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PubMed2001

Evaluation of formalin-fixed paraffin-embedded tissues from vaccine site-associated sarcomas of cats for polyomavirus DNA and antigen.

OBJECTIVE: To determine whether vaccine site-associated sarcomas (VSS) from cats contain polyomavirus antigen or DNA. SAMPLE POPULATION: 50 formalin-fixed paraffin-embedded tissue blocks of VSS from cats. PROCEDURE: Sections from each tissue block were evaluated for polyomavirus antigen by use of an avidin-biotin-complex immunohistochemical staining method, using rabbit anti-murine polyomavirus polyclonal antiserum as the primary antibody. The DNA was extracted from sections of each tissue block, and a polymerase chain reaction assay was performed, using primers designed to amplify regions of the bovine polyomavirus genome and consensus polyomavirus primers designed to detect unknown polyomaviruses. RESULTS: Polyomavirus antigen and DNA were not detected in any of the VSS. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that polyomaviruses likely do not have any direct involvement in the pathogenesis of VSS in cats.

Animals↗

PubMed2001

Evaluation of formalin-fixed paraffin-embedded tissues from vaccine site-associated sarcomas of cats for papillomavirus DNA and antigen.

OBJECTIVE: To determine whether vaccine site-associated sarcomas (VSS) from cats contain papillomavirus antigen or DNA. SAMPLE POPULATION: 50 formalin-fixed paraffin-embedded tissue blocks of VSS from cats. PROCEDURE: Sections from each tissue block were evaluated for papillomavirus antigen by use of an avidin-biotin-complex immunohistochemical staining method, using rabbit anti-bovine papillomavirus type-1 antibody. The DNA was extracted from sections of each tissue block, and polymerase chain reaction assays were performed, using primers designed to amplify regions of the E5 gene of bovine papillomavirus and consensus primers designed to amplify a region of the L1 gene of animal papillomaviruses. Sections from 20 of the tissue blocks were evaluated by use of nonradioactive in situ hybridization for bovine papillomavirus DNA. RESULTS: Papillomavirus antigen and DNA were not detected in any of the VSS. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that papillomaviruses likely do not have any direct involvement in the pathogenesis of VSS in cats.

Animals↗

PubMed1979

Sensitivity and background staining of the unlabeled antibody enzyme (PAP) method. new approaches to the investigation of the effects of non-immune serum, primary antiserum concentration and pronase digestion using formalin fixed paraffin embedded material.

The effect of primary antibody concentration, non-immune serum, and pronase digestion on the stainability of immunoglobulin containing cells (plasma cells) and background stainability was investigated using the peroxidase-antiperoxidase (PAP) method and antigens consisting of formalin fixed paraffin embedded maxillary mucosa, colonic mucosa, inflammatory synovia and soft-tissue plasmocytoma. The effect of primary anti-serum concentration and non-immune serum on cell and background stainability was smaller, but pronase digestion clearly improved positive stainability and reduced background stainability.

Animals↗

PubMed1989

Identification of cryptosporidium in paraffin-embedded tissue sections with the use of a monoclonal antibody.

Recently a monoclonal antibody has been developed against the oocyst form of Cryptosporidium species for use in detecting the organism in fecal material. The authors undertook this study to determine if this antibody could be used in identifying Cryptosporidium species in paraffin-embedded tissue sections. Three biopsies showing Cryptosporidiosis as diagnosed by characteristic appearance by light and electron microscopy were selected from the surgical pathology files at the authors' institution. Paraffin sections were examined with the use of both an indirect immunofluorescence and an avidin-biotin-peroxidase technique. In all the cases, prominent staining of oocysts was seen; however, intracellular trophozoites did not react with the antibody. The authors conclude that this antibody can be used to identify Cryptosporidium species oocysts in tissue sections and that the antibody appears to be specific for the oocyst form only.

Adult↗

PubMed1991

Application of DNA flow cytometry from paraffin-embedded tissue to the diagnosis of mycosis fungoides.

