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Results for “Paraffin Embedding”

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PubMed1993

Molecular diagnosis of trisomy 18 using DNA recovered from paraffin embedded tissues and possible implications for genetic counselling.

Severe malformations pointing to trisomy 18 were observed in two fetuses. No chromosome analysis had been performed. To confirm the diagnosis of trisomy 18, DNA was isolated from formalin fixed and paraffin embedded tissues. Molecular analysis by typing chromosome 18-specific (CA)n-repeats unequivocally showed the presence of trisomy 18 in both cases.

Chromosomes, Human, Pair 18↗

PubMed1981

Toxoplasma gondii infection of the central nervous system. Use of the peroxidase-antiperoxidase method to demonstrate toxoplasma in formalin fixed, paraffin embedded tissue sections.

The peroxidase-antiperoxidase immunohistochemical technique was employed to stain formalin fixed, paraffin embedded tissue sections from three cases of encephalitis caused by Toxoplasma gondii. We studied two cases of congenital infection and one case of acquired toxoplasmosis occurring in an immunocompromised host. The peroxidase-antiperoxidase method was exquisitely sensitive and highly specific and stained both the encysted and tachyzoite forms of the organism, as well as allowing for easy identification of infected cells. In two cases of necrotizing encephalitis--one congenital, the other acquired--widespread dissemination of the Toxoplasma organism throughout the neural parenchyma was visualized using the peroxidase-antiperoxidase stain. Brain biopsy material that had been obtained eight days prior to death in the case of adult acquired toxoplasmosis did not contain any of the characteristic tissue cysts and was not diagnostic for toxoplasmosis by conventional staining techniques. However, peroxidase-antiperoxidase staining of tissue sections from this biopsy unequivocally demonstrated both free tachyzoites and multiple infected cells. Further application of the peroxidase-antiperoxidase method should increase our understanding of the pathology and pathogenesis of toxoplasmic encephalitis as well as allowing timely diagnosis in cases presenting with neurologic symptomatology.

Animals↗

PubMed1982

Demonstration of monoclonal lymphoplasmacytic proliferations by immunofluorescence on routine formalin-fixed, paraffin-embedded tissue.

This study explores the use of immunofluorescence on routine formalin-fixed paraffin-embedded tissue to distinguish monoclonal from polyclonal lymphoplasmacytic proliferations. Sixteen tissues containing plasma cell or lymphocyte and plasma cell proliferations were studied. Five-micron sections were deparaffinized in xylene, rehydrated, and treated with 0.1% trypsin for two hours. After washing with phosphate-buffered saline, separate sections were stained with fluorescein-conjugated antibody to IgG, IgA, IgM, IgD, and IgE, and a single section was stained with both fluorescein-conjugated anti-kappa and rhodamine-conjugated antilambda. The latter section was useful to distinguish nonspecific adsorption of the fluorochromes. Where possible, results were correlated with immunoelectrophoretic studies of serum and urine. Eleven specimens with monoclonal and four specimens with polyclonal lymphoplasmacytic proliferations were readily identified, including a case of giant lymph node hyperplasia with a monoclonal IgDK plasma cell component (confirmed by specific absorption studies). Identification of monoclonality by immunofluorescence preceded immunoelectrophoretic identification in one case. One other case gave equivocal results by fluorescence. Further, the method worked on formalin-fixed decalcified tissues, although a somewhat heavier background staining was noted. This method offers a simple, reliable technique to establish or confirm the diagnosis of monoclonal lymphoplasmacytic lesions.

Adult↗

PubMed1990

Detection of hepatitis B virus DNA in formalin-fixed, paraffin-embedded liver tissue by the polymerase chain reaction.

DNA isolated from formalin-fixed, paraffin-embedded liver tissues from nine patients with hepatocellular carcinoma and six control patients was screened for hepatitis B virus (HBV) DNA with surface (S) and core (C) gene-specific primers by a modification of the polymerase chain reaction--southern blot technique (PCR-SB). PCR-SB results were correlated with histologic, immunohistochemical, and serologic findings. All cases with an established HBV etiology were positive by PCR-SB, as were three cases with negative immunohistochemistry and serology. Often there was selective amplification with one primer set and, in two cases, smaller than expected HBV amplification products suggesting internal deletions. The presence of a potent PCR inhibitor in nucleic acid preparations from tissue blocks that can be removed by Sephadex G-50 chromatography was confirmed. PCR-SB will be a powerful method for the diagnosis and follow-up of patients with HBV infection and may provide new insights into viral hepatocarcinogenesis.

