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PubMed1987

Monoclonal antibodies marking T lymphocytes in paraffin-embedded tissue.

The authors investigated the ability of 70 monoclonal antibodies obtained from the Third International Workshop on Human Leukocyte Antigens (Oxford, 1986) to mark T lymphocytes in B5-fixed paraffin-embedded tissue. No staining occurred with 65 of the antibodies; however, 5 antibodies marked small lymphocytes in the T-cell areas of human tonsil. Two antibodies which strongly labeled lymphocytes, UCHL1 and T2/48, were used to examine 106 cases of non-Hodgkin's lymphoma, 29 cases of Hodgkin's disease, and a variety of normal and neoplastic tissues. UCHL1 and T2/48 each marked 86% (37/43) of B5-fixed T-cell lymphomas. Only 50% of formalin-fixed T-cell lymphomas were marked with these antibodies. UCHL1 marked 1.8% (1/56) of the B-cell lymphomas, compared with T2/48, which marked 19.6% (11/56) of the B-cell lymphomas. T2/48 had the interesting attribute of marking cells of the follicular mantle-zone and intermediate lymphocytic lymphoma, suggesting that the antibody recognizes a B-cell differentiation antigen. No Reed-Sternberg cells, epithelial neoplasms, sarcomas, neurogenic tumors, or normal nonlymphoid tissue were marked by either antibody. These antibodies successfully mark T cells in paraffin tissue sections and should aid in the investigation and characterization of abnormal lymphoid proliferations, "undifferentiated" malignant neoplasms, and immunologically mediated disorders.

Antibodies, Monoclonal↗

PubMedDate not supplied

delta Antigen in hepatitis B: immunohistology of frozen and paraffin-embedded liver biopsies and relation to HBV infection.

The finding that the recently described hepatitis B (HB)-associated delta antigen (delta Ag) is preserved in pronase-treated, formalin-fixed paraffin sections allowed a combined prospective and retrospective immunohistological study of its occurrence in 571 liver biopsies. Among 116 frozen biopsies (69 HBAg seropositive, 47 HBAg seronegative) and 455 paraffin-embedded biopsies (296 HBAg seropositive, 159 HBAg-negative), delta Ag was found in 10 HBAg seropositive patients. With the exception of 1 patient with chronic persistent HB, all had chronic-active HB and none had acute HB; 5 patients were i.v. drug abusers. In follow-up biopsies, the delta Ag persisted with HBsAg for as long as 6 years. The expression of delta Ag showed similarities to the HBcAg system including nuclear localization, mixed nuclear cytoplasmic expression, and coexistence with anti-delta in blood. The findings are compatible with the hypothesis that delta Ag represents a transmissible, defective viral agent which requires HBV as a helper and may modulate, but not terminate, ongoing HBV infection.

Antigen-Antibody Complex↗

PubMed1988

Immunohistochemical analysis of estrogen receptors (ER) using formalin-fixed paraffin-embedded breast cancer tissue: correlation with clinical endocrine response.

A new immunohistochemical assay utilizing anti-ER monoclonal antibodies (H 222, Abbott) for detection of ER in formalin-fixed paraffin-embedded breast cancer tissue has been clinically evaluated. It is shown that tumors containing immunoreactive epithelial cells are much better candidates for hormonal manipulation than those without. In addition, primary tumors and corresponding regional lymph node metastases contain qualitatively the same ER pattern. The potentials and limitations of the present method are discussed in relation to other well-known ER assays.

Breast Neoplasms↗

PubMed2006

Histological and genotypical characterization of feline cutaneous mycobacteriosis: a retrospective study of formalin-fixed paraffin-embedded tissues.

Twenty-nine cases presumptively diagnosed as feline cutaneous mycobacteriosis were evaluated microscopically with haematoxylin and eosin and modified Fite's stained sections using archived formalin-fixed paraffin-embedded tissue specimens. Lesions were characterized histologically as feline leprosy (7 cases lepromatous and 16 cases tuberculoid) or atypical mycobacteriosis (3 cases); three cases did not fit these criteria and were classified as 'miscellaneous'. Actinomycetales-specific polymerase chain reaction (PCR) of variable regions 1, 2 and 3 of the 16S ribosomal RNA (rRNA) gene and subsequent sequence analysis of the amplicons were performed to identify the species of mycobacteria associated with each case. Together, this study identified 10 different Actinomycetales organisms with greater than 98% nucleotide sequence identity to named species, nine were of the genus Mycobacterium and eight were associated with feline leprosy (both lepromatous and tuberculoid). Based on this study, we conclude that feline cutaneous mycobacteriosis should be considered as a syndrome with varied clinical and histological presentations associated with a variety of different Mycobacterium species, organisms other than Mycobacterium sp. may be associated with feline cutaneous mycobacteriosis lesions, and molecular diagnostic techniques can be an important tool for identifying agents associated with lesions of feline cutaneous mycobacteriosis.

Animals↗

PubMed1995

Immunohistochemical detection of Tritrichomonas foetus in formalin-fixed, paraffin-embedded sections of bovine placenta and fetal lung.

An immunohistochemical technique using a monoclonal antibody was evaluated as a diagnostic tool to specifically label Tritrichomonas foetus in formalin-fixed, paraffin-embedded sections of placenta and fetal lung from bovine abortions. Trichomonads were demonstrated in tissues from each of 12 abortions due to T. foetus and none of 15 abortions due to other or unidentified causes. Moderate to marked background staining occurred only in severely autolyzed tissues from T. foetus-infected fetuses. The antibody faintly labeled 1 of 3 other species of trichomonads (Trichomonas gallinae) but did not label other protozoa, bacteria, or fungi tested.

Abortion, Veterinary↗

PubMed2000

[Estrogen and progesterone receptors of primary breast carcinoma and their axillary lymph node metastases--immunohistochemical investigations of routine formalin-fixed paraffin-embedded tissue].

OBJECTIVE: The estrogen- (ER) and progesterone receptors (PR) of 27 pre- and 33 postmenopausal primary breast carcinomas with 320 axillary lymphonodal metastases were investigated in formalin-fixed paraffin-embedded tissue. MATERIAL AND METHODS: Special interest was devoted to the comparison of the receptor equipment between the axillary lymphonodal metastases and the appertained primary carcinoma. ER-antibody D5 and PR-antibody NCL-PGR were used for investigation. RESULTS: ER-positive carcinomas of premenopausal women and ER-respectively PR-positive carcinomas of postmenopausal women corresponded significantly more frequent to receptor-positive lymphonodal metastases. Carcinomas with receptor-positive metastases were mostly ER-positive and PR-negative. About all receptor-positive metastases had the same combination of receptors. In cases of high numbers of lymphonodal metastases these were in general more often receptor-negative. Respectively, the receptor-positive metastases numbered lower than the receptor-negative. Immunohistochemical receptor-negative primary carcinomas can inherit receptor-positive lymphonodal metastases. CONCLUSIONS: Immunohistochemical investigations render it possible to determine ERs and PRs not only in primary carcinomas but in lymphonodal metastases, too. Eventually the prognostic factors "ER" and "PR" will be used more often in the determination of therapeutic decisions.

Adult↗

PubMed2004

Optimization of in situ hybridization assay using non-radioactive DNA probes for the detection of canine herpesvirus (CHV) in paraffin-embedded sections.

Two non-radioactive probes using digoxigenin or biotin were developed for detecting canine herpesvirus (CHV) and compared for their sensitivities by in situ hybridization (ISH) in formalin fixed, paraffin embedded sections, which has been used routinely in veterinary fields. Sections of the CHV-infected cell preparation were subjected to several different ISH protocols using digoxigenin- or biotin-labeled probe respectively. Results were compared for the hybridization and background signal intensities. The best result was obtained by the optimized ISH protocol using digoxigenin-labeled probe for detection of CHV DNA. The optimized ISH assay, which developed in this study, may be a valid tool for the study of pathogenesis and diagnosis of CHV infection.

Animals↗

PubMed1991

Immunohistochemical assay of neu/c-erbB-2 oncogene product in paraffin-embedded tissues in early breast cancer: retrospective follow-up study of 245 stage I and II cases.

The expression of neu oncoprotein was assayed by immunohistochemistry (IHC) on 245 paraffin-embedded, Stage I and II breast cancers from patients treated at the City of Hope between the years 1980 and 1987. Only cases showing membrane staining were scored as positive. Fifty-four (22%) of the tumors stained positively for neu. Probability of disease-free survival (DFS) and overall survival (OS) was compared based on neu positivity and other prognostic factors. Overall, DFS and OS did not differ significantly among neu-positive and neu-negative cases. However, when only cases with favorable (Stages I and II) nuclear grade were analyzed, OS and DFS were significantly lower in neu-positive cases, with a 9-fold increase in risk of death and a 3-fold increase in risk of relapse. Our findings suggest that immunohistologic study of neu oncoprotein may help to define patients at greater risk among low-stage/low-nuclear-grade patients with breast cancer, a group hitherto recognized as having a good prognosis.

Adult↗

PubMed1993

bcl-2 rearrangement in Hodgkin's disease. Results of polymerase chain reaction, flow cytometry, and sequencing on formalin-fixed, paraffin-embedded tissue.

We examined 81 cases of Hodgkin's disease for evidence of the t(14;18) translocation, using the polymerase chain reaction assay on lysates of formalin-fixed, paraffin-embedded tissue. Seven of 74 amplifiable cases (9%) were positive for the translocation, which involves the bcl-2 oncogene and the immunoglobulin heavy chain gene. Two of these cases were sequenced and the breakpoints had the same pattern found in follicular lymphoma. The nuclei from one of the cases were sorted into large and small subpopulations. The t(14;18) signal was more intense in the large nucleus subpopulation, which contained a greater proportion of Reed-Sternberg-like nuclei. These results are consistent with the hypothesis that Reed-Sternberg cells carry the translocation, but they do not exclude the possibility that the translocation is found in cells representing the reactive component of Hodgkin's disease. The results also demonstrate that routinely processed material is suitable for polymerase chain reaction-based analysis of translocations, although the sensitivity is reduced 10- to 100-fold, compared with fresh tissue.

Base Sequence↗

PubMed1985

Optimal immunoreactivity of keratin proteins in formalin-fixed, paraffin-embedded tissue requires preliminary trypsinization. An immunoperoxidase study of various tumours using polyclonal and monoclonal antibodies.

The effect of preliminary trypsinization on the immunoreactivity of keratin proteins in formalin-fixed, paraffin-embedded tissues of a variety of tumors (squamous cell carcinomas, adenocarcinomas, mesotheliomas, and transitional cell carcinomas) was evaluated. Three types of trypsin (Type II and Type IX porcine trypsin and Type III bovine trypsin) and varying concentrations of trypsin were assessed. Immunoreactivity of keratin proteins was determined using rabbit anti-keratin antibodies and monoclonal antibodies (combination of AE1 and AE3) and immunoperoxidase techniques. Preliminary trypsinization was mandatory for optimal immunoreactivity of keratin proteins using either polyclonal or monoclonal antibodies. Excellent results were obtained using Type II porcine trypsin at concentrations of 25 mg/dl for 30-45 min or 50 mg/dl for 20 min, at 37 degrees C. Trypsin treatment with excessive concentrations of enzyme and/or extended incubation times promoted tissue digestion and in some cases, yielded decreased immunoreactivity and altered staining patterns.

Adenocarcinoma↗

PubMedDate not supplied

In situ detection of reovirus in formalin-fixed, paraffin-embedded chicken tissues using a digoxigenin-labeled cDNA probe.

An in situ hybridization (ISH) technique using a digoxigenin (DIG)-labeled cDNA probe detected avian reovirus (ARV) RNA in formalin-fixed, paraffin-embedded chicken tissues. Tissues were collected 3 and 10 days following inoculation with the R-2 or the S1133 strain of ARV. The cDNA clone HJp1, located on the S1 gene segment of the ARV S1133 strain, was used to prepare a nonradioactive probe. The ISH assay localized ARV RNA in infected tissues including heart, liver, intestine, pancreas, and tendon. No positive-stained cells occurred in sections from uninfected chickens.

Animals↗

PubMed1989

Expression of endogenous receptors for neoglycoproteins, especially lectins, that allow fiber typing on formaldehyde-fixed, paraffin-embedded muscle biopsy specimens. A glycohistochemical, immunohistochemical, and glycobiochemical study.

A panel of biotinylated (neo)glycoproteins was used for specific detection of endogenous sugar receptors, especially lectins, in formaldehyde-fixed, paraffin-embedded muscle biopsy specimens from human deltoid, quadriceps, and biceps muscles, tibial and quadriceps muscles of rat, and bovine masseter muscle. The glycohistochemical probes used consisted of conjugates of a labeled, histochemically inert carrier protein and various covalently linked, histochemically crucial sugar moieties. Specific binding of alpha-L-fucoside, beta-D-galactoside, beta-D-xyloside, and alpha-D-mannoside to muscle sections was detected, showing no species-specific differences. The presence of receptors for the N-acetylated sugars in natural glycoconjugates, and for sugars with a phosphate group, i.e., mannose-6-phosphate and galactose-6-phosphate, was demonstrated glycohistochemically. However, these binding specificities revealed species-specific differences, e.g., the absence of N-acetyl-D-galactosamine-specific receptors or galactose-6-phosphate-specific receptors in rat muscle. Other charged sugars included glucuronic acid and sialic acid, which bound only to ox and rat muscle or failed to reveal their respective receptors in all types of muscle investigated. This different extent of staining with anionic probes served as a further control to ascertain carbohydrate binding specificity. Positive glycohistochemical reaction developed within sarcomeres only at the level of A-bands. Granular staining was observed in the sarcoplasm among the myofibrils and also in the subsarcolemmal regions. Differences in expression of glycohistochemically detectable sugar receptors were noted between type 1, type 2A, and type 2B fibers. The molecular properties of one type of glycohistochemically detectable sugar receptor were inferred both immunohistochemically and biochemically. An antiserum against an endogenous beta-galactoside-specific lectin from muscle tissue localized this lectin within sections consistently similar to (neo)glycoproteins, detecting beta-galactoside-specific receptor(s). This similarity of binding patterns strongly supports the assumption that (neo)glycoproteins with beta-galactoside termini indeed bind to the respective endogenous lectin. The lectin-specific antiserum enabled us to ascertain that glycohistochemical fiber typing corresponds to enzyme histochemical typing. Moreover, biochemical purification using affinity chromatography and subsequent affinity elution revealed only the immunohistochemically detectable beta-galactoside-specific lectin. Consequently, use of a panel of neoglycoproteins, when frozen sections for histochemical analysis are not available, co

Acetylation↗

PubMed1979

Effect of fixation on the antigenicity of human lactoferrin in paraffin-embedded tissues and cytocentrifuged cell smears.

Immunoperoxidase techniques were used to study the preservation of the antigenicity of human lactoferrin (LF) of polymorphonuclear neutrophils (PMN) and various exocrine glandular cells in paraffin-embedded tissue blocks and cytocentrifuged cell smears. Tissues fixed in Carnoy's fluid in contrast to other fixatives used, showed good preservation of LF antigenicity irrespective of the fixation time. Cell smears fixed in Carnoy's fluid showed diffuse nuclear and cytoplasmic staining for LF, although morphologic integrity was poorly preserved. Granular cytoplasmic staining for LF with no staining of nuclei was seen in cell smears fixed in buffered formol acetone for 2--10 min. The nature of nuclear LF staining and future applications of the present methods are discussed.

Antigens↗

PubMed1989

Rapid diagnosis of Aujeszky's disease in pigs by improved in situ hybridization using biotinylated probes on paraffin-embedded tissue sections.

A practical in situ hybridization (ISH) method has been developed for the rapid diagnosis of pseudorabies virus (PRV) infection in pigs. The method utilizes routine formalin fixed and paraffin-embedded tissue sections, non radioactive biotin-labelling, simple hybridization procedure and diaminobenzidine (DAB) silver enhancement detection system. The rapid (ISH) method showed concordance with the direct filter hybridization (DFH) and with the peroxidase-antiperoxidase (PAP) assays. By the simplified ISH, diagnosis can be made within two days.

Animals↗

PubMed1977

Combined Nomarski interference contrast and immunofluorescent study of neuropathological specimens: CSF sediments and paraffin embedded brain tissues.

The direct immunofluorescent technique may be easily improved by the use of the Nomarski optics. This contrast allows accurate identification of fluorescent CSF cells and structures in formalin fixed paraffin embedded brain tissues; in the latter, the combined optical procedure is fruitfull in order to avoid fluorescent artifacts misinterpretation. Furthermore, it is emphazised that the conditions in which routine neuropathological specimens are removed and stored usualy does permit the application of the immunofluorescent technique.

Brain↗

PubMed1998

Pretreatment with restriction enzyme or bovine serum albumin for effective PCR amplification of Epstein-Barr virus DNA in DNA extracted from paraffin-embedded gastric carcinoma tissue.

An association between Epstein-Barr virus (EBV) and gastric carcinoma has been studied through the EBV genome present in the carcinoma cells. Recently, we found that EBV DNA in paraffin-embedded gastric carcinoma tissue was detected effectively by PCR after pretreatment of the extracted DNA with a restriction enzyme, BamHI or EcoRI. Here, we show that the PCR amplification was also enhanced by pretreatment of the DNA with other restriction enzymes or with bovine serum albumin and several other proteins. Treatment with these proteins may remove a PCR inhibitor(s) in the DNA samples extracted from the paraffin blocks.

Animals↗

PubMed1985

Use of freeze-dried paraffin-embedded sections for immunohistologic staining with monoclonal antibodies.

Most diagnostically valuable monoclonal antibodies recognize antigens that do not survive conventional tissue processing. The use of frozen tissue sections for immunohistologic studies overcomes this obstacle but introduces a number of practical problems, e.g., the necessity to store material in the frozen state, poor morphologic preservation, etc. In the present paper we report that antigenic denaturation during conventional tissue processing appears to occur during exposure to aldehyde-containing fixatives and to alcohol but not as a result of heating or exposure to melted paraffin wax. In consequence, we have developed a technique by which tissue is freeze-dried and then embedded directly in paraffin wax. All but one of the 40 monoclonal antibodies investigated stained the freeze-dried paraffin sections with an intensity equal to or greater than that observed on frozen sections. There was less diffusion artifact and less background staining than in cryostat sections, and cellular morphology was better preserved. One of the most important advantages of this new method is that antigens in freeze-dried paraffin-embedded tissue are stable, and tissue blocks may be handled in the same manner as conventional paraffin blocks. An additional finding was that, once the tissue has been freeze-dried, paraffin embedded, and sectioned, the antigens it contains are resistant to fixatives (e.g., formol, formol sublimate, alcohols) which would very rapidly cause their destruction in frozen sections.

Antibodies, Monoclonal↗

PubMed1987

Glycoconjugates in retinoblastoma. A lectin histochemical study of ten formalin-fixed and paraffin-embedded tumours.

The binding of eleven biotin- or peroxidase-coupled lectins with different carbohydrate specificities to tumour tissue and remaining morphologically normal retina was studied in ten formalin-fixed and paraffin-embedded human eyes with retinoblastoma. In undetached retinas, outer and inner segments of photoreceptors bound concanavalin A (ConA) as well as Lens culinaris (LCA), wheat germ (WGA) Ricinus communis (RCAI) and peanut (PNA) agglutinins. Both nuclear and plexiform layers bound ConA, LCA and, in some specimens, WGA and RCAI. These results agree with those obtained with normal adult human retina, the main difference being that PNA labelled some rods in addition to cones in the retinoblastoma eyes. Flexner-Wintersteiner rosettes reacted with ConA and LCA, and often with WGA, PNA and RCAI. Undifferentiated retino-blastoma cells always bound ConA and LCA, and in some tumours WGA, PNA and RCAI. Pretreatment with neuraminidase increased the number of cells that bound PNA and RCAI, but diminished binding of WGA. Pokeweed mitogen and Bandeiraea simplicifolia I, Dolichos biflorus, soybean, Ulex europaeus I and Lotus tetragonolobus agglutinins labelled only vascular endothelial cells. Retinoblastoma cells most closely resembled photoreceptor cells in their lectin-binding patterns.

Carbohydrates↗