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PubMed1993

Progesterone receptors in routinely paraffin-embedded primary breast carcinomas and lymph node metastases.

Described here is an immunohistochemical technique using the commercially available monoclonal progesterone receptor (PR) antibody KD 68 in routinely fixed and paraffin-embedded breast carcinomas and lymph node metastases. The authors' technique is compared with several incubation variations. The method applying the primary antibody in a dilution of 1:10 overnight followed by a biotinylated second antibody showed the best results when Triton X-100 was added to the buffer. Using this method, comparison with the results on frozen sections of 34 breast carcinomas yielded a significant concordance of 94%. Correlation between the results on paraffin sections and those obtained by the standard dextran-coated charcoal cytosol assay was 80%. The value of the method for predicting endocrine therapy response was shown in 20 patients. Thus the reliability of the method has been demonstrated and was applied on 151 lymph node metastases and the corresponding primary breast carcinomas from 50 patients. Generally PR content in the metastases was lower than in the primary tumors (p < 0.001). This finding indicates that evaluation of PR in lymph node metastases should be included in the decision for endocrine therapy of breast cancer.

Breast Neoplasms↗
PubMed1994

Distribution of anti-CD68 (EBM11) immunoreactivity in formalin-fixed, paraffin-embedded bovine tissues.

A commercially acquired anti-human macrophage antibody (anti-CD68; EBM11) was used in an immunocytochemical technique to detect macrophages in formalin-fixed, paraffin-embedded tissues from cattle, pigs, humans, rats, turkeys, dogs, and cats. In healthy cattle, the antibody labeled alveolar macrophages, pulmonary intravascular cells (presumably intravascular macrophages), and macrophage-like cells in other tissues. In bovine lungs infected with Pasteurella haemolytica, EBM11 antibody labeled 95% of alveolar macrophages and macrophages within alveolar septa but only 0-2% of streaming or "oat" leukocytes. Alveolar macrophages were also stained by EBM11 in pigs but not in rats, turkeys, dogs, and cats. The antibody also stained macrophage aggregates in the mesenteric lymph nodes and intestinal lamina propria of Mycobacterium paratuberculosis-infected cattle. This study shows that the anti-CD68 (EBM11) antibody is a useful marker of macrophages in normal bovine tissues or tissues from areas of acute or chronic inflammation that have been routinely processed. The study also adds strength to the growing evidence suggesting that streaming leukocytes seen in pneumonic pasteurellosis are neutrophils.

Adult↗
PubMedDate not supplied

Immunohistochemical demonstration of p53 protein in colorectal adenomas and adenocarcinomas. Reliable application of the heat-induced antigen retrieval method to formalin-fixed, paraffin-embedded material.

Ninety-nine polypoid neoplasms and eight advanced adenocarcinomas of the colon were studied immunohistochemically for p53 protein expression. For reproducible antigen retrieval, formalin-fixed, paraffin-embedded archival sections were heated at 90 degrees C for 120 min in 0.01 mol/L phosphate-buffered saline, pH 7.2, prior to immunostaining. Proliferating cell nuclear antigen served as a positive control marker for effective antigen retrieval. The 99 polyps were categorized into 24 high-grade adenomas, 60 non-invasive cancer-in-adenomas (CIA), and 15 CIA with stromal invasion. All the polyps contained portions of low-grade adenoma. Positive nuclear staining of p53 protein was observed in none of the non-neoplastic mucosa, nine (9%) of 99 low-grade adenomas, 17 (71%) of 24 high-grade adenomas, 46 (77%) of 60 non-invasive CIA, 10 (67%) of 15 invasive CIA, and five (63%) of eight advanced carcinomas. When the antigen retrieval treatment was omitted, the positivity rates were 0, 2, 17, 35, 40, and 63%, respectively. When the antigen-retrieved staining pattern was classified into (i) 'sparse' (< 25% of the nuclei of neoplastic glands labeled), 'scattered' (25-75%) and 'dense' (> 75%); or (ii) 'focal' (the positively labeled glands occupying < 25% of the tumor area), 'intermediate' (25-75%) and 'diffuse' (> 75%), the sparse and focal patterns predominated in high-grade adenomas and non-invasive CIA with low-grade atypia, while the dense and diffuse patterns predominated in invasive CIA and all the advanced carcinomas revealed the dense and diffuse patterns. Non-invasive CIA with high-grade atypia belonged to an intermediate type between the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenocarcinoma↗
PubMed1996

Improvement of the method for demonstration of nucleolar organizer regions (NORs) in formalin fixed and paraffin embedded tissue using microwave pretreatment.

For demonstration, accurate evaluation and measurements of nucleolar organizer regions (NORs) in nuclei, a good quality reaction of silver staining is required. On formalin fixed and paraffin embedded tissue this can be achieved only, if special procedures are used. We managed to get sharply delineated, dark brown or black dots representing AgNORs (silver stained NORs), that are easy to count and measure, with microwave pretreatment of tissue sections before incubation in silver solution.

Animals↗
PubMed2005

Immunohistochemical detection of immunoglobulins and complements in formaldehyde-fixed and paraffin-embedded renal biopsy tissues; an adjunct for diagnosis of glomerulonephritis.

BACKGROUND: The present study was undertaken to demonstrate the deposition of immunoglobulins or complements in formaldehyde-fixed and paraffin-embedded renal biopsy tissues through the unmasking of antigens with microwave treatment plus protease digestion or trypsin digestion. METHODS: Biopsy samples from patients with IgA nephritis (n = 7), lupus nephritis (7), membranous nephropathy (7) and mesangiocapillary glomerulonephritis (3) were used. Antigen unmasking was performed with (i) microwave treatment plus protease digestion for 10, 30 or 60 min, or (ii) digestion with 0.25% trypsin for 60 or 120 min. RESULTS: Microwave treatment plus protease digestion for 30 or 60 min and trypsin digestion for 120 min provided good results for the unmasking of immunoglobulins in glomeruli with structural preservation. The IgA deposits in IgA nephritis and IgG deposits in lupus nephritis and membranous nephropathy were clearly revealed in more than 80% of cases by both pretreatments. Microwave treatment plus protease digestion for 30 min revealed the deposition of C3 in all cases of mesangiocapillary glomerulonephritis and lupus nephritis and was superior to trypsin digestion. Characteristic patterns of C3 deposition were observed for these forms of glomerulonephritis, although C3 deposits in membranous nephropathy were detected in only 50% of cases. It was not possible to unmask all of the antigens in the glomeruli, especially those with weak immunofluorescence. CONCLUSION: Microwave treatment plus protease digestion is effective for the unmasking of antigens in paraffin sections and as useful for the diagnosis of immune-mediated glomerulonephritis as trypsin digestion.

Biopsy↗
PubMed1996

Evaluation of antibodies reactive with porcine lymphocytes and lymphoma cells in formalin-fixed, paraffin-embedded, antigen-retrieved tissue sections.

OBJECTIVES: To determine whether antibodies raised against human or porcine lymphocytes are reactive with porcine lymphocytes and lymphoma cells in formalin-fixed, paraffin-embedded, antigen-retrieved tissue sections, and to determine the conditions suitable for antigen retrieval. DESIGN: We evaluated reactivities of 27 anti-lymphocyte antibodies with porcine tissue sections, including those of lymphomas, treated with 11 antigen-retrieval methods. ANIMALS: Swine. PROCEDURE: We used 19 anti-human and 8 anti-porcine lymphocyte antibodies. For antigen retrieval, we tested 11 methods: heating with 6 soaking solutions in a microwave oven, heating with 2 commercially available soaking solutions in a water bath, and enzyme digestion with 3 proteases. After these treatments, sections were immunostained along with untreated sections. RESULTS: Of 27 anti-lymphocyte antibodies tested, CDw75 (clone LN-1), CD79 alpha (mb-1, clone HM57),CD79 beta (B29, clone B29/123), HLA-DR (clone TAL 1B5), and polyclonal CD3 were strongly reactive with porcine lymphoid tissues, including lymphomas, when sections were deparaffinized and heated in 0.1M Tris-HCl buffer (pH 8.0) in a microwave oven or with antigen-retrieval solution in a water bath. All patterns of positive staining were essentially the same as those previously described in human beings. CONCLUSION: These 5 antibodies, with the antigen retrieval sequences, are helpful in identifying, distinguishing, and characterizing lymphocyte subsets in archival sections of normal and pathologic porcine lymphoid tissues including lymphomas. In addition, our findings indicated that antigen retrieval by microwave heating of the sections can appreciably expand the range of antibodies useful in paraffin immunohistochemistry, including those that otherwise work only on sections from frozen tissues or from an antigen-derived species.

Animals↗
PubMed1986

Monoclonal antibody labeling of mononuclear cell surface antigens in formaldehyde-fixed paraffin-embedded cutaneous tissue.