Base Sequence↗

PubMedDate not supplied

Effect of microwave oven heating times on androgen receptor antigen retrieval from paraffin-embedded prostatic adenocarcinoma.

The aim of this study was to investigate the effect of microwave oven heating for antigen retrieval on the immunoreactivity of human prostate carcinoma androgen receptor (AR) in tissue sections. Formalin-fixed, paraffin-embedded tissue sections were microwaved at 5-min intervals for a total of 15, 20, 25, 30, 35 minutes at maximum power (700W). The monoclonal antibody F39.4.1 directed against human AR was used at a 1:10 dilution. Without microwave oven heating, prostatic tissue did not exhibit any AR immunoreactivity. Moderate positivity appeared after three 5-minute cycles of microwave heating. The intensity of immunoreactivity improved progressively with heating times of 20 and 25 min up to an optimum time of 30 minutes, when nuclear staining was most intense with the absence of background staining and without loss of morphological details. While antigen retrieval is effective in restoring antigenicity in a variety of setting, the length of time prostate tissue is exposed to microwave radiation is critical in order to obtain optimal AR immunostaining. AR immunostaining reliably permitted evaluation of the distribution and intensity of positively stained nuclei and the distinction of the various cell types in archival material.

Adenocarcinoma↗

PubMed2006

Protein extraction from formalin-fixed, paraffin-embedded tissue sections: quality evaluation by mass spectrometry.

A satisfactory protocol of protein extraction has been established based on the heat-induced antigen retrieval (AR) technique widely applied in immunohistochemistry for archival formalin-fixed, paraffin-embedded (FFPE) tissue sections. Based on AR, an initial serial experiment to identify an optimal protocol of heat-induced protein extraction was carried out using FFPE mouse tissues. The optimal protocol for extraction of proteins was then performed on an archival FFPE tissue of human renal carcinoma. FFPE sections were boiled in a retrieval solution of Tris-HCl containing 2% SDS, followed by incubation. Fresh tissue taken from the same case of renal carcinoma was processed for extraction of proteins by a conventional method using radioimmunoprecipitation assay solution, to compare the efficiency of protein extraction from FFPE tissue sections with extraction from fresh tissue. As a control, further sections of the same FFPE sample were processed by the same procedure without heating treatment. Evaluation of the quality of protein extracted from FFPE tissue was done using gel electrophoresis and mass spectrometry, showing most identified proteins extracted from FFPE tissue sections were overlapped with those extracted from fresh tissue.

Animals↗

PubMed1998

Tyrosinase immunoreactivity in formalin-fixed, paraffin-embedded primary and metastatic melanoma: frequency and distribution.

Monoclonal antibody T311 specifically detects tyrosinase protein expression. Tyrosinase-derived peptides are recognized by CD8+ T-cells and applied in immunotherapy. We examined formalin-fixed paraffin-embedded tissue of 50 melanoma (primary n=31, metastatic n=19) and 41 control cases (junctional, dermal, compound, Spitz, Reed, balloon-cell nevi) by immunochemistry using the alkaline phosphatase-anti-alkaline phosphatase method after antigen retrieval. Staining with mAb T311 showed a sensitivity of 94% for melanoma with a very high specificity for melanocytic cells. Immunopositivity (94% of melanomas overall) correlated inversely with clinical stage: clinical stage I and stage II showed 100%, stage III and stage IV 86% immunoreactivity each. Staining changed from an exclusively homogeneous pattern in early stages to a more heterogeneous pattern in later stages. Melanocytic control tissue like nevi of different subtypes all showed weak to moderate, homogeneous immunoreactivity with polarity towards the epidermis. RT-PCR ELISA analysis of short-term melanoma cell cultures displayed mRNA expression in only half of the originally immunopositive tumors only, suggesting rapid mRNA expression loss in culture. mAb T311 allows detection of melanoma-associated tyrosinase protein expression and thus profiling of melanomas using routine archival tissue suited for immunotherapy approaches involving tyrosinase derived epitopes.

Antibodies, Monoclonal↗

PubMed1997

Quantitative analysis of mitochondrial DNA deletion in paraffin embedded muscle tissues from patients with KSS and CPEO.

The percentage of common deletion of mitochondrial DNA (mtDNA) was determined quantitatively by a PCR-based, non-radioactive method in DNA extracted from formalin-fixed, paraffin-embedded skeletal muscle tissues from two patients with Kearns Sayre syndrome (KSS) and one with chronic progressive external ophthalmoplegia (CPEO). The method involved PCR cycle titration of wild-type and deleted mtDNA in parallel, staining of gel bands with the sensitive fluorescence dye SYBR Green I, and quantitation of intensity on a computer screen by the NIH image program. We determined 75% and 71% common deletion of mtDNA in the KSS patients and 35% in the CPEO patient.

