteristics, the common form of acute "lymphoblastic" leukaemia and acute myeloblastic leukaemia.

Anatomical Distribution of T and B Lymphocytes Identified by Immunohistochemistry in the Chicken Spleen

Antisera against chicken bursal and thymic lymphocytes were raised in rabbits. They were made specific for the target T or B cells by appropriat absorption and then used for immunohistochemical identification of T and B lymphocytes in tissue sections of chicken spleen, applying the unlabeled antibody enzyme method. Lymphocyte reacting with anti-chicken-thymus globulin predominated in the periarteriolar lymphatic sheaths and in the red-pulp cords. A few of these lymphocytes were localized between the periellipsoid lymphatic tissue and in germinal centers. The periellipsoid lymphocytes around the Schweigger-Seidel sheaths consisted of lymphocytes positive with anti-chicken-bursa-cell globulin. The majority of lymphocytes in the germinal centers were labeled also by anti-chicken bursa-cell globulin. Age-dependent changes in the anatomical localization of T and B lymphocytes are described between late embryonic development and an age of 2 years. The different conditions of avian and mammalian T and B cell distribution in the spleen were considered.

HODGKIN'S DISEASE IN THE FIRST DECADE OF LIFE

This report is based on a study of 35 children with Hodgkin's disease. The longest survival periods were 9.8 years in a patient who has expired and 9 years in a patient who is still alive. Fourteen patients are still alive at the time of submission of this paper; the survival periods range from 0.2 to 9 years; 11 are known dead and their survival periods range from 0.3 to 9.8 years. The outcome in 10 cases is not known. A striking predominance of males was noted (91%). In 30 of 31 classifiable cases, the histologic sections showed either nodular sclerosing Hodgkin's disease or Hodgkin's disease with lymphocytic predominance. Regardless of classification, a striking preponderance of mature lymphocytes in tissue sections was evident in most cases. These findings suggest that a particular host response characterizes the disease in the young.