Immunoassay of lymphocyte subsets in ovine palatine tonsils

Abstract

By means of immunohistochemistry (IHC) and triple color flow cytometry (FCM), five commercial antibodies (anti-CD2, CD4, CD8, CD21, and CD45) were evaluated to quantify and localize the B- and T-lymphocytes in the ovine palatine tonsil. The results of both techniques were compared and evaluated. For the immunohistochemical analysis, three fixation methods were evaluated for their suitability to localize the different lymphocyte populations: 3.5% formaldehyde, zinc salts-based fixative and cryopreservation. The anti-CD45 antibody showed a positive reaction after all three fixation methods. The four other antibodies tested (anti-CD2, CD4, CD8 and CD21) were compatible with zinc salts-based fixation and cryopreservation. The CD21+ B-lymphocytes were localized in the tonsillar lymphoid follicles, while the CD2+ T-lymphocytes were abundant in the interfollicular regions and rare within the lymphoid follicles. The CD8+ T-cells were concentrated adjacent to the follicles, while the CD4+ T-cells were localized in the interfollicular zones as well as in the follicles. Both by immunohistochemistry and flow cytometry, a quantification of the different lymphocyte subsets was made. When comparing the results, a reversed B/T cell ratio was noticed. Possible explanations for this observation are discussed.