Evaluation of tissue preparation methods and paired immunofluorescence staining for immunocytochemistry of lymphomas

Abstract

Eight cross-linking fixatives were tested for preservation of extracellular or intracellular IgG, IgA, IgM, IgD, kappa and lambda light chains, J chain and secretory component. Most of the selected fixatives have been used in recent immunohistochemical studies of lymphoproliferative processes and comprised routine formalin, glutaraldehyde(1%)-formalin, Baker's formalin-calcium, formalin-sublimate, acetic acid(2%)-formalin-saline, Bouin's fluid, Susa fixative, and carbodiimide. The results obtained in artificial test substrates with defined amounts of IgG or IgA and in biological substrates (colon mucosa, tonsils, and different types of lymphomas) were compared by immunofluorescence with the antigenic preservation afforded by fixation in cold 96% ethanol (with or without inclusion of a pre-fixation 48 h washing period). An antigen concentration at least an eight-fold higher was necessary for detection with most other fixatives. Bouin's and Susa fixatives were peculiar in that they required antigen concentration 150 times higher for detection of IgG but only 3-8 times higher for IgA. Light chains were relatively well preserved by all fixatives except glutaraldehyde. For all cross-linking fixatives, the extent of antigenic masking depended on the concentration of environmental proteins, and the efficiency of unmasking with pronase or trypsin, therefore, varied with the location in the tissue. The J chain was particularly vulnerable to degradation during proteolytic treatment. The extensive masking of extracellular immunoglobulin in formalin-fixed tissue afforded a relatively good signal-to-noise ratio for immunoglobulin-producing cells when kappa and lambda chains were traced. Thus, differentiation between polyclonal and monoclonal B-cell processes on the basis of cytoplasmic labelling was often better in undigested sections. However, the light-chain type of membrane immunoglobulin could usually not be determined in directly fixed tissue. Ethanol fixation preceded by washing in saline afforded such determination and also preserved certain T-cell and HLA-DR antigens as well as diffuse alpha-naphthylbutyrate esterase. Reactive and malignant macrophages could further be traced by their cytoplasmic expression of L1 antigen, both in formalin- and ethanol-fixed material.

The prognostic value of the monoclonal antibodies HMFG1 and HMFG2 in breast cancer

“…In some cases extracellular staining was observed both in tubules and between cells. Staining of the intracytoplasmic vacuoles was also considered extracellular, as electron microscopy has shown that the surface of the inner membrane of the vacuole has features typical of the exterior surface of the cell membranes (Battifora, 1975 (Brandtzaeg & Rognum, 1982). The change in the immunohistochemical staining pattern from extracellular in normal and benign breast tissue to mixed with varying amounts of intracellular positivity in malignant breast tissue has been observed using other antibodies Foster et al, 1982) and lectins (Franklin, 1983).…”

K-ras mutations and HLA-DR expression in large bowel adenomas

“…In pathological conditions, such as inflammation, dysplasia and carcinoma, the epithelium may express varying amounts of these antigens (Rognum et al, 1983(Rognum et al, , 1987Ruiz-Cabello et al, 1988). The presence of HLA-DR on the surface membrane of the neoplastic cells is a prerequisite for recognition by activated CD4+ T cells, and it has been shown that the growth of colorectal cancer cells expressing class II following y-interferon treatment is inhibited by activated CD4+ T-helper cells , indicating a direct cytotoxic effect (Gjertsen et al, personal communication).…”

“…HLA-DR immunohistochemistry A murine monoclonal antibody to a non-polymorphic human HLA-DR antigen, clone L234 (IgG2a, Beckton Dickinson, Mountain View, CA, USA) was applied (1:20 for 20 h) in an indirect three-step immunofluorescence method (Brandtzaeg and Rognum, 1983), including affinity purified biotinylated horse anti-mouse IgG (0.05 g IgGI-1, 3 h) and fluorescein isothiocyanate (FITC)-labelled avidin (0.05 g 1-', 30 min), both purchased from Vector Laboratories (Burlingame, CA, USA).…”

Is heterogeneous expression of HLA-DR antigens and CEA along with DNA-profile variations evidence of phenotypic instability and clonal proliferation in human large bowel carcinomas?

“…One section from each series was stained by a trichrome routine method (HAS) containing haematoxylin, azofloxin, and saffron (Stave & Brandtzaeg, 1977 (Rognum et al, 1980). The red signal from this conjugate was enhanced by combining the FITC-labelled avidin with a TRITClabelled swine anti-rabbit IgG (Brandtzaeg & Rognum, 1983 (Rognum et al, 1980). Both the tumour and the adjacent transitional mucosa were evaluated.…”