The distinction of mycosis fungoides from benign inflammatory lesions is sometimes difficult by conventional histological techniques. Aneuploidy, a feature often associated with malignant tumors, can be assessed even in tissue routinely processed in paraffin using the flow cytometric technique of Hedley and associates. In many tumor systems, there are significant diploid clones. We have evaluated the flow cytometric DNA ploidy of paraffin-embedded tissue in the diagnosis and prognosis of mycosis fungoides (MF). We studied 22 cases of MF and 10 control cases of inflammatory skin lesions with epidermal involvement. Aneuploidy was found in 27% of the MF cases, but in none of the controls (ED). Aneuploid features were seen in 23% of tissues from early stage disease. Aneuploidy did not correlate with atypia, epidermotropism, or number of mitoses. There was a trend towards showing adverse outcome in those patients with aneuploid lesions. The detection of aneuploidy might be helpful for early diagnosis of MF.

Aneuploidy↗

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↗

PubMed1986

Identification of Brucella abortus in formalin-fixed, paraffin-embedded tissues of cows, goats, and mice with an avidin-biotin-peroxidase complex immunoenzymatic staining technique.

An avidin-biotin-peroxidase complex immunoenzymatic staining technique was evaluated for light microscopic detection of Brucella organisms in formalin-fixed, paraffin-embedded tissues. Tissues from cows, goats, and mice inoculated with B abortus strain 2308 were examined, using rabbit antiserum to Brucella cell surface protein as primary antibody. Stained organisms were identified histologically in tissue sections containing B abortus, as detected by bacteriologic examination of duplicate nonprocessed tissue samples. Bacillus sp, Corynebacterium pyogenes, Escherichia coli, Listeria monocytogenes, Pasteurella haemolytica, P multocida, Salmonella typhimurium, Staphylococcus sp, and Streptococcus sp did not stain with this system, using anti-Brucella cell surface protein primary antibody, thus indicating specificity for Brucella organisms.

Animals↗

PubMed1994

Immunoperoxidase staining of hepatitis C virus in formalin-fixed, paraffin-embedded needle liver biopsies.

The localization of hepatitis C virus (HCV) in the liver has not been well clarified. We report successful indirect immunoperoxidase staining of the HCV core antigen using polyclonal antibodies raised in rabbits and conventional formalin-fixed, paraffin-embedded needle biopsy sections of liver. The core antigen was distributed in a fine granular pattern diffusely, perisinusoidally, or focally within the hepatocellular cytoplasm of livers from patients with HCV infection. The staining tended to show a more heterogeneous pattern in terms of intensity and distribution in cases of more advanced disease. Hepatocellular carcinoma cells were also frequently stained. HCV immunostaining will provide important information on the pathogenesis and treatment of HCV-related liver diseases.

Adult↗

PubMed2005

Technology insight: Application of molecular techniques to formalin-fixed paraffin-embedded tissues from breast cancer.

Breast cancer is a heterogenous disease in terms of both clinical behavior and molecular characteristics. To develop prognostic and predictive markers for breast cancer, it would be useful to be able to analyze formalin-fixed paraffin-embedded tissue (FPET) collected and banked from completed clinical trials. RNAs extracted from FPETs are chemically modified and fragmented, and are therefore not ideal substrates for gene-expression profiling assays. However, methods are being developed to optimize the use of such RNAs for high-throughput gene expression profiling assays. For microarray analysis, existing methods may be adequate for fresh FPET, but they do not work well with older FPET. For older samples, real-time reverse transcription-polymerase chain reaction is the method of choice for gene-expression profiling.

Biopsy↗

PubMed1987

NCL-5D3: a new monoclonal antibody recognizing low molecular weight cytokeratins effective for immunohistochemistry using fixed paraffin-embedded tissue.

Production of a new monoclonal antibody designated NCL-5D3 is described. The antibody recognizes several low molecular weight cytokeratins, in particular cytokeratin Moll number 8 as determined by immunoblotting studies, and is highly effective for immunocytochemistry using routinely processed paraffin-embedded material. Staining is enhanced by prior treatment of the sections with trypsin. Assessment using a wide variety of normal and neoplastic tissue indicates reactivity with all tissues of simple or glandular epithelial origin, and in addition with many squamous carcinomas. Thus the antibody should prove of value in diagnostic histopathology.

Antibodies, Monoclonal↗

PubMed1994

Detection of herpes simplex virus type 2 Bgl II N fragment in paraffin-embedded cervical tissue sections using nested polymerase chain reaction.

