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At least 721 records · Page 40Linked to original sources
PubMed2000

Evaluation of formalin-fixed paraffin-embedded tissues obtained from vaccine site-associated sarcomas of cats for DNA of feline immunodeficiency virus.

OBJECTIVE: To evaluate the use of a polymerase chain reaction (PCR) method for detection of feline immunodeficiency virus (FIV) DNA, using formalin-fixed paraffin-embedded (FFPE) tissues, and to use this method to evaluate tissues obtained from vaccine site-associated sarcomas (VSS) of cats for FIV DNA. SAMPLE POPULATION: 50 FFPE tissue blocks from VSS of cats and 50 FFPE tissue blocks from cutaneous non-vaccine site-associated fibrosarcomas (non-VSS) of cats. PROCEDURE: DNA was extracted from FFPE sections of each tumor and regions of the gag gene of FIV were amplified by a PCR, using 3 sets of primers. Sensitivity of the method was compared between frozen and FFPE tissues, using splenic tissue obtained from a cat that had been experimentally infected with FIV. RESULTS: We did not detect FIV DNA in VSS or non-VSS tissues. Sensitivity of the PCR method was identical for frozen or FFPE tissues. CONCLUSIONS AND CLINICAL RELEVANCE: It is possible to detect FIV DNA in FFPE tissues by use of a PCR. We did not find evidence to support direct FIV involvement in the pathogenesis of VSS in cats.

Animals↗
PubMed2026

Spatial Total RNA Sequencing of Formalin-Fixed Paraffin-Embedded Tissue by spRandom-seq.

The molecular pathogenesis of infectious diseases and cancer is orchestrated by nanoscale of host and microbial RNA transcripts within the tissue microenvironment. Nevertheless, spatially resolving the comprehensive transcriptional landscape within complex clinical tissues, like formalin-fixed paraffin-embedded (FFPE) specimens, still poses a formidable challenge. Here, we present spRandom-seq, a random primer-based spatial total RNA sequencing technology designed to spatially resolve complete transcriptomes from host, bacteria, and even nanoscale viruses in FFPE tissues. Capitalizing on the random primer design, our technology not only facilitated the discovery of specific lncRNAs and alternative splicing events in mouse brain and olfactory bulb, but also delineated pronounced spatial heterogeneity in clinical FFPE sections-across distinct tumor regions in breast cancer and microbial infection sites in Klebsiella pneumoniae-infected tissues. Importantly, integrated analysis of host and viral RNAs in FFPE samples from hepatitis B virus (HBV)‑positive hepatocellular carcinoma (HCC) demonstrated that complement and coagulation pathways were specifically activated across expansive HBV‑infected tumor areas, which also exhibited an increased burden of copy number variations (CNVs). Owing to its compatibility with existing spatial transcriptomics platforms and minimal operational complexity, spRandom-seq represents a practical and scalable approach for clinical pathology applications and infection diagnostics.

Paraffin Embedding↗
PubMed2002

Evaluation of formalin-fixed paraffin-embedded tissues from feline vaccine site-associated sarcomas for feline foamy virus DNA.

OBJECTIVE: To evaluate a group of vaccine site-associated sarcomas (VSS) for the presence of feline foamy virus (FeFV) DNA, using polymerase chain reaction (PCR) methods. SAMPLE POPULATION: 50 formalin-fixed paraffin-embedded (FFPE) tissue blocks from VSS of cats. PROCEDURE: DNA was extracted from FFPE sections of each tumor, and regions of the gag and pol genes of FeFV were amplified by use of PCR methods, using 1 primer set for each region. Sensitivity of the method was compared between fresh and FFPE cells, using mouse kidney tissue that was injected with FeFV-infected cultured cells and using agarose-cell pellets. Results-Feline foamy virus DNA was not detected in VSS tissues. Sensitivity of the method was 10 times greater in fresh versus FFPE mouse tissues. Sensitivity of the method in fresh FeFV-infected cultured cells versus FFPE agarose-cell pellets was equal when fixation was 24 or 48 hours and 10 times greater when fixation was 72 hours or 1 week. CONCLUSIONS AND CLINICAL RELEVANCE: A PCR-based method can be successfully applied to FFPE tissues for FeFV DNA detection. Results suggest there is no direct FeFV involvement in the pathogenesis of VSS in cats.

