Aims-To investigate the effect of tissue preparation on immunostaining and to establish whether there is a standard tissue preparation schedule that allows optimal demonstration of all antigens. Methods-Blocks of tonsil were subjected to variations to a standard fixation, processing, and section preparation schedule. The sections were stained with five antibodies-L26 (CD20), UCHL1 (CD45RO), CD3, vimentin, and antikappa light chain-using the streptavidinbiotin immunostaining technique. When further investigation was necessary, other tissues and antibodies were used and where weak immunostaining was obtained the use of microwave pretreatment to improve staining was tested. Results-Several factors involved in fixation were found to affect immunoreactivity. These included the duration, pH, and type of fixative used. In tissue processing only temperature and the duration of the dehydration and wax infiltration steps affected immunoreactivity. Of all the factors investigated, the temperature and duration of the section drying had the greatest effect. In contrast, long term storage of cut sections before immunostaining had no effect on the reactivity of the antibodies tested. Antibodies were found to be affected by alterations to tissue preparation by varying degrees, UCHL1 and vimentin being the most susceptible to changes in fixation and L26 to changes in processing. Where weak staining occurred, microwave pretreatment was generally found to eliminate-the problem. Conclusions-There is no standard tissue preparation schedule for the optimal demonstration of all antigens. Factors involved in all aspects of tissue preparation can affect immunoreactivity, so it is important that precise details of the preparation schedule are given when reporting immunocytochemical studies, rather than using the general term "routinely fixed and processed". ( Clin Pathol 1997;50:422-428)
Proteolytic enzymes, protease and trypsin have recently been introduced to reduce the inconsistency hitherto encountered in the unlabelled antibody--enzyme method using PAP. This study investigated factors determining the optimum conditions for use of such enzymes in order to establish which one is most suitable. Trypsin was the most effective enzyme; however, its activity decreased over 3 h, a feature paralleled immunocytochemically. Method and duration of fixation appears to influence the required time of exposure to trypsin in order that consistent immunostaining may be produced. Treatment of sections with trypsin prior to the use of the unlabelled antibody--enzyme method using PAP renders the technique reliable, provided the enzyme is used in a carefully controlled manner.
Using routine histology, resin embedded sections and immunohistochemical techniques on formalin-fixed, paraffin processed tissue, 66 cases of primary gastrointestinal lymphoma have been classified. This study necessitated the development of reliable criteria to separate lymphomas of true histiocytic origin from those of lymphocytic origin. Among the morphologic properties of malignant histiocytes were complex pleomorphic nuclei, abundant well delineated cytoplasm and phagocytosis. These cells were shown to contain all major immunoglobulin chains, C3, lysozyme and in some cases alpha 1 antitrypsin. Malignant lymphomas derived from histiocytes could be divided into two groups: malignant histiocytosis of the intestine (MHI), a recently described diffuse pleomorphic lymphoma associated with villous atrophy of the small intestine, and histiocytic lymphoma (HL) which forms solid tumor masses in a similar manner to lymphocyte derived tumors. Immunohistochemical studies of lymphocyte derived tumors were negative apart from one case with plasmacytoid differentiation. Of the 66 cases, 50% were of histiocytic origin (33% MHI, 17% HL) and 41% of lymphocyte origin, there was one case of Hodgkin's disease and five cases were unclassified. The role of the histiocyte in gastrointestinal mucosa deserves further study.
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