Adult↗

PubMed1997

Trivariate flow cytometric analysis of paraffin-embedded lung cancer specimens: application of cytokeratin subtype specific antibodies to distinguish between differentiation pathways.

The aim of the present study was to investigate whether trivariate FCM analysis, for the simultaneous detection of two different CK subtypes in combination with DNA content, can be applied to paraffin embedded samples of different types of non-small cell lung cancer in order to evaluate the cell cycle of individual sublines. Single cell suspensions were prepared from 50 microm thick paraffin sections of 22 lung carcinomas by pepsin digestion and immunostained with CK-antibodies which were chosen to distinguish glandular differentiation (adenocarcinomas) and squamous differentiation. There was a good correlation between the immunocytochemical results of the different CK antibodies in tissue sections and in the corresponding single cell suspensions. Gating for CK-positivity revealed a higher S-phase fraction as compared to the ungated cell population. The tumor cells in adenocarcinoma cases were specifically recognized by CK7 antibodies, while well-differentiated squamous cell carcinomas were specifically stained for CK14 and/or CK17. In poorly differentiated squamous cell carcinomas simultaneous expression of CK7 and CK17 was detected in a subpopulation of the tumor cells, next to cells positive for CK7 or CK17 alone. The trivariate FCM analysis allowed the separate estimation of ploidy status and cell cycle parameters in the three different cell populations of these, apparently (phenotypically) heterogeneous, malignancies.

Adenocarcinoma↗

PubMed1996

Immunohistochemical detection of P-glycoprotein in formalin-fixed and paraffin-embedded normal and neoplastic canine tissues.

Expression of P-glycoprotein, a phylogenetically conserved integral plasma membrane protein, is implicated as one of the most important factors contributing to tumor cell multidrug resistance. Formalin-fixed, paraffin-embedded normal and neoplastic canine tissues were studied using an avidin-biotin complex technique employing three murine monoclonal antibodies (C494, C219, JSB-1) to different epitopes of the P-glycoprotein molecule. Evaluation of immunostaining of normal canine tissues revealed positive labeling detected by each antibody in the liver, proximal renal tubular epithelium, adrenal cortex, colonic epithelium, and capillary endothelial cells of the brain. A total of 166 tumors of epithelial or mesenchymal origin were evaluated for P-glycoprotein immunoreactivity. Hepatomas (4/4), colorectal adenomas (7/7), colorectal carcinomas (4/4), adrenal cortical adenomas (3/3), hemangiopericytomas (15/15), apocrine gland adenocarcinomas (4/5, 80%), and transitional cell carcinomas (2/2) consistently labeled with at least one of the antibodies. Histiocytomas (0/10), cutaneous plasma cell tumors (0/10), fibromas (0/3), fibrosarcomas (0/4), and leiomyomas (0/4) were uniformly negative with all antibodies. Malignant lymphomas (6/22, 27.3%), malignant melanomas (4/13, 30.8%), leiomyosarcomas (3/6, 50%), mammary gland carcinomas (12/19, 63.2%), mammary gland adenomas (3/9, 33.3%), squamous cell carcinomas (8/10, 80%), basal cell tumors (5/7, 71.4%), apocrine gland adenomas (1/2, 50%), cholangiocarcinomas (2/3, 66.7%), and thyroid gland carcinomas (2/4, 50%) gave variable results. The antibodies C494, JSB-1, and C219 labeled 66/166 (39.8%), 53/166 (31.9%), and 38/166 (22.9%) of all tumors studied, respectively. A total of 26/166 (15.7%), 22/166 (13.3%), and 37/166 (22.6%) of tumors were labeled by all three, just two, or one antibody alone, respectively. The antibody C494 was the only antibody labeling 28/166 (16.9%) of the cases. JSB-1 alone labeled 9/166 (5.4%) of the tumors. C219 failed to label any tumors not also labeled by either C494 or JSB-1. Labeling by C494 was more intense and specific than labeling by the other two antibodies. Results indicate that P-glycoprotein can be detected in routinely processed canine tissues. The detection of P-glycoprotein within canine liver, kidney, adrenal gland, and colon and within tumors arising from these tissues is consistent with that reported in the literature for human tissues. Variable labeling results of other tumors such as malignant lymphoma and mammary gland carcinomas also is consistent with reports of human studies. Detection of multidrug resistance markers such as P-glycoprotein in canine tissues may provide additional information upon which to base a prognosis or to design treatment regimens for canine tumors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2005

Discriminating nevus and melanoma on paraffin-embedded skin biopsies using FTIR microspectroscopy.

