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Results for “Paraffin Embedding”

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PubMed1993

Epidermal growth factor receptor expression in squamous cell lung carcinomas: an immunohistochemical and gene analysis in formalin-fixed, paraffin-embedded material.

Epidermal growth factor (EGF) and its receptor (EGFr) constitute an important and well-characterized mitogenic system in various ectodermal tissues. We evaluated the expression of EGFr and examined possible EGFr gene alterations in 18 formalin-fixed, paraffin-embedded squamous cell lung carcinomas (SCLC) by an immunohistochemical assay, Southern blotting and differential polymerase chain reaction (DPCR). The immunohistochemical study employing the F4 and EGF-R1 monoclonal antibodies, directed against the intra- and extra-cellular portion of the receptor respectively, showed EGFr over-expression in 89% of the SCLC cases examined. All cases showed positive immunostaining for both antibodies, thus excluding the possibility of truncated receptors. In addition, analysis of the EGFr gene was carried out by Southern blotting and DPCR on paraffin extracted DNA from the same carcinoma cases. We found amplification of the EGFr gene in 5/18 (27%) SCLCs. All 5 positive cases showed EGFr over-expression, suggesting a possible correlation between the presence of EGFr gene amplification and over-expression of receptor protein. No correlation was observed among EGFr staining, EGFr gene amplification and differentiation of carcinomas. In addition, Southern blot analysis with HER-A2, a probe which hybridizes a sequence of the receptor's intracellular domain, revealed three novel EcoRI restriction fragment patterns. We suggest that these patterns correspond to EcoRI polymorphic sites of the receptor's tyrosine kinase domain.

Base Sequence↗

PubMed1994

c-erbB-2 amplification in breast cancer: detection in formalin-fixed, paraffin-embedded tissue by in situ hybridization.

We describe a sensitive and practical in situ hybridization method, using a digoxigenin-labeled probe, for the detection of c-erbB-2 amplification in breast cancer in formalin-fixed, paraffin-embedded tissue sections. Forty-six primary breast carcinomas were studied. Nuclear hybridization signal was observed in 36 of 46 carcinomas. Signal was confined to malignant cells. Normal breast epithelium and stromal and inflammatory cells were uniformally negative. DNase predigestion, no-probe preparations, and competitive hybridization confirmed the specificity of the reaction. The hybridization reaction was localized to multiple discrete foci in tumor cell nuclei, suggesting multiple sites of gene copy and transcriptional activity in the nucleus. Considerable cell-to-cell variation in hybridization signal was evident within individual tumors and positive reactions were observed in several cases in which amplification could not be detected by either Southern or slot blot analysis. The high sensitivity and specificity of the reaction and its use in a tissue-based system will allow the study of a range of possible precursor lesions of breast cancer for evidence of c-erbB-2 amplification.

Breast Neoplasms↗

PubMed1985

Extraction of cells from paraffin-embedded tissue sections for single-cell DNA cytophotometry.

A simple technique is presented for the isolation of cells from paraffin-embedded tissues for Feulgen DNA cytophotometric investigations. Tissue fragments from paraffin blocks were deparaffinized in xylene, rehydrated and refixed in a formalin solution and incubated in a solution of 0.5 pepsin in 0.25% hydrochloric acid. After filtration through a 70 micron mesh and centrifugation, the cells were smeared upon a glass slide. Comparison between the results obtained with freshly prepared imprints and with pepsin-extracted cells of the same tumor showed that the extraction technique does not influence the Feulgen reaction or the DNA distribution pattern. Investigations carried out on bladder and embryonal carcinomas have demonstrated that the method permits an analysis of histologically or histochemically identified tumor cells within individual tissue areas.

Cell Separation↗

PubMed1998

Melan A/MART-1 immunoreactivity in formalin-fixed paraffin-embedded primary and metastatic melanoma: frequency and distribution.

Monoclonal antibody (MAb) A103 specifically detects Melan A/MART-1 protein expression. Melan A/MART-1-derived peptides are recognized by CD8+ T-cells and are used in immunotherapy. We examined formalin-fixed paraffin-embedded tissue from 57 melanomas (34 primary, 23 metastatic) and 39 control cases (junctional, dermal, compound, Spitz, Reed and balloon-cell naevi) using the alkaline phosphatase and anti-alkaline phosphatase immunochemical method after antigen retrieval. Immunoreactivity was rated as low, medium or high, and staining pattern as homogeneous or heterogeneous. Staining with MAb A103 showed a sensitivity of 88% for melanoma, with a very high specificity for melanocytic cells. Immunopositivity decreased along with clinical stage, with stage I showing 100%, stage II 88%, stage III 90% and stage IV 75% immunoreactivity. Staining changed from an exclusively homogeneous pattern in the early clinical stages to a more heterogeneous pattern in the later stages. Melanocytic control tissues consisting of naevi of different subtypes all showed weak to moderate homogeneous immunoreactivity, with polarity towards the epidermis. Analysis of short-term melanoma cell cultures using reverse transcription-polymerase chain reaction (RT-PCR) enzyme-linked immunosorbent assay (ELISA) demonstrated mRNA expression in only one third of the originally immunopositive tumours, suggesting rapid mRