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PubMed1983

Immunoreactivity of paraffin-embedded normal tissues and mesenchymal tumors for smooth muscle myosin.

Immunohistochemical study of smooth muscle myosin, a protein distinct from skeletal, cardiac, or nonmyogenous myosins in paraffin-embedded normal tissues and benign and malignant mesenchymal tumors revealed its strong expression in normal smooth muscle, capillary endothelium, and pericytes. All benign smooth muscle tumors with exception of gastric leiomyomas and few other leiomyomas of the gastrointestinal tract displayed strong or moderate immunoreactivity. On the other hand, strong or moderate immunoreactivity was detected in only eight of 28 spindle-cell leiomyosarcomas, as well as in 13 out of 27 malignant fibrous histiocytomas and three out of nine malignant hemangiopericytomas, while epithelioid leiomyosarcomas, fibrosarcomas, malignant schwannomas, and synovial sarcomas were negative or only weakly positive. Our results demonstrate that, while smooth muscle myosin is a very good marker of normal smooth muscle and benign smooth muscle tumors, it is expressed in diagnostically significant amounts in less than a third of spindle-cell leiomyosarcomas and none of the studied epithelioid leiomyosarcomas.

Female↗

PubMed1987

A study of free radicals in paraffin embedded and deparaffinized human heart muscle tissue using electron spin resonance (ESR).

Free radicals (spectroscopic splitting factor; g factor = 2.003-2.005) were investigated in formol-fixed, paraffin embedded heart-muscle tissue sections using electron spin resonance (ESR) spectra. Changes in signal amplitude, g factor and line width were registered during deparaffinization, chloroform-methanol extraction, vapour treatment and bromination. An attempt was made to identify the source of the ESR signals by a correlation between the signal amplitude and number of fluorescent and/or Sudan-black-positive granules counted in the tissue sections. An increase in signal amplitude, g value and line narrowing were characteristic of the ascorbyl radical after deparafinization in air. Vapour treatment revelated that the broader signal has lower g factor, a characteristic that is tentatively assigned to oxidized lipids. The bromination resistant minor fraction of free radical centres with small g factor might be associated with the pigment content of the samples.

Adult↗

PubMed1991

PCR amplification from paraffin-embedded tissues. Effects of fixative and fixation time.

The polymerase chain reaction (PCR) DNA amplification method is a powerful new tool for the retrospective analysis of paraffin-embedded tissue (PET). The technique has afforded the sensitive and specific detection of nucleic acid sequences associated with genetic and infectious diseases. However, PET processing conditions vary in their suitability for amplification. The authors have examined the effects of 11 fixatives at three fixation times. The effect of fixation was measured by the ability of the DNA in a treated tissue to serve as a template for the amplification of DNA fragments that ranged from 110 to 1,327 base pairs in length. Specimens fixed in acetone or 10% buffered neutral formalin were found to be best suited for subsequent analysis by PCR. A second group of fixatives, including Zamboni's, Clarke's, paraformaldehyde, formalin-alcohol-acetic acid, and methacarn, compromised amplification efficiency. Tissues treated with Carnoy's, Zenker's, or Bouin's, respectively, were even less desirable for amplification analysis.

Acetone↗

PubMed1986

Simultaneous nuclear antigen and DNA content quantitation using paraffin-embedded colonic tissue and multiparameter flow cytometry.

The simultaneous quantitation of nuclear antigens and DNA content is presented using monoclonal antibodies and flow cytometric analysis, with paraffin-embedded human colonic pathology specimens utilized as source material. The monoclonal antibodies evaluated were shown by immunogold electron microscopy to recognize nuclear proteins preferentially associated with interchromatin (p105) and heterochromatin (p34) regions. Indirect immunofluorescence analysis of p105 revealed two distinct G1-G0 cell subpopulations in cells from normal colonic epithelium and colonic adenocarcinomas. In addition, enhanced levels of both p105 and p34 were observed in aneuploid DNA content stemlines, relative to diploid cells. Cell-sorting experiments performed on cells sorted on the basis of p105 and DNA contents reveal the capability of this method for identifying morphologically heterogeneous cell subpopulations. Other data suggest that p105 is differentially expressed in well-differentiated versus poorly differentiated tumor regions. The potential utility of this approach for the retrospective study of proliferation-associated antigens and protooncogene protein products is discussed.

Adenocarcinoma↗

PubMed1985

A simple micromethod for collagen and total protein determination in formalin-fixed paraffin-embedded sections.

A simple, sensitive, and quantitative procedure is described for the measurement of collagen and protein content in tissue sections prepared from formalin-fixed paraffin-embedded samples. The method can detect as little as 5.7 micrograms of collagen per mg of protein. It is based on the selective binding of Sirius red F3BA and Fast green FCF to collagen and noncollagenous components, respectively, when the sections are stained with both dyes dissolved in aqueous saturated picric acid. Both dyes are eluted readily and simultaneously with NaOH-methanol and the absorbances obtained at 540 and 605 nm can be used to determine the amount of collagen and protein. The color equivalence of each dye was determined after destaining the sections and measuring the collagen content by hydroxyproline analysis and the amount of protein by the micro-Kjeldahl procedure. When several sections prepared from five rat tissues were analyzed first by the dye binding method and then by the chemical procedure, comparable results were obtained. This method could be of use in measuring collagen in tissue specimens and could be helpful in assessing the degree of fibrosis in tissue samples and in evaluating the effects of antifibrogenic drugs currently in use.

Animals↗

PubMed2004

The HOPE-technique permits Northern blot and microarray analyses in paraffin-embedded tissues.

There is an increasing demand for tissue samples that, after having been used for conventional histologic examination, are also suited for molecular analyses. As to formalin-fixed, paraffin embedded (FFPE) tissue, the latter applications are very limited. The HOPE (Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) technique comprises a new protection-solution with an organic buffer, with acetone as the only dehydrating agent, and pure paraffin of 52-54 degrees C melting temperature, allowing for all pathologic routine investigations. In contrast to FFPE tissue, the HOPE-technique allows for the application of molecular methods, such as high molecular DNA and RNA isolation, which can be used for PCR and reverse transcription PCR (RT-PCR). In this study, we investigated whether RNA from HOPE-fixed tissue samples is suitable for Northern blot and microarray analyses. RNAs of two HOPE-fixed breast cancer specimens of different histologic grade were used to carry out an array experiment. It turned out that RNA from HOPE-fixed tissue is of high quality and can be successfully used for array experiments. In addition, by detecting GAPDH and high mobility group protein gene B1 (HMGB1)-specific transcripts, we were able to demonstrate that RNA from HOPE-fixed tissue can also be used for Northern blot hybridization.

Blotting, Northern↗

PubMed1984

Immunohistochemical demonstration of plasma protein in squamous epithelium of formalin-fixed, paraffin-embedded oral mucosa.

The distribution of immunoglobulins and fibrinogen in normal and inflamed oral mucosa was examined by direct immunofluorescence using enzyme-treated sections of formalin-fixed, paraffin-embedded tissue. Plasma proteins were found inter- and intracellularly in the surface epithelium in most of the normal and all of the inflamed mucosal specimens. This diffusion of plasma proteins into the surface epithelium was shown to contribute to the appearance of edematous epithelial cells and eosinophilic bodies ("keratin pools"). The results clearly demonstrate that oral epithelium is not to be regarded as a covering membrane isolated from the underlying connective tissue. Furthermore, the study confirms that immunohistochemical studies can be performed on enzyme-pretreated sections of routinely fixed and embedded tissue although it is emphasized that the method is not of universal applicability.

Blood Proteins↗

PubMed1987

Fixation, processing, and immunochemical reagent effects on preservation of T-lymphocyte surface membrane antigens in paraffin-embedded tissue.

Fixatives, fixation additives, paraffin processing reagents, and immunochemical reagents were investigated for effects on preservation of T-lymphocyte surface membrane antigens CD3, CD4, and CD8 in human tonsil. Individual reagent effects were assessed in frozen sections by use of monoclonal antibodies and this information was used to optimize T-cell immunostaining in paraffin sections. Harmful factors were fixation delay, fixation at acid pH, fixation and processing at temperatures above 4 degrees C, hot paraffin wax, proteolytic enzymes, methanolic hydrogen peroxide, Triton X-100, and prolonged iodine treatment. Optimal T-cell demonstration in paraffin sections followed tissue fixation in periodate-lysine-paraformaldehyde dichromate at 4 degrees C, pH 7.5; processing through isopropanol, then xylene or chloroform, at 4 degrees C; and embedding in low melting point wax at 45-50 degrees C. Graded antigen stability occurred: CD3 most stable, CD8 least, and CD4 intermediate. CD4 and CD8 antigen preservation in paraffin sections required critical optimal tissue handling. CD3 was more stable and was also demonstrated in tissue fixed in commercial formalin, glutaraldehyde, and Bouin's fluid when fixation and processing conditions were optimized for pH and temperature. Of the fixation additives studied, polyethylene glycol and several potassium and magnesium salts enhanced immunostaining, whereas calcium chloride and lidocaine were deleterious.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1992

Relationship between histopathologic typing and morphonuclear assessments of 238 thyroid lesions. Digital cell image analysis performed on Feulgen-stained nuclei from formalin-fixed, paraffin-embedded materials.

A digital cell image processor was used to compute 15 parameters on Feulgen-stained thyroid nuclei from 238 archival, i.e., formalin-fixed, paraffin-embedded thyroid lesions. The morphonuclear parameters were related to morphometric (nuclear area), densitometric (nuclear DNA content), and textural (chromatin pattern characteristics) features. With respect to development of a malignant condition, their variations were compared with the World Health Organization's International Histological Classification of thyroid tumors. The relationship between morphonuclear parameter assessments and tumor size and the presence or absence of metastasis at the time of diagnosis was also investigated: no relationship with respect to cytomorphonuclear assessments was found. Nuclear area and nuclear DNA content discriminated between simple multinodular goiters and multinodular goiters with adenomatous hyperplasia. In the same way, the mean parameter values describing the chromatin pattern of multinodular goiters were significantly distinct from those describing the chromatin pattern of adenomas and carcinomas. Furthermore, chromatin pattern descriptions made it possible to discriminate between cell nuclei from papillary carcinomas and follicular carcinomas, and further between follicular carcinomas and follicular adenomas. A marked variation was observed within individual cases of multinodular goiters, adenomas, and carcinomas, a feature suggesting that morphonuclear assessment is of limited value for the diagnosis of individual clinical cases. In contrast, these results show that morphometric, densitometric, and textural nuclear assessments are useful aids for thyroid tumor typing, adding interesting results with respect to thyroid tumor progression. Indeed, the primary findings demonstrate that nodules with adenomatous hyperplasia from multinodular goiter lesions and microvesicular adenomas more closely resemble follicular carcinomas than do simple multinodular goiters and normomacrovesicular adenomas. Such data might reflect a biologic progression from benign to malignant follicular thyroid lesions.

Adenoma↗

PubMed1992

Distribution of cytokeratin polypeptides in syringomas. An immunohistochemical study on paraffin-embedded material.

The distribution of cytokeratin (CK) polypeptides expressed in syringomas (12 cases) was compared with that in normal eccrine sweat ducts using immunohistochemical techniques on paraffin-embedded tissue. Intradermal and intraepidermal segments of the eccrine duct showed reactivity with an antibody to CK1/5/10/11 in all cell layers, whereas CK19 expression was restricted to the luminal cell layer. CK14 was expressed in all cells of the eccrine duct except for the peripheral cells of the intraepidermal duct. Expression of CK5/6 was seen in the basal cells of the dermal duct and of the lower intraepidermal duct (sweat duct ridge) exclusively. Reactivity with an antibody to CK1 was found in the intermediate cells of the uppermost part of the eccrine dermal duct. In addition, this antibody gave a strong staining of the peripheral cells of the intraepidermal duct, leaving basal cells of the sweat duct ridge and luminal cells unstained. In syringoma, CK distribution was essentially comparable with that found in the uppermost part of the dermal duct and in the sweat duct ridge. Namely, ductal luminal cells expressed CK1/5/10/11, CK19, and variably CK14. Intermediate cells of ductal structures and solid nests were homogeneously stained by antibodies to CK1 and CK1/5/10/11, whereas CK14 was expressed heterogeneously. The basal or outermost layer of ductal structures and solid nests was reactive with antibodies to CK1/5/10/11, CK5/6, and CK14. With regard to CK expression, the results indicate that syringoma represents a tumor differentiating toward both the uppermost part of the dermal duct and the lower intraepidermal duct (sweat duct ridge) of the eccrine sweat gland.

Adenoma↗

PubMed1990

Indirect immunoperoxidase and immunofluorescence staining of Bordetella bronchiseptica in formalin-fixed, paraffin-embedded lung tissues of rabbits.

Indirect immunoperoxidase (IP) and immunofluorescence (IF) techniques were developed, and used for the identification of B. bronchiseptica in formalin-fixed, paraffin-embedded lung tissues of 26 rabbits displaying focal chronic interstitial pneumonia, vascular changes, and chronic bronchitis and bronchiolitis. Labelled B. bronchiseptica organisms were observed at the level of the bronchial and bronchiolar mucosa, both in areas affected by chronic bronchitis-bronchiolitis and in zones that were free of lesions. B. bronchiseptica was not observed in areas of focal chronic interstitial pneumonia, nor in vessels exhibiting inflammatory changes. B. bronchiseptica was observed also in a few rabbits that were culturally negative for this agent. The results obtained suggest that B. bronchiseptica could have been related to the lesions of chronic bronchitis and bronchiolitis observed, but not to the changes of focal chronic interstitial pneumonia and vasculitis, as areas affected by these alterations did not exhibit B. bronchiseptica immunoreactivity.

Animals↗

PubMed1991

Detection of Epstein-Barr virus DNA in formalin-fixed paraffin-embedded tissue of nasopharyngeal carcinoma using polymerase chain reaction and in situ hybridization.

The polymerase chain reaction method for amplification of DNA in formalin-fixed, paraffin-embedded tissue sections was used to detect Epstein-Barr virus DNA in nasopharyngeal carcinomas from Japanese patients. Thirty-one cases of nasopharyngeal carcinoma and 8 cases of lymph node metastasis of nasopharyngeal carcinoma were studied. Detection rates of Epstein-Barr virus in various types of nasopharyngeal carcinoma according to the World Health Organization classification were as follows: 10 of 10 undifferentiated carcinomas, 8 of 13 nonkeratinizing carcinomas, and 5 of 7 keratinizing carcinomas. Eight lymph node metastases, for which the primary was positive for Epstein-Barr virus, also contained Epstein-Barr virus DNA. By in situ hybridization using a biotinylated Epstein-Barr virus probe, it was clearly demonstrated that Epstein-Barr virus DNA was localized in the nuclei of the neoplastic cells. The clinical features of nasopharyngeal carcinoma with or without Epstein-Barr virus were not different. These results demonstrate that nasopharyngeal carcinoma in Japanese patients is closely associated with Epstein-Barr virus infection, similar to nasopharyngeal carcinoma of other endemic and nonendemic areas.

Carcinoma↗

PubMedDate not supplied

Modifications to the in situ TUNEL method for detection of apoptosis in paraffin-embedded tissue sections.

The in situ detection of cells undergoing apoptosis is increasingly important in the analysis of injury and degeneration in the central nervous system. Limited information is presently available on the quantification of apoptosis in paraffin-embedded brain tissue sections, a technique which would be most useful in the evaluation of archival tissue for diagnostic and experimental purposes. In this report, optimized conditions for tissue digestion and permeabilization using Proteinase K and Triton X and a quantification method for apoptosis detection are described using brain sections from aluminum maltolate-treated aged and young rabbits as compared to untreated matched controls. This method provides optimal staining of apoptotic cells without the problem of tissue destruction, and should prove useful in evaluating the process of apoptosis in neurodegenerative disorders.

Aging↗

PubMed1992

[Detection of clonal immunoglobulin rearrangements in paraffin embedded tissues by PCR].

A polymerase chain reaction based method was established using immunoglobulin VH and VK gene rearrangements as markers to detect clonal B cell populations in paraffin embedded sections. Family specific VH and VK primers are used in separate reactions together with the corresponding J primers to amplify rearranged VH and VK genes from genomic DNA. This allows to distinguish clonal from polyclonal B cell populations in most of the cases. The method may be helpful in routine diagnosis of B cell NHL and some morphological and immunohistochemical difficult cases.

Base Sequence↗

PubMed1983

Improvement of the butyl methacrylate-paraffin embedment.

The excellent butyl methacrylate-paraffin method as an embedment for light microscopy has been technically improved. More uniform and reproducible polymerization has been obtained by using a vacuum oven to degas the polymerizing mixture and to replace the air with nitrogen at 650 Torr. The amount of benzoyl peroxide required must be determined for each batch of butyl methacrylate. A teflon lined, reusable metal mold and a method of one-step blocking of tissues in preparation for sectioning are also described.

Fixatives↗

PubMed1994

[Detection of mycobacterial DNA from paraffin-embedded tissue in lung and lymphoid epithelial granulomas].

Standard techniques for the detection of mycobacteria in granulomatous diseases can be inadequate. We analysed 71 formalin fixed and paraffin-embedded tissue blocks from 68 non-immunocompromised patients with caseating, non-caseating, scarred, and miliary granulomas of lung and lymph nodes. A reamplification PCR protocol was established to detect a 123 bp product of the repetitive insertion sequence IS986/IS6110. After exclusion of 6 PCR-negative cases with clinical sarcoidosis 97% of lung tissue blocks with more than 10% caseating and non-caseating granulomas contained mycobacterial DNA. By routine microbiology mycobacteria could be detected in 78% of the patients. Scarred granulomas were PCR-negative. All miliary granulomas were PCR-positive. Lymph nodes showed comparable results. We think that this method facilitates aetiologic analysis of granulomatous diseases especially when the suspicous tissue is fixed and microbiology is not available.

DNA, Bacterial↗

PubMed1989

Interphase cytogenetics in paraffin embedded sections from human testicular germ cell tumor xenografts and in corresponding cultured cells.

A protocol was developed that allows determination of chromosome aberrations in interphase nuclei from paraffin embedded human tissues. As a model system tissue sections from xenografts derived from three testicular germ cell tumors (two teratocarcinoma and one embryonal carcinoma), as well as sections from normal intestine and testicular tissues, were hybridized with a biotin-labeled DNA probe specific for 1q12. For comparison, isolated nuclei in suspension and metaphase spreads from in vitro cell lines derived from the respective xenografts were assayed. Our results show that interphase tumor cells with aberrant numbers of chromosome 1 can be reliably detected both in vitro and in vivo. Subclones with two and three labeled chromosomes, respectively, could be defined and quantitatively evaluated. Present and future possibilities to pinpoint specific chromosome aberrations directly in cells present in body fluids or in tumor tissues are discussed.

Animals↗

PubMed1998

A quick method to extract DNA from paraffin-embedded tissues.

BACKGROUND: The amplification power of the polymerase chain reaction (PCR) technique has had great impact in molecular analysis, and DNA extraction is a common requirement in retrospective studies utilizing PCR as a tool. Conventional methods used in deparaffinization, harvesting and purification of DNA from paraffin-embedded tissue are time consuming and cause a significant loss in the yield of DNA. METHODS: We utilized heating-melting-cooling removal of paraffin, digestion of the sample with proteinase K and purification of the extracted DNA by a microconcentrator. The products, after PCR amplification of the p53 gene exon 8, were used to make a comparison between our method and the conventional xylene-phenol-choloform method. RESULTS: The amplified products from our method were superior to that of the conventional method. CONCLUSION: The method we propose has a better recovery of DNA and is more time efficient.

DNA↗