Animals↗
PubMed1993

An improved strategy and a useful housekeeping gene for RNA analysis from formalin-fixed, paraffin-embedded tissues by PCR.

Specific amplification of nucleic acid sequences by PCR has been extensively used for the detection of gene rearrangements and gene expression. Although successful amplification of DNA sequences has been carried out with DNA prepared from formalin-fixed, paraffin-embedded (FFPE) tissues, there are only a few reports regarding RNA analysis in this kind of material. We describe a procedure for RNA extraction from different types of FFPE tissues, involving digestion with proteinase K followed by guanidinium-thiocyanate acid phenol extraction and DNase I digestion. These RNA preparations are suitable for PCR analysis of mRNA and even of intronless genes. Furthermore, the universally expressed porphobilinogen deaminase mRNA proved to be useful as a positive control because of the lack of pseudogenes.

Base Sequence↗
PubMed2001

Detection of novel gene expression in paraffin-embedded tissues by isotopic in situ hybridization in tissue microarrays.

Correlating altered gene expression patterns with particular disease states is a critical step in understanding disease processes and developing treatment strategies. Many thousands of novel gene sequences have recently been annotated in public and private databases and are now available for analysis. Tissue-specific expression patterns of these sequences can be evaluated physically on DNA arrays and other high throughput assays, or virtually by bioinformatics mining of expressed sequence tag (EST) databases. As a secondary screening tool, in situ hybridisation (ISH) not only confirms tissue specificity, but also reveals what is often valuable information about cell-type expression patterns of nov16l sequences. Due to their availability and long-term stability at room temperature, formalin-fixed paraffin-embedded clinical specimens provide an invaluable resource for evaluating expression patterns of novel human genes. We describe a high-throughput approach for identifying and quantifying the expression of novel genes in paraffin-embedded human tissues using isotopic in situ hybridisation and tissue microarrays (TMA).

Blotting, Northern↗
PubMed2001

In situ hybridization and reverse transcription-polymerase chain reaction for cyclin D1 mRNA in the diagnosis of mantle cell lymphoma in paraffin-embedded tissues.

Mantle cell lymphoma (MCL) is characterized by the chromosomal translocation t(11;14), which involves rearrangement of the bcl-1 proto-oncogene to the immunoglobulin heavy chain gene and results in overexpression of cyclin D1 mRNA. In this study, we evaluated the diagnostic relevance of three methods that may be helpful in the diagnosis of MCL: in situ hybridization (ISH) and a stringent reverse transcriptase-polymerase chain reaction (RT-PCR) protocol for cyclin D1 mRNA, and immunohistochemistry for cyclin D1 protein. The study group included 37 paraffin-embedded specimens (25 from lymph nodes and 12 from extranodal tissues) from 30 patients. MCL diagnosis was performed according to the Revised European-American Classification of Lymphoid Neoplasms. Twenty-nine patients with non-MCL lymphoproliferative disorders comprised the control group. Biotin-labeled ISH was performed in 28 cases of MCL, 24 (86%) of which were found to be positive. As shown by ISH in extranodal tissues, cyclin D1 mRNA was present not only in neoplastic lymphoid cells, but in other cell types as well. For this reason, RT-PCR results were considered reliable for MCL diagnosis only on informative material (from tissues that do not normally express cyclin D1); this method was evaluated as positive in 16 of 18 (89%) MCL cases. Cyclin D1 immunopositivity was present in 20 of 29 (69%) MCL cases. No members of the control group were found to express cyclin D1 mRNA by either ISH or RT-PCR under the stringent conditions used. In conclusion, stringent RT-PCR for cyclin D1 expression can be helpful in MCL diagnosis in paraffin-embedded material from lymph nodes. ISH is a sensitive method for cyclin D1 mRNA detection; its sensitivity is superior to that of cyclin D1 immunohistochemistry and similar to that of the stringent RT-PCR used. ISH is very specific as well, clearly more specific than RT-PCR, because it allows the correlation of molecular findings with morphology. This method can be applied on all types of paraffin-embedded tissues and provides an accurate tool for MCL diagnosis.