The influence of the sequential stages of conventional formaldehyde fixation and paraffin embedding of cutaneous tissue on monoclonal antibody labeling of cell surface antigens is described. The effects of variation in fixation time, dehydration, clearing, wax embedding, and enzyme treatment of cutaneous sections were examined. By curtailing fixation time, using cold ethanol dehydration, and limited cold clearing with xylene, immunoreactivity of several important monoclonal antibodies was retained. Wax embedding could be achieved at 58 degrees C for 1 h or by using low-melting-point wax at 42 degrees C for 3 h. Thus was derived an optimal processing procedure which afforded good tissue morphology and allowed reliable reproducible labeling by monoclonal antibodies to cell surface antigens.

Antibodies, Monoclonal↗
PubMed2001

[Extraction of RNA from paraffin embedded tissues and analysis of p53 gene expression in colonic cancer].

We examined, immunohistochemically and molecular biologically, p53 gene expression in 10 patients with colonic cancer. RNA was extracted from paraffin embedded normal and colonic cancer tissues by using RNA isolator kit and proteinase K. The most effective time and concentration of proteinase K for RNA extraction was 24 hours and 100 micrograms/ml, respectively. P53 gene expression was analyzed by ABI PRISM 7700 Sequence Detection System(ABI 7700 System, Perkinelmer). Gene expression level in each sample was estimated on the basis of the standard curve of ABI 7700 System. Human G3PDH gene was used as the internal control. Immunohistochemically, the tumor cells in all examined cases showed a strong positivity for anti-p53 gene antibody. In ABI 7700 System, expression of p53 gene in the malignant tissues revealed a high level in only 2 cases that had a clinical stage IV, however, in remaining 8 cases a clinical stage was I to III and expression level of p53 was relatively lower. These results suggest that colonic cancer cells show mutant-p53 gene expression, and a ratio of mutant- to wild-p53 gene may have something to do with a relationship between gene expression and clinical stage.

Adenocarcinoma↗
PubMed2004

Application of combined immunofluorescence and fluorescence in situ hybridization on paraffin-embedded sections to characterize T-cell lymphoma with EBV-infected B-cell blasts.

Combined immunofluorescence (IF) and fluorescence in situ hybridization (FISH) on formalin-fixed, paraffin-embedded tissue sections were used to examine lymph node tissue from two patients diagnosed with T-cell lymphoma with Epstein-Barr virus (EBV)-infected B-cell blasts. The majority of cells within the samples comprised T-cells staining positively for CD3. In addition, both patients had a population of large pleiomorphic cells that were positive for the B-cell marker CD20 and for EBV LMP-1. Standard PCR clonality testing of the nodes revealed both immunoglobulin heavy chain (IGH) and T-cell receptor (TCR) clonal rearrangements in one patient, although in the other case monoclonality was demonstrated only for TCRG. Cytogenetics of cultured lymphocytes from nodal tissue revealed two apparently unrelated abnormal clones in both patients. Combined IF and FISH revealed that these phenomena reflected two abnormal populations of B- and T-cells rather than reactive B-cell hyperplasia or biphenotypic evolution from a common ancestral lymphoma. True B-cell malignancy probably emerged within a preexisting but unrelated T-cell lymphoma. This is the first study to relate the phenotype of the abnormal cells in such cases to specific clonal populations of cells, and it demonstrates a method that may easily be introduced into a diagnostic cytogenetics laboratory with access to standard pathology laboratory resources.

Antigens, CD20↗
PubMed1994

[Study of B-lymphocyte clonality using in vitro gene amplification (PCR) in paraffin embedded samples].

Family-specific primers were used in polymerase chain reaction (PCR) to analyse clonality of immunoglobulin heavy chain (IgH) gene rearrangement. DNA templates were extracted from formalin-fixed paraffin-embedded biopsies from B-cell lymphomas (n = 19), T-cell lymphomas (n = 3), reactive lymphadenopathies (n = 20) and negative controls (n = 2). PCR was also performed on DNA extracted from fragments of the same biopsies that were either fixed in Bouin's solution (n = 24) or snap-frozen (n = 22). The latter were also studied for IgH gene rearrangement by the Southern blot technique. Polyclonal or clonal fragments were obtained from all frozen biopsies. No amplification was observed in 3 out of 44 formalin fixed specimens and 18 out of 24 Bouin fixed specimens. A clonal amplification was only observed in 16 out of 19 formalin-fixed B-cell lymphoma specimens. Therefore this technique allows to study B-cell clonality from formalin-fixed, embedded material.

Acetates↗
PubMed2006

Real-time quantitative RT-PCR shows variable, assay-dependent sensitivity to formalin fixation: implications for direct comparison of transcript levels in paraffin-embedded tissues.

Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) is a versatile tool for precise quantification of gene expression. Formalin-fixed and paraffin-embedded (FFPE) tissue is well suited for qRT-PCR, if RNA extraction is optimized and small amplicon sizes are used. However, little is known whether individual assays may show variable sensitivity to fixation. This is of great importance, if a direct comparison of different transcripts is performed within the same sample, such as for mRNA splice variants. We established a cell culture model to test for and quantify differences in performance of individual qRT-PCR assays on FFPE as compared with fresh material, using TaqMan methodology. RNA was isolated from 7 different cell lines either directly or after preparation of a FFPE cell block. RNA from both sources was reverse transcribed and gene expression quantified using 13 different TaqMan assays. All assays allowed highly reproducible target quantification, using both fresh and FFPE-derived cDNA. The 13 assays showed an average Ct difference of 3.2 between fresh and FFPE cells, if identical amounts of cDNA were used as template. However, the Ct shifts varied from 1.8 to 5.1 for individual assays, indicating variable resistance to fixation. These Ct shift differences were statistically highly significant in 27/78 (35%) of all possible combinations of assays. Because the Ct shift remained constant for each assay, they could be used for calculation of correction factors which rendered FFPE-derived expression data highly comparable to those obtained from fresh material, and as a consequence among each other. Thus, a standardized assessment of qRT-PCR assay efficiencies in FFPE allows for precise intraindividual comparison of mRNA species, such as splice variants with different biologic functions, in archival tissues.

Biological Specimen Banks↗
PubMed2005

Array-based comparative genomic hybridization from formalin-fixed, paraffin-embedded breast tumors.

Identification of prognostic and predictive genomic markers requires long-term clinical follow-up of patients. Extraction of high-quality DNA from archived formalin-fixed, paraffin-embedded material is essential for such studies. Of particular importance is a robust reproducible method of whole genome amplification for small tissue samples. This is especially true for high-resolution analytical approaches because different genomic regions and sequences may amplify differentially. We have tested a number of protocols for DNA amplification for array-based comparative genomic hybridization (CGH), in which relative copy number of the entire genome is measured at 1 to 2 mb resolution. Both random-primed amplification and degenerate oligonucleotide-primed amplification approaches were tested using varying amounts of fresh and paraffin-extracted normal and breast tumor input DNAs. We found that random-primed amplification was clearly superior to degenerate oligonucleotide-primed amplification for array-based CGH. The best quality and reproducibility strongly depended on accurate determination of the amount of input DNA using a quantitative polymerase chain reaction-based method. Reproducible and high-quality results were attained using 50 ng of input DNA, and some samples yielded quality results with as little as 5 ng input DNA. We conclude that random-primed amplification of DNA isolated from paraffin sections is a robust and reproducible approach for array-based CGH analysis of archival tumor samples.

Breast Neoplasms↗
PubMed2006

Characterization of anti-TIMP-1 monoclonal antibodies for immunohistochemical localization in formalin-fixed, paraffin-embedded tissue.

The aim of this study was to evaluate seven anti-TIMP-1 (tissue inhibitor of metalloproteinase-1) monoclonal antibodies by immunohistochemical (IHC) staining of formalin-fixed, paraffin-embedded (FFPE) tissue. Detection of the TIMP-1 protein was studied by IHC in FFPE human archival normal and neoplastic samples. Indirect IHC technique was used, and the seven antibodies (clones VT1, VT2, VT4, VT5, VT6, VT7, and VT8) were tested in various concentrations using different pretreatment protocols. All seven VT antibodies specifically immunostained the cytoplasm of islets of Langerhans cells in normal pancreas, epithelial cells of hyperplastic prostate, tumor cells of medullary thyroid carcinoma, and fibroblast-like cells of malignant melanoma. Specificity of the anti-TIMP-1 antibodies was confirmed by several controls, e.g., Western blotting on proteins extracted from FFPE tissue showed that the VT7 antibody reacted specifically with a protein band of approximately 28 kDa, corresponding to the molecular mass of TIMP-1. However, sensitivity varied with the different antibodies. Use of heat-induced epitope retrieval (HIER) and the VT7 clone applied at low concentrations demonstrated more intense immunoreactivity with the TIMP-1-positive cell types compared to the other six clones. Furthermore, when tested on a range of normal and neoplastic endocrine tissues, the VT7 clone demonstrated immunoreactivity with all neuroendocrine cell types. In conclusion, all seven antibodies detected TIMP-1 protein in various normal and neoplastic FFPE tissues, but one clone, VT7, was superior for IHC staining of TIMP-1 in FFPE tissue sections when using HIER.

