Siamak S. Tabibzadeh

Immunohistologic analysis of lymphoid cells by a rapid double staining method

The development of monoclonal antibodies has allowed characterization of subpopulations of lymphoid cells and of their in situ distribution in tissues. The feasibility of simultaneous localization of two surface antigens was studied by double staining with monoclonal antibodies to B cells, T cell subsets and follicular dendritic reticulum cells (DRC) using the avidin-biotin-complex (ABC) method in sections of human lymphoid tissues (tonsil, lymph node, spleen) and a non-lymphoid tissue, endometrium. Color mixture was avoided when an additional incubation with avidin-biotin-labeled peroxidase and subsequent development in the respective substrate of the first sequence preceded the second staining sequence using the primary antibodies at optimal concentrations. The antigenic profiles were portrayed by contrasting and distinct colors of the reaction products. It was observed that T lymphocytes of the cytotoxic/suppressor and helper/inducer phenotypes were topographically associated with each other and with B cells in B and T cell areas of lymphoid tissues as well as in lymphocytic aggregates in endometria. Subpopulations of these cells were mantled by processes of DRCs in lymphoid follicles. The findings indicate that the double ABC staining method can be used for simultaneous demonstration of two surface antigens in tissue sections.

Applicability of imprints to immuno-ultrastructural studies of lymphoid tissues.

In order to evaluate the applicability of imprints to immuno-ultrastructural studies of lymphoid tissues, we compared distribution pattern and morphology of B cells, T cells, T-cell subsets, and follicular dendritic reticulum cells (DRC) at the light and ultrastructural level in imprints and sections of tonsils and lymph nodes. The surface antigenic profile of lymphoid cells was revealed with monoclonal antibodies in an avidin-biotin-peroxidase complex (ABC) method. Distribution of lymphoid cells in coherent areas of imprints recapitulated their disposition in sections of respective lymphoid tissues. Preservation of microanatomical relationships and ultrastructure of lymphoid cells in imprints allowed evaluation of associations and fine structural detail of lymphoid cells. Morphologic configurations of B cells in imprints, confined to round aggregates, were similar to fine structural morphology of B cells in mantle zones (MZ) and germinal centers (GC). Processes of DRCs in imprints formed conformations resembling their meshwork within follicles and mantled lymphoid cells. In imprints and sections, lymphocytes of cytotoxic/suppressor phenotype had a large amount of cytoplasm with many organelles. In contrast, cells of helper/inducer phenotype displayed a high nucleocytoplasmic (N/C) ratio and small numbers of organelles. Thus, imprints represent an easy, fast, and reliable method that lends itself to immunoultrastructural studies of lymphoid tissues.