FTIR microspectroscopy, in combination with cluster analysis, has been used to characterise skin tissues, in order to discriminate cancerous from non-cancerous ones. The main objective of this in vitro study was to demonstrate the applicability of infrared spectral imaging to separate, on paraffinised biopsies, pigmented nevi (benign skin lesions) from melanomas (malignant skin lesions). Infrared spectra were collected from paraffin-embedded samples of nevi and melanomas, without deparaffinisation. Despite the important contribution of the paraffin in these spectra, it was possible to find meaningful and discriminating spectral regions. Spectral imaging was first performed to localize different skin layers (dermis and epidermis). Spectra extracted from the images were subjected to hierarchical classification algorithm, which allowed the discrimination of melanomas from the nevi, using selected spectral windows that correspond to vibrations of DNA and melanin content. The diversity of skin lesions and direct accessibility to the skin make this organ an interesting field of investigation using this technique.

Biopsy↗

PubMed1994

PCR-RFLP studies on chromosome 3p in formaldehyde-fixed, paraffin-embedded cervical cancer tissues.

Loss of heterozygosity (LOH) has been extensively studied on the short arm of chromosome 3, and functional proofs have been obtained defining a tumor-suppressor locus at 3p21-22. We examined 31 paraffin-embedded cervical cancer samples for LOH, using 5 PCR-primer pairs, located around 3p21. Allele loss was found in 19 out of the 27 informative samples (70%) while 13 out of 23 informative samples (56%) had LOH located at 3p21-22. More of the human papillomavirus (HPV)-positive samples had LOH compared to the HPV-negative samples, giving only a weak association between loss of allele and HPV integration. Modifications of the DNA in the formaldehyde-fixed samples were detected, and further studies will be required to clarify how such artifacts may affect restriction fragment length polymorphism (RFLP) studies on fixed tissues.

Alleles↗

PubMed1995

Jacalin, another marker for histiocytes in paraffin-embedded tissues.

Jacalin is a lectin which reacts with D-galactose. We have tested jacalin on 75 samples of different formalin- and alcohol-fixed tissues. A consistent cytoplasmic stain of the histiocytes was observed in paraffin-embedded tissues in all cases studied of reactive sinus histiocytosis, macrophages in clear centres of follicular hyperplasia, in tuberculosis granulomas and in osteoclast-like giant cells in a breast carcinoma. We failed to find any clear binding of jacalin to the cells of eosinophilic granulomas, giant cell tumors of tendon sheath, pleomorphic malignant fibrous histiocytomas, Hodgkin's disease, melanomas, nevi or signet ring cell carcinomas of the breast and stomach. It seems that jacalin is a good marker for free histiocytes/macrophages, not for fixed histiocytes and tumors related to them. This lectin might play a role in differential diagnosis with histiocyte mimicking processes.

Breast Neoplasms↗

PubMed1999

Distribution of sialic acid-dependent carbohydrate epitope in thyroid tumors: immunoreactivity of FB21 in paraffin-embedded tissue sections.

The reactivity of monoclonal antibody FB21, which recognizes a sialic acid-dependent carbohydrate epitope, was tested with 94 non-neoplastic and neoplastic thyroid tissue specimens using immunohistochemical methods on formalin-fixed, paraffin-embedded tissue sections. These specimens included 11 cases of adenomatous goiter, three cases of Basedow's disease, 30 cases of follicular adenoma, 20 cases of papillary carcinoma, 15 cases of follicular carcinoma, six cases of medullary carcinoma, and nine cases of anaplastic carcinoma. FB21 reacted with 14 of 15 cases of follicular carcinoma that showed a microfollicular or trabecular pattern, and with nine of 20 cases of papillary carcinoma. A positive reaction was found on the cell surface membranes or apical parts of neoplastic follicles. FB21 also reacted with five of 30 cases of follicular adenoma. These cases showed a follicular pattern and positive staining pattern similar to that in follicular carcinoma. Adenomatous goiters, Basedow's disease, medullary carcinomas, and anaplastic carcinomas were negative for FB21 reactivity. Although the different reactivities of FB21 with papillary carcinoma and follicular carcinoma remain to be investigated, the high frequency of reactivity with FB21 suggests that it may be useful as a complement to morphological diagnosis in follicular carcinoma.

Adenoma↗

PubMed1997

[Flow cytometric quantification of numerical chromosome aberrations in non-small cell lung carcinomas using formalin-fixed paraffin-embedded tissue].

Fluorescence in situ hybridization with biotinylated repetitive DNA probe specific for the centromeric region of chromosome 17 (p17H8: Oncor) was applied to suspended nuclei which were isolated by Shutte's method from formalin-fixed paraffin-embedded tissue. The tissues were obtained from surgically resected specimens from nine patients with non-small cell lung carcinoma. The isolated nuclei were prepared with 0.05% pepsin/0.1NHCl for 15 minutes at 37 degrees C. Subsequently, these were immersed in 70% acetic acid for 10 seconds at room temperature. After heat denature with hybridization mixture which contained 3 mu 1 DNA probe for 10 minutesat 70 degrees C, 1 x 10(6) nuclei were incubated overnight at 37 degrees C. After washing with 60% formamide/2 x SSC, the hybridized probes were labeled by FITC conjugated avidin. A number of centromeric signals of chromosome 17 wasevaluated by fluorescence microscopy (BH-2, Olympus). Furthermore, a probe-related FITC intensity was quantified using flow cytometry (FACScan, Becton Dickinson). As the results, there was good correlation between a relative fluorescence intensity determined by flow cytometry and a relative fluorescence signal by fluorescence microscopy (p < 0.05).

Aged↗

PubMed1995

Use of light scatter when recording a DNA histogram from paraffin-embedded tissue.

The quality of a DNA histogram as recorded in a flow cytometer can often be improved by gating on forward and orthogonal light scatter. This may be helpful when measuring DNA histograms from formalin-fixed, paraffin-embedded material. We report that nuclei from many human tumours scatter more light orthogonally than those from normal stroma. Gating on light scatter enabled the DNA histogram from the tumour to be recorded with reduced contamination from normal cells. In studies of human mammary carcinomas, we have found that this method has the potential to improve the estimation of S phase and the measurement of DNA index (DI) when the DI is close to 1 (near diploid).

Breast Neoplasms↗

PubMed1997

MAGE-3 immunoreactivity in formalin-fixed, paraffin-embedded primary and metastatic melanoma: frequency and distribution.

Monoclonal antibody 57B specifically detects MAGE-3 gene protein expression. MAGE-derived peptides are recognized by CD8+ T cells and applied in immunotherapy. We examined formalin-fixed, paraffin-embedded tissue of 61 melanoma (primary, n = 40; metastatic, n = 21) and 46 control cases (junctional, dermal, compound, Spitz, Reed, and balloon-cell nevi) by immunohistochemistry using the alkaline phosphatase anti-alkaline phosphatase method after antigen retrieval. Immunoreactivity was rated positive at 20 positive cells per tumor or more. Staining pattern was homogeneous, scattered, or focal. All control samples and internal controls were immunonegative. Staining with monoclonal antibody 57B showed a specificity of 100% with a sensitivity of 44%. Immunopositivity (overall, 44% of melanomas) increased along with tumor, node, and metastasis stage; pT1 showed 13%, pT2 22%, pT3a 29%, pT3b 45%, pT4 100%, pTxN1 60%, and pTxNxM1a 63% of samples positive. The staining pattern was homogeneous on pT1 to pT3a tumors, homogeneous or focal in pT3b and pT4a, and homogeneous, focal, or scattered in pTxN1 and pTxNxM1a. The frequency of immunopositivity relates well to data on mRNA expression using reverse transcriptase polymerase chain reaction in a subgroup analyzed by both methods. Monoclonal antibody 57B can be used to allow profiling of melanomas using routine archival tissue, when considering immunotherapeutic approaches involving MAGE-3-derived epitopes.

Animals↗

PubMed1998

Detection of Jembrana disease virus in spleen, lymph nodes, bone marrow and other tissues by in situ hybridization of paraffin-embedded sections.

Jembrana disease virus (JDV) is a lentivirus that causes an acute, severe disease syndrome in infected Bali cattle in Indonesia. An in situ hybridization technique was developed that detected JDV genomic RNA in formalin-fixed paraffin-embedded tissue sections, using a digoxigenin-labelled riboprobe. Large numbers of JDV-infected cells were demonstrated in many tissue sections from experimentally infected animals early in the disease course, which was consistent with the extremely high circulating viraemia previously reported to occur during the febrile phase. The number of infected cells was consistently highest in sections of spleen, followed by many other tissues including lymph nodes, lungs, bone marrow, liver and kidney. Infected cells were also identified in the general circulation and within unusual intravascular lesions in lung sections. The relatively high level of infection found in bone marrow suggested that its involvement may be important in the disease pathogenesis, as it is with other lentiviruses.

Animals↗