To study the presence of transforming sequence Bgl II N of HSV-2 in cervical cancer tissues, we developed the nested polymerase chain reaction (PCR) for detecting such a sequence in paraffin-embedded cervical tissue sections. Samples derived from 46 patients with premalignant and malignant lesions were tested. The sequence was found in 20-25% of total cases tested but not observed in any of the normal healthy controls. This study also indicates that for the detection of HSV-2 Bgl II N sequence in cervical tissue, the nested PCR may be more reliable than the in situ hybridization method.

Adenocarcinoma↗

PubMed1990

Human papillomavirus infection and filaggrin expression in paraffin-embedded biopsy specimens of extragenital Bowen's disease and genital bowenoid papulosis.

Cutaneous Bowen's disease (BD) and genital bowenoid papulosis (BP) are considered as precancerous or cancerous lesions that are sometimes infected with human papillomavirus (HPV). We studied retrospectively paraffin-embedded sections of 11 samples of cutaneous BD and 6 samples of genital BP from the general population for HPV infection and filaggrin expression. Using in situ hybridization with biotinylated probes of HPV types 1, 2, 5, 6, 11, 16, and 18, under stringent and/or non-stringent conditions and a streptavidin-alkaline-phosphatase complex for hybrid detection, HPV DNA was detected in 6/17 cases (5 BD and 1 BP). Positive nuclei were located in intermediate or upper epithelial cell layers. HPV 16 was found in 2 cases of BD but associated either with HPV 2 or 18. Three additional lesions reacted only under non-stringent conditions; HPV could not be typed with the probes used. The positive case of BP reacted with the four probe types 1, 2, 16, 18 and was negative with HPV 6 or 11. Viral antigen was not detected by indirect immunofluorescence with a rabbit antiserum directed to group-specific viral capsid antigen. Differentiation disorders were observed in the intermediate and upper cell layers of these specimens, as shown by a reduced expression of filaggrin/profilaggrin, a marker of terminal differentiation, in extragenital BD (7/11 cases), and an increased expression in genital BP (4/5 cases) although viral DNA was not always detectable. This study shows that in situ hybridization is a valuable technique for HPV DNA detection and its typing in BD and BP lesions on deparaffinized sections. The positive nuclei were located in the cell layers that exhibited abnormal expression of differentiation. There is no relation between the HPV infecting type and the filaggrin expression.

Aged↗

PubMed2006

Ewing's sarcoma of bone: the detection of specific transcripts in a large, consecutive series of formalin-fixed, decalcified, paraffin-embedded tissue samples using the reverse transcriptase-polymerase chain reaction.

AIMS: (i) To report on the routine use of the reverse transcriptase-polymerase chain reaction (RT-PCR) technique on decalcified or non-decalcified, formalin-fixed, paraffin-embedded tissue (FFPET) for translocation detection, with particular emphasis on improved RNA extraction methodology and the use of PCR primers designed to generate small amplicons. (ii) To report on the relative incidences of translocation types and transcript variants in a large, single institution series of Ewing's sarcoma of bone. METHODS AND RESULTS: Using RT-PCR to detect specific transcript variants, we analysed FFPET from 54 consecutive cases of Ewing's sarcoma of bone. We used 'gold standard' detection methods on corresponding fresh and fresh frozen tissue to validate the technique. We have demonstrated the effective use of RT-PCR on decalcified and non-decalcified FFPET samples for sarcoma-specific translocation detection (96% sensitivity, 100% specificity). Tissue decalcification did not affect the detection rate. The relative incidence of Ewing's sarcoma-specific translocation types and transcript variants was entirely consistent with previously published data. CONCLUSIONS: With equal effectiveness, RT-PCR can be applied to both acid decalcified and non-decalcified FFPET for (Ewing's sarcoma) translocation detection and the technique can be introduced into routine practice in histopathology departments.

Adolescent↗

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↗

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↗

PubMed2003

Nested PCR assays for detection of Blastomyces dermatitidis DNA in paraffin-embedded canine tissue.

A Blastomyces dermatitidis nested PCR assay targeting the gene encoding the Wisconsin 1 (WI-1) adhesin was developed and compared with a nested PCR targeting the 18S rRNA gene (rDNA) of members of the family ONYGENACEAE: We examined 73 paraffin-embedded tissue samples obtained from nine dogs which died of blastomycosis and nine dogs which succumbed to lymphosarcoma according to autopsy findings; amplifiable canine DNA was extracted from 25 and 33 specimens from the two groups, respectively. The B. dermatitidis PCR amplified DNA from 8 of 13 tissue samples in which yeast cells were detected by microscopy. Sequencing revealed that all PCR products were homologous to the B. dermatitidis WI-1 adhesin gene. No PCR product was amplified from 12 microscopically negative biopsy specimens from dogs with blastomycosis or from 33 biopsy specimens from dogs with lymphosarcoma. The 18S rDNA PCR amplified DNA from 10 and 9 tissue samples taken from dogs which died of blastomycosis and lymphosarcoma, respectively. Only six products were identified as being identical to B. dermatitidis 18S rDNA; they were exclusively obtained from specimens positive by the B. dermatitidis nested PCR. For specificity testing, 20 human biopsy specimens proven to have histoplasmosis were examined, and a specific H. capsulatum product was amplified by the 18S rDNA PCR from all specimens, whereas no product was obtained from any of the 20 samples by the B. dermatitidis PCR assay. In conclusion, the PCR targeting a gene encoding the unique WI-1 adhesin is as sensitive as but more specific than the PCR targeting the 18S rDNA for detection of B. dermatitidis in canine tissue.

Animals↗

PubMed1987

Detection of albumin mRNAs in rat liver by in situ hybridization: usefulness of paraffin embedding and comparison of various fixation procedures.

Our aim was to define optimal conditions for efficient and reproducible albumin mRNA detection in rat liver by in situ hybridization. We used an albumin-specific [3H]-labeled cDNA probe with a specific activity of 6-8.10(6) cpm/microgram DNA. In situ hybridization is as efficient on paraffin sections as on cryostat sections for detecting albumin mRNAs. Perfusion fixation with a 4% paraformaldehyde solution results in homogeneous RNA retention within tissue blocks, in contrast with immersion fixation, which yields heterogeneous RNA preservation. Comparison of immersion fixation with three different fixatives (paraformaldehyde, ethanol-acetic acid, and Bouin's fixative) shows that the highest level of hybridization signal is obtained with paraformaldehyde. Ethanol-acetic acid and Bouin's fixative appear less efficient for albumin mRNA detection. Loss of mRNAs within liver tissue blocks over time is largely although not completely prevented by paraffin embedding.

Albumins↗

PubMed2003

Chronic myeloid leukemia following therapy with imatinib mesylate (Gleevec). Bone marrow histopathology and correlation with genetic status.

We evaluated bone marrow pathologic features and cytogenetic and molecular genetic status of 13 patients with interferon-resistant, chronic-phase chronic myeloid leukemia (CML), treated with imatinib mesylate (Gleevec). All had morphologic evidence of CML in the blood and bone marrow and were positive for bcr-abl by reverse transcriptase-polymerase chain reaction, fluorescence in situ hybridization (FISH), or both. Follow-up marrow biopsies, interphase FISH for bcr-abl, and conventional cytogenetics were performed at 3-month intervals (up to 24 months) after therapy initiation. All patients exhibited a reduction in bone marrow cellularity with decreases in myeloid/erythroid ratios at 3 to 6 months after therapy. The percentage of bcr-abl-positive cells by FISH decreased in all patients (pretherapy median, 73%; 3 months median, 47%). Cytogenetic and FISH data defined 2 groups after 6 months of follow-up: 5 patients became negative for bcr-abl by FISH; 8 remained positive, 4 of whom developed signs of clonal cytogenetic evolution. Patients who became negative for bcr-abl had no morphologic evidence of CML at 15 to 24 months of follow-up, whereas patients who remained positive redeveloped morphologic features of CML as cellularity increased. Some bcr-abl-positive patients showed signs of progression, including 2 patients who developed myeloid blast phase. Although all patients demonstrated an initial decrease in bone marrow cellularity after imatinib mesylate therapy, continued follow-up showed that histopathologic findings correlated with genetic response.

Antineoplastic Agents↗