Soil ameba infection. Specific indirect immunoenzymatic (peroxidase) staining of formalin-fixed paraffin sections.

This report describes the use of the immunoenzymatic (peroxidase) method to identify the species and to stain distinctively the amebas in formalin-fixed paraffin-mounted sections. This permits the use of hematoxylin and eosin counterstaining. The method, now well developed by others for many purposes, is an alternative to immunofluorescence and seems to offer a number of advantages and a lesser number of disadvantages.     

The tumour-associated antigen MAGE-1 is detectable in formalin-fixed paraffin sections of malignant melanoma

The MAGE-1 gene encodes a protein encompassing a HLA-A1-restricted target epitope for cytolytic T lymphocytes. Monoclonal antibodies directed against the MAGE-1 protein were tested for usage in immunohistology of routine pathology material. Seven formalinfixed, paraffin-embedded malignant melanomas were studied by the Avidin-Biotin complex (ABC) method with or without different antigen retrieval methods. Native, frozen tissues from the same tumours were used to validate the results by immunohistochemistry on frozen sections, by PCR for mRNA and by protein demonstration in tissue extracts using western blotting. Of 4 monoclonal antibodies tested, mAB 34B and mAB 77B were highly efficient in detecting MAGE-1 protein in deparaffinised sections with the regular ABC method after microwave pretreatment. In a series of an additional 28 patients 75% expressed MAGE-1, 50% in a substantial proportion. Follow-up studies in 6 patients indicate that the expression pattern remains stable but may change substantially within a short range. Immunohistology is thus a rapid and well-established method that might be used to select and monitor HLA-A1 positive patients with malignant melanoma and other candidate tumours for MAGE-1-directed immuno-therapy.     

p53 immunostaining in the distinction between benign and malignant mesothelial proliferations using formalin-fixed paraffin sections.

The distinction between reactive mesothelium and mesothelioma in pleural biopsy specimens is notoriously difficult, and conventional immunohistochemical markers have provided no relief. The object of this study was to examine the frequency of immunohistochemically detectable p53 overexpression in routinely processed, paraffin-embedded tissue from pleural mesotheliomas and from pleura showing reactive mesothelial hyperplasia, using a polyclonal antibody to formalin-resistant p53 epitopes, and to consider the diagnostic utility of this antibody in the distinction between mesothelioma and reactive mesothelium in pleural biopsy specimens. Immunostaining was enhanced by pepsin predigestion prior to the application of the primary antibody. Positivity occurred in 10/16 epithelial mesotheliomas, 9/19 biphasic mesotheliomas, 2/12 sarcomatous mesotheliomas but in none of 20 reactive pleura. Immunostaining was particularly intense in some of the biopsy specimens, which may be due to the rapidity with which these small pieces of tissue were fixed. In conclusion, this study suggests that p53 immunostaining can help to distinguish epithelial or biphasic mesothelioma from reactive mesothelial hyperplasia in formalin-fixed, paraffin-embedded pleural biopsy specimens.     

Lectin binding to serous ovarian tumours.

Sections of normal ovarian surface epithelium, benign serous cystadenomas, borderline serous cystadenomas and serous cyst-adenocarcinomas were stained with a pattern of lectins (Con A, WGA, SBA, DBA, UEA I, PNA and RCA I) to determine the different glycoproteins and their cellular changes. The epithelial cells stained with Con A, WGA, UEA I and RCA, although the intensity of the staining was generally higher in the malignant tumours. PNA stained only the malignant cells of the cystadenocarcinoma and DBA only the benign epithelial cells. These findings show that ovarian epithelial cells contain different glycoconjugates and that malignant transformation is accompanied by changes in the composition of these glycoconjugates.     

Demonstration of light chain monotypia in B cell non-Hodgkin's lymphomas using unfixed freeze-dried and formalin-fixed trypsinised paraffin sections.

Immunohistological light chain analysis was carried out in 55 patients with non-Hodgkin's lymphoma of B cell origin. Unfixed freeze-dried paraffin sections were used to detect surface Ig and formaldehyde-fixed trypsinised sections to detect cytoplasmic Ig. Cytoplasmic Ig was seen inconstantly in freeze-dried paraffin sections. There was complete agreement with regard to the type of light chain between freeze-dried and formaldehyde-fixed trypsinised sections. Immunohistology showed the monotypic immunoglobulin light chains in 83% of cases when unfixed freeze-dried paraffin sections were used for sIg demonstration while only in 45% of cases when formaldehyde-fixed material was used for cIg detection. The efficiency of sIg demonstration in unfixed freeze-dried paraffin sections was comparable with that based on unfixed cryostat sections or on cell suspensions when the lymphocytic lymphoma/CLL group was excluded from the evaluation. The relatively low frequency of monotypic sIg positivity obtained in this group (12/19) is due to the low density of surface Ig in CLL lymphocytes.     

Aberrant MT2 positivity distinguishes follicular lymphoma from reactive follicular hyperplasia in B5- and formalin-fixed paraffin sections.

Often, it is difficult to distinguish follicular lymphoma from reactive follicular hyperplasia histologically. Immunotypic evidence of monoclonality cannot be demonstrated routinely or reliably in routine paraffin-embedded sections. To determine whether a panel of monoclonal antibodies reactive with lymphoid cells in paraffin-embedded sections might be useful in distinguishing these confusing proliferations, the authors examined 45 follicular lymphomas and 30 follicular hyperplasia with the following antibodies; L26, B2, MT1, MT2, and UCHL-I. All sections were routine paraffin preparations from formalin- or B5-fixed tissue and were immunostained with the avidin-biotin immunoperoxidase technique. Ninety-two percent of the B5-fixed and 77% of the formalin-fixed lymphomas were MT2 positive. None of the reactive hyperplasias stained positively, and none of the other antibodies demonstrated consistent differences between these benign and malignant proliferations. MT2 marks interfollicular T cells and mantle-zone B cells in normal lymph nodes but does not mark normal germinal centers; this staining pattern is retained in reactive hyperplasia. However, paradoxically, in most follicular lymphomas the neoplastic germinal centers show aberrant MT2 positively. The authors conclude that MT2 may be useful in distinguishing follicular lymphoma from follicular hyperplasia in paraffin sections.     

Direct immunofluorescence of skin using formalin-fixed paraffin-embedded sections.

The technique of direct immunofluorescence has been applied to skin biopsy specimens fixed in formalin and embedded in paraffin wax. The results have been compared with those obtained by using snap-frozen biopsy specimens from the same patients. Trypsinisation of the dewaxed material allowed subsequent detection of immunoglobulins, complement, and fibrinogen. When compared to the fluorescence in the snap-frozen specimens, the staining in the paraffin sections was less bright and there was a higher rate of negatives. Even so, it was possible to establish the diagnosis in most cases of pemphigus, pemphigoid, and lupus erythematosus.     

Detection and quantification of hippocampal synaptophysin messenger RNA in schizophrenia using autoclaved,formalin-fixed,paraffin wax-embedded sections

Most in situ hybridization histochemistry studies of messenger RNA in human brain have been carried out on frozen tissue. Recently, autoclaving has been reported to enable routinely processsed material to be used for in situ localization of messenger RNA. We have investigated whether autoclaving also permits in situ hybridization histochemistry to be used quantitatively. To do this, we targeted synaptophysin messenger RNA with a ³⁵S-labelled oligonucleotide probe in autoclaved, formalin-fixed, paraffin wax-embedded sections of the hippocampal formation of 11 schizophrenics and 11 controls. We compared the results with those seen on frozen sections from adjacent blocks, which had been used previously to demonstrate a loss of the messenger RNA in schizophrenia. Synaptophysin messenger RNA was readily detected in the autoclaved sections. The hybridization signal correlated strongly with that seen in the frozen sections. We found a similar pattern and magnitude of decreased synaptophysin messenger RNA in schizophrenia in the autoclaved sections as we had in the frozen sections, including the selective preservation of synaptophysin messenger RNA in CA1. The reduction of synaptophysin messenger RNA was replicated when six subjects with schizophrenia not included in the earlier study were considered separately.We conclude that autoclaving renders formalin-fixed, paraffin wax-embedded sections of human brain suitable for quantitative in situ hybridization histochemistry. This has considerable implications, given the wider availability, better morphology and easier handling of fixed than frozen human brain tissue. Using this material, we confirmed the finding of decreased synaptophysin messenger RNA in the hippocampal formation in schizophrenia, furthering the evidence for synaptic pathology in this region in the disorder.     

Immunohistochemical localization of CA 125 antigen in formalin-fixed paraffin sections of ovarian tumors with the use of Pronase

The OC 125 monoclonal antibody was used to localize CA 125 antigen in routine paraffin sections of ovarian tumors with the use of a modified avidin-biotin-peroxidase complex (ABC) technic. Pretreatment of the paraffin sections with Pronase allowed subsequent detection of CA 125 antigen. OC 125 stained 4 (80%) of 5 benign and borderline serous ovarian tumors, 12 (86%) of 14 serous adenocarcinomas, and 3 (23%) of 13 benign and malignant mucinous ovarian tumors. The pattern of distribution of CA 125 antigen was mostly at the intraluminal and peripheral cell surfaces, while intracytoplasmic staining also was seen. Overall, CA 125 antigen detectability rate in paraffin sections was found to be compatible with those reported in frozen sections. The method allows retrospective immunohistochemical examination of a large number of cases with ovarian tumors.     

Histomorphometry in paraffin sections of thyroid tumors.

Planimetric features of cell nuclei in paraffin-embedded histological sections of benign and malignant thyroid tumors, as well as normal thyroid tissue as control, were determined by means of a semiautomatic system. The main aim was to objectify possible quantitative differences between adenomas and carcinomas of the thyroid gland, which had recently been reported by several authors. For each nuclear profile, the area, the maximum diameter as well as two form factors were calculated. Statistical analyses of morphometric differences between normal controls, oxyphilic adenomas and carcinomas, and between follicular adenomas and carcinomas were performed using the T-test, a multivariate test, and a discriminant analysis. The tests revealed significant differences between controls and all other groups. The most striking result, however, was the total discrimination between follicular adenomas and carcinomas, with no false reclassification. Carcinomas had a higher mean nuclear area and diameter and a lower form factor. A similar reliability of discrimination could be obtained by comparing these morphometric values in oxyphilic adenomas and carcinomas. When using a test set of 9 cases (4 adenomas, 5 carcinomas), only one adenoma was falsely reclassified as a carcinoma by the discriminant analysis. Our results thus allow the conclusion that planimetric nuclear measurements indeed seem to be useful for the objectivation of cytomorphologic differences between adenomas and carcinomas of the thyroid gland.     

Lectin binding to the human retina

Formalin-fixed, paraffin-embedded tissue sections of the human retina were stained with different fluorescein isothiocyanate-conjugated lectins. The lectins used were concanavalin A (Con A), Triticum vulgaris (WGA), glycine maximum (SBA), Dolichos biflorus (DBA), Ulex europaeus (UEA I), Arachis hypogaea (PNA), and Ricinus communis (RCA I). Con A stained both the inner and outer segments of the rods and cones, whereas WGA stained the inner and outer segments of the rods and the outer segments of the cones. PNA selectively stained only the inner segments of the cones. In addition, Con A and WGA stained neuron cytoplasm and nerve fibers in different layers of the retina. The results obtained differ in some important aspects from those previously obtained in the frog and monkey retina; this finding may be due to species differences. The results of lectin staining in the normal human retina may form the basis for future studies of retinal diseases.     

Lectin binding by Giardia lamblia.

Surface carbohydrates of Giardia lamblia were examined using six plant lectins chosen because of their specificity for major carbohydrates moieties. The binding to axenically grown G. lamblia trophozoites was assessed in both a quantitative microagglutination assay and a fluorescence assay. Of the six lectins tested, wheat germ agglutinin (WGA) agglutinated the highest percentage (22.9 +/- 3.7%) of live trophozoites, and fluorescein-labeled WGA (100 micrograms/ml) bound to 98 +/- 5% of them. Since the carbohydrate specificity of WGA includes both N-acetyl-D-glucosamine (GlcNAc) and sialic acid, inhibition experiments were performed. GlcNAc inhibited he binding of WGA to G. lamblia in both assays to a greater extent than did sialic acid. Binding of WGA was not altered by prior treatment of trophozoites with neuraminidase, suggesting that WGA was binding to GlcNAc moieties on G. lamblia and not to sialic acid. The remaining five lectins either bound nonspecifically or exhibited low percentages of binding. The apparent presence of GlcNAc but not sialic acid or other exposed surface carbohydrates may be important in the interaction of G. lamblia with its human host.     

Lectin binding to human gastric adenocarcinomas and adjacent tissues.

The binding of lectins to paraffin sections of nine gastric carcinomas and adjacent mucosa was examined by fluorescence microscopy. A battery of nine lectins was employed, and both intestinal and diffusely infiltrating tumors were tested. Wheat germ agglutinin and Ricinus communis agglutinin I appeared to bind to both mucus and nonmucus glycoproteins; these lectins labeled tumor cells, benign epithelial cells, and nonepithelial tissues strongly and consistently. Peanut agglutin, soybean agglutinin, Dolichos biflorus agglutinin, Bandeiraea simpifolica agglutinin, and Ulex europaeus agglutinin I bound extensively to mucosubstances in vacuoles and apices of benign epithelial cells but often bound to tumor cells focally and in some cases not at all. Neuraminidase digestion enhanced lectin staining in some tumors; but in others, especially those of the diffusely infiltrating type, neuraminidase digestion did not enhance the staining of tumor cells. The results suggest that the decrease in the proportion of tumor cells labeling with lectin relative to superficial epithelial cells can be due either to the oversialylation of mucoproteins or to the loss of glycosylating enzyme activity. Concanavalin A did not bind to mucosubstances in the vacuoles or apices of benign epithelium, but bound to mucus vacuoles of metaplastic epithelium and to coarse cytoplasmic granules in two of the tumors examined. This suggests either the abnormal addition of mannose to mucus glycoprotein or the production of a distinct glycoprotein by some gastric tumors.     

Detection of estrogen receptors with monoclonal antibodies in routinely processed formalin-fixed paraffin sections of breast carcinoma. Use of DNase pretreatment to enhance sensitivity of the reaction

This study describes an improved immunohistochemical method for the sensitive and specific identification of estrogen receptors (ERs) in paraffin sections from formalin-fixed and routinely processed breast carcinoma tissues, using DNase pretreatment to expose nuclear antigenic sites and commercially available immunoreagents (including monoclonal antibody) in kit form. Results were compared with dextran-coated charcoal cytosolic assay (DCC) and with conventional immunohistochemistry on frozen sections. Sensitivity and specificity for determinations on paraffin sections were 88% and 86%, respectively, and statistical analysis showed very good agreement between DCC and paraffin sections (kappa = 0.805). The DNase technic on paraffin sections allows excellent correlation between histologic characteristics and ER status and reduces DCC sampling error resulting from stromal dilution and tumor variability. This method offers a reliable and reproducible alternative when tissue is not suitable or unavailable for DCC or frozen tissue analysis and can be used for retrospective studies on stored tissue blocks.     

Cross-reactive antigenicity of nucleoproteins of lyssaviruses recognized by a monospecific antirabies virus nucleoprotein antiserum on paraffin sections of formalin-fixed tissues

Diagnosis of rabies is routinely confirmed by detection of rabies virus antigens in acetone-fixed frozen brain tissues or imprint smears using an immunofluorescence method with commercial antirabies virus antibodies. Since recent molecular analyses disclosed wide heterogeneity in the genome sequences of rabies virus strains and related lyssaviruses, it is necessary to confirm the presence of common epitopes in these lyssaviruses. In this study we confirmed the presence of cross-reactive antigens of various lyssaviruses in paraffin sections of formalin-fixed tissue using a monospecific rabbit antiserum prepared by immunization with a recombinant nucleoprotein of rabies virus. By immunohistochemical application, the antigen was detected predominantly in the cytoplasm of neurons in the brains of mice infected with rabies virus, Duvenhage virus, Mokola virus and European bat lyssavirus-1, while no cross-reaction was observed in uninfected humans and animals including dogs, bats, and raccoons. In addition, we examined one autopsy case that was infected in a rabies-endemic nation and developed the clinical manifestation of rabies after returning to Japan in 1970, and found that the antigen was well preserved in paraffin sections of formalin-fixed tissues. Thus, this suggests that the lyssavirus-specific antigen is recognized by the monospecific antibody against rabies virus nucleoprotein, and that this cross-reactive antigen is detectable on formalin-fixed paraffin-embedded tissues by immunohistochemical analysis.