Cyclin D1↗
PubMed1989

Enzyme histochemical demonstration of alkaline phosphatase activity in plastic-embedded tissues using a Gomori-based cerium-DAB technique.

We describe a new method for light microscopic demonstration of alkaline phosphatase (ALP) activity in plastic-embedded sections. Rat tissues were fixed in acetone (-20 degrees C), infiltrated in glycol methacrylate (GMA), and embedded at 0 degrees C. Sections were cut at 1 and 2 microns, dried at room temperature, and incubated in the conventional Gomori medium. Cerium chloride was used to convert calcium phosphate into cerium phosphate, which was subsequently converted into cerium perhydroxide. The slight yellow precipitate of cerium perhydroxide was amplified using 3,3'-diaminobenzidine tetrahydrochloride (DAB). For comparison, tissue sections were processed according to the calcium-cobalt method. The method described combines exact localization of ALP activity with optimal preservation of tissue morphology.

3,3'-Diaminobenzidine↗
PubMed1985

An inexpensive device for freeze drying and plastic embedding tissues at low temperatures.

The preparation of biological tissues for electron microscopy by rapid freezing retains the original localization of ions and molecules. A reproducible freezing regime was established by quenching tissues in liquid propane according to the method of Elder et al. (1981). Tissue was thereafter freeze dried in a custom built freeze drying device with a liquid nitrogen cooled stage to prevent ice recrystallization during drying. The device was also designed to allow the vacuum embedding of tissue in low temperature resin such as Lowicryl and polymerization in situ. This paper describes the design of the device and an example of its use in the freeze drying of cartilage. The results show that minimal ice damage occurs to the chondrocytes and that intracellular organelles are clearly visible. The regime described may prove a useful and pragmatic alternative to cutting tissue in the frozen state. Translocation of elements is unlikely except perhaps in the case of very labile elements such as Na and K, but this remains to be fully elucidated.

Animals↗
PubMed2001

Automated immunohistochemical staining of formalin-fixed and paraffin-embedded tissues using a catalyzed signal amplification method.

An immunohistochemical assay using catalyzed signal amplification (CSA), which is based on the peroxidase catalyzed deposition of biotinylated tyramide, is a highly sensitive method to visualize weak immunohistochemical signals originating from rare antigens or masked antigens in formalin-fixed, paraffin-embedded (FFPE) tissues. However, CSA methods are hampered by poor reproducibility and the complexity of their staining procedures. In this study, we aimed to apply the CSA procedure to a capillary gap-based, automated immunostainer, TechMate Horizon, to perform immunohistochemical signal amplification effectively and reproducibly. A variety of cellular antigens previously considered to be undetectable in FFPE human specimens were selected and examined with the automated immunostainer. Compared with the manual CSA staining method that takes more than 2 hours, the automated CSA method took less than 2 hours to complete. The staining results from the automated CSA method presented higher reproducibility, as well as lower background owing to well-regulated, punctual staining and washing at every step of the procedure. Conclusively, the automation of the CSA method enabled us to perform the time-consuming and complicated CSA amplification technique with minimal effort in an accurate, consistent, and reproducible manner.

Antigens↗
PubMed2005

Apolipoprotein D expression in primary brain tumors: analysis by quantitative RT-PCR in formalin-fixed, paraffin-embedded tissue.

Apolipoprotein D (apoD) expression has been shown to correlate both with cell cycle arrest and with prognosis in several types of malignancy, including central nervous system astrocytomas and medulloblastomas. ApoD expression was investigated by real-time quantitative RT-PCR using RNA extracted from 68 formalin-fixed, paraffin-embedded brain specimens. Glyceraldehyde phosphate dehydrogenase was used as an internal control. Quantitation was achieved on all specimens. Sixteen poorly infiltrating WHO grade I glial neoplasms (i.e., pilocytic astrocytomas and gangliogliomas) showed an average 20-fold higher apoD expression level compared with the 20 diffusely infiltrating glial neoplasms (i.e., glioblastoma, anaplastic astrocytoma, oligodendrogliomas; p=0.00004). A small number of exceptions (i.e., two high-expressing glioblastomas and three low-expressing gangliogliomas) were identified. Analyzed as individual tumor groups, poorly infiltrating grade I pilocytic astrocytomas and gangliogliomas differed significantly from each tumor type within the diffusely infiltrating higher-grade category (p<0.05 for each comparison) but not from each other (p>0.05). Conversely, each individual tumor type within the diffusely infiltrating category differed significantly from both pilocytic astrocytomas and gangliogliomas (p<0.05) but did not vary from other infiltrating tumors (p>0.05). Ependymomas, non-infiltrating grade II neoplasms, expressed levels of apoD similar to or lower than levels expressed by the diffusely infiltrating gliomas. Ten medulloblastomas with survival longer than 3 years averaged slightly higher apoD expression than four fatal medulloblastomas; however, this result was not statistically significant and individual exceptions were notable. In 17 of the medulloblastomas, MIB-1 proliferation rates quantitated by image cytometry did not correlate with apoD expression. In addition, apoD expression was 5-fold higher in the slowly proliferating grade I glial neoplasms compared with non-proliferating normal brain tissue (p=0.01), suggesting that apoD expression is not simply an inverse measure of proliferation. ApoD expression measured by quantitative RT-PCR may be useful in the differential diagnosis of primary brain tumors, particularly pilocytic astrocytomas and gangliogliomas.

Apolipoproteins↗
PubMed1996

Hepatitis C and B virus infections in hepatocellular carcinoma. Analysis of direct detection of viral genome in paraffin embedded tissues.

BACKGROUND: Although there have been many seroepidemiologic studies on hepatitis C virus (HCV) infection and hepatocellular carcinoma (HCC) occurrence, the actual role of HCV in hepatocarcinogenesis is unknown. METHODS: We have previously reported on a highly sensitive method of detecting and identifying sequences of RNA genome in formalin fixed, paraffin embedded (FFPE) tissue by polymerase chain reaction (PCR) assay. Using this method, we carried out a retrospective study to determine the prevalence of HCV and hepatitis B virus (HBV) genomes in FFPE specimens from 102 Japanese patients with HCC. RESULTS: HCV-RNA was detected by nested PCR reverse transcription (RT) in 64 of the 102 patients (62.7%), and 78.1% (50/64) of those HCV-RNA-positive patients had HCV genotype II. HCV-RNA was present in 54 of 70 (77.1%) anti-HCV-positive patients, and also in 5 of 20 (25%) anti-HCV-negative patients. HBV-DNA was detected by nested PCR in FFPE liver specimens from 21 of 102 (20.6%) patients. HBV-DNA positivity was consistent with seropositivity for serum HBV markers in 17 of these 21 patients (80.9%). HBV-DNA was present in FFPE samples from 2 patients who were seronegative for HBV markers, and in 1 patient who was seropositive for anti-HBs. Double infection of these two viruses was found in 6 patients (5.9%). Three patients (2.9%) were negative for both hepatic viral genomes and serum viral markers. CONCLUSIONS: The precise prevalence of HCV and/or HBV infection among HCC patients can be determined by studying routinely-processed FFPE HCC samples preserved for up to 11 years using the technique of nested PCR. HCV-RNA was detected in the majority of our HCC cases; type II was the most common genotype of HCV encountered. The incidence of HCV-associated HCC was three times greater than that of HBV. Thus, the hepatitis virus infection most frequently associated, and probably ectologically implicated, with HCC in Japanese people is HCV infection.

Adult↗
PubMed1992

Immunohistochemical and histochemical profile of Aschoff bodies in rheumatic carditis in excised left atrial appendages: an immunoperoxidase study in fresh and paraffin-embedded tissue.

We have evaluated the nature of Aschoff cells within Aschoff bodies seen in 35 of 100 excised left atrial appendages from cases of rheumatic mitral stenosis who underwent closed mitral valvotomy. These were tested using a panel of monoclonal and polyclonal antisera by the indirect immunoperoxidase staining for leucocyte common antigen, macrophage, desmin, vimentin, alpha-1-antitrypsin, alpha-1-antichymotrypsin, lysozyme, acid phosphatase and nonspecific esterase. The Aschoff cell gave strong reactivity with monoclonal antisera to vimentin, macrophage and variable reaction with polyclonal antisera known to recognise macrophages/histiocytes in tissues, namely alpha-1-antitrypsin, alpha-1-antichymotrypsin and lysozyme. These were also strongly positive for acid phosphatase and nonspecific esterase. The Aschoff cell lacked affinity for desmin and only an occasional cell in 4 out of 20 and 6 out of 35 cases showed a weak reaction with myoglobin and leucocyte common antigen, respectively. Intense consistent reactivity with several histiocytic markers affirms the genesis of these cells from macrophages/histiocytes and not muscle cells; a controversy which must be laid to rest!