Antibodies, Monoclonal↗
PubMed1996

Immunohistochemical detection of estrogen and progesterone receptors performed with an antigen-retrieval technique on methacarn-fixed paraffin-embedded breast cancer tissues.

Immunohistochemical detection of estrogen and progesterone receptors (ER and PgR, respectively) was performed in 67 cases of Japanese female invasive breast carcinoma on methacarn-fixed paraffin-embedded sections using monoclonal antibodies against ER (1D5) and PgR (10A9) with an antigen-retrieval technique based on microwave exposure in citrate buffer solution. Staining localized in nuclei and specimens containing > or = 20% specifically stained tumor cell nuclei were considered ER- or PgR-positive; the positive rate was 37% (25/67) for ER and 45% (30/67) for PgR. The immunohistochemical (IHC) results were compared with cytosolic receptors obtained from tissue homogenates as measured by dextran-coated charcoal (DCC) assay, in which tumors were considered ER- and/or PgR-positive when receptor concentration was greater than 3 or 5 fmol/mg cytosolic protein, respectively, and agreement between the two methods was 71.6% for ER and 80.6% for PgR. The sensitivity and specificity were 53.5 and 91.7% for ER-IHC, and 77.4 and 83.3% for PgR-IHC, respectively. In relation to age of the patient and stage of the cancer, ER immunoreactivity correlated with patient's age (> or = 55 vs 55, P = 0.032), and proportionally increased with aging (P = 0.0084) using the Kruskal-Wallis statistics. PgR-immunoreactivity correlated with nodal involvement (P = 0.031) by the TNM system. However, no correlations were found between the results of the DCC assay and any of the clinical parameters examined. Thus, immunohistochemical assay may provide valuable information in predicting prognosis and response to endocrine therapy.

Acetates↗
PubMed1986

Extraction of nuclei from selected regions in paraffin-embedded tissue.

A method for the extraction of nuclei from selected regions in paraffin-embedded tissue is described. Fifty-micrometer sections are cut, dewaxed, and rehydrated. For the final handling, the sections are manually transferred from one tray to another. The sections are put on a slide under a dissection microscope and the region of interest is isolated by scraping off the irrelevant region with a scalpel. An optimal number of single nuclei is obtained by incubation in a protease solution with intermediate syringing. The nuclei are washed and can be used for flow cytometry. Resuspension of the nuclei in foetal calf serum and cytocentrifugation results in preparations suited for image analysis. DNA cytometric measurements of nuclei in a carcinoma in situ and an invasive carcinoma region in breast tissue present in the same tissue block and in a severe dysplasia/carcinoma in situ (CIN III) region of the cervix are presented.

Breast Neoplasms↗
PubMed1991

Analysis of cell cycle kinetics using flow cytometry from paraffin-embedded tissues in endometrial adenocarcinoma.

Flow cytometric analysis of cell cycle kinetics from paraffin-embedded tissues was performed to evaluate the DNA heterogeneity and risk factor of endometrial adenocarcinoma. We studied 54 tumor samples and 6 controls with normal endometrium, and all samples were measured for S-phase fraction (SPF) and proliferative index (PI). Intratumor variations of SPF and PI values were evaluated at 3 different sites of the tumor; (1) primary endometrial lesion, (2) the site of myometrial invasion, and (3) metastatic lesions. SPF and PI values of endometrial adenocarcinoma were significantly higher than those of normal endometrium. There was a significant correlation between the PI value and the histological grade. SPF and PI values at the primary endometrial lesion had a significant correlation with the prognosis of this cancer at stage III or IV. SPF and PI values which were measured at the site of myometrial invasion tended to be lower than those from other sites. Our results indicate that the SPF and PI values of endometrial lesions are one of the important prognostic factors of endometrial adenocarcinoma at stage III or IV. In addition, DNA syntheses of endometrial adenocarcinoma cells display intratumor heterogeneity.

Adenocarcinoma↗
PubMed1980

Diagnosis of hairy-cell leukaemia by tartrate-resistant acid phosphatase activity in paraffin-embedded tissue sections.

Tartrate-resistant acid phosphatase activity may be demonstrated in paraffin-embedded liver and spleen specimens as well as in decalcified bone marrow biopsies after fixation in a mixed glutaraldehyde-formaldehyde-calcium acetate solution. This technique may routinely be applied to haematologically relevant material and aids in the differential diagnosis of hairy-cell leukaemia.

Acid Phosphatase↗