Antibodies, Monoclonal↗
PubMed1999

Detection of COL1A1-PDGFB fusion transcripts in dermatofibrosarcoma protuberans by reverse transcription-polymerase chain reaction using archival formalin-fixed, paraffin-embedded tissues.

The reciprocal translocation t(17;22)(q22;q13) and a supernumerary ring chromosome, r(17;22), derived from the translocation, have been shown to be highly characteristic of dermatofibrosarcoma protuberans (DFSP). Its consequence is a fusion of two genes, a collagen type I alpha 1 gene (COL1A1) and platelet-derived growth factor B-chain gene (PDGFB). The COL1A1-PDGFB fusion gene, is expected to be a diagnostic molecular assay. However, previous studies on this subject were mostly based on frozen tissue specimens or cultured tumor cells. In this present study, the investigators conducted a reverse transcription (RT)-polymerase chain reaction (PCR) assay to detect the COL1A1-PDGFB fusion transcripts using archival formalin-fixed, paraffin-embedded tumor specimens from 12 patients with DFSP. To amplify the fusion transcripts, a specific COL1A1 forward and PDGFB reverse primers were designed for single step PCR. The COL1A1-PDGFB fusion transcripts could be detected in 10 of 12 paraffin-embedded DFSP tumor specimens (83%). Subsequent sequence analysis using the PCR products confirmed that the detected messages were derived from gene fusions composed of PDGFB exon 2 and different regions of the COL1A1 gene (exon 8, 10, 22, 24, 32, 38, 45 or 46). Two samples of Bednar tumor included in this series also contained the fusion transcripts. In sample of DFSP with fibrosarcomatous transformation, the COL1A1-PDGFB could not be detected in the fibrosarcoma areas of the third recurrence, though the chimeric transcripts were identified in the ordinary DFSP areas of the first recurrence. No fusion transcripts could be amplified in non-DFSP lesions, including 10 dermatofibromas and 9 malignant fibrous histiocytomas. These results indicate that this molecular assay could be applied to archival formalin-fixed, paraffin-embedded tumor tissues as a diagnostic aid for DFSP.

Adolescent↗
PubMed2005

A practical approach to the clinical diagnosis of Ewing's sarcoma/primitive neuroectodermal tumour and other small round cell tumours sharing EWS rearrangement using new fluorescence in situ hybridisation probes for EWSR1 on formalin fixed, paraffin wax embedded tissue.

BACKGROUND: Over 90% of Ewing's sarcoma/primitive neuroectodermal tumour (ES/PNET) cases have the t(11;22) chromosomal rearrangement, which is also found in other small round cell tumours, including desmoplastic small round cell tumour (DSRCT) and clear cell sarcoma (CCS). Although this rearrangement can be analysed by fluorescence in situ hybridisation (FISH) using routinely formalin fixed, paraffin wax embedded (FFPE) tissues when fresh or frozen tissues are not available, a sensitive and convenient detection method is needed for routine clinical diagnosis. AIMS: To investigate the usefulness of newly developed probes for detecting EWS rearrangement resulting from chromosomal translocations using FISH and FFPE tissue in the clinical diagnosis of ES/PNET, DSRCT, and CCS. METHODS: Sixteen ES/PNETs, six DSRCTs, and six CCSs were studied. Three poorly differentiated synovial sarcomas, three alveolar rhabdomyosarcomas, and three neuroblastomas served as negative controls. Interphase FISH analysis was performed on FFPE tissue sections with a commercially available EWSR1 (22q12) dual colour, breakapart rearrangement probe. RESULTS: One fused signal and one split signal of orange and green, demonstrating rearrangement of the EWS gene, was detected in 14 of 16 ES/PNETs, all six DRSCTs, and five of six CCSs, but not in the negative controls. CONCLUSIONS: Interphase FISH using this newly developed probe is sensitive and specific for detecting the EWS gene on FFPE tissues and is of value in the routine clinical diagnosis of ES/PNET, DSRCT, and CCS.

Adolescent↗
PubMed2004

Analysis of p53 mutations in cells taken from paraffin-embedded tissue sections of ductal carcinoma in situ and atypical ductal hyperplasia of the breast.

Mutations in the p53 tumor suppressor gene are frequent in breast tumors but the implication of p53 mutations in breast cancer development remains poorly understood. In this study, we applied laser capture microdissection (LCM) microscope to histologically review and sample cells from paraffin-embedded breast tissue sections obtained from six cases of ductal carcinoma in situ (DCIS) and ten cases of atypical ductal hyperplasia (ADH). p53 mutations were detected, using single stranded conformational polymorphism (SSCP) and sequencing, in cell samples of three cases with DCIS and five cases with ADH. p53 mutations are therefore present in DCIS and ADH of the breast, considered as pre-malignant precursors to breast cancer, and some of them may represent early events in breast cancer development.

Breast↗
PubMed2003

Feasibility of T-cell receptor gamma (TCRgamma) gene rearrangement on formalin-fixed, paraffin-embedded tissues by PCR assays.

INTRODUCTION: T- and B-lymphocytes are involved in recognition of foreign antigen by the specificity of their surface T-cell receptor and immunoglobulin, generated by gene rearrangement. Each T- and B-lymphocyte carries unique rearranged TCR or immunoglobulin gene, which has been applied to detect clonal from non-clonal T- and B-cell proliferation. METHODS: Paraffin-embedded biopsy tissues of 85 T-, 24 B-cell non-Hodgkin's lymphomas (NHL) of various subtypes, and seven reactive lymphoid hyperplasia were retrieved from the archives for determining the feasibility of TCRgamma gene rearrangement analysis by PCR assays in our laboratory. DNA was extracted by Proteinase K digestion. The analyses were performed by five PCR assays, and analysed on polyacrylamide gel. RESULTS: Clonal TCRgamma gene rearrangement was demonstrated in 69/85 (81.2%) of the cases. Selective rearrangement of specific Vgamma segment was observed, especially in peripheral T-cell lymphoma-unspecified and nasal NK/T-cell lymphoma. Clonal TCRgamma rearranged band was also demonstrated in 4/24 (16.7%) and 2/7 (28.6%) of B-NHL and reactive lymphoid tissues respectively. CONCLUSION: PCR assays were able to demonstrate clonal TCRgamma gene rearrangement in a high proportion of T-NHL. However, the PCR results should be interpreted carefully. A neoplasm should only be considered as T-cell type if it does not express any B-cell marker because TCRgamma is not lineage specific as shown by the presence of clonal TCRgamma gene rearrangement in B-NHL. Hence, the results for TCR gene rearrangement should always be interpreted in conjunction with histology and immunophenotyping.

B-Lymphocytes↗
PubMedDate not supplied

Microwave stabilization versus chemical fixation. A morphometric study in glycolmethacrylate- and paraffin-embedded tissue.

A morphological and morphometrical study was performed on testicular cells after microwave stabilization of the tissue while immersed in phosphate buffered saline (PBS), 0.9 NaCl or Tris-HCl. Fixation in Carnoy's fluid without irradiation was chosen as a control chemical fixation method. After microwave stabilization or chemical fixation, the testes were embedded in paraffin or in plastic (glycolmethacrylate). An excellent morphology, comparable to that after chemical fixation in Carnoy's fluid, was observed in the plastic sections of tissue irradiated in PBS or NaCl, even when the sections were subsequently treated with an aggressive reagent at high temperature, required for the Feulgen reaction. The nuclear area of the microwave-stabilized Sertoli cells was 37-46% smaller in haematoxylin-eosin stained, paraffin sections in comparison with that in the glycolmethacrylate sections. The microwave-stabilized, paraffin-embedded tissue was much more vulnerable to the hot HCl treatment of the Feulgen staining than the chemically fixed tissue, resulting in an additional 10-20% decrease in nuclear size. The latter finding is particularly important for quantitative microscopy, where the Feulgen staining method is often employed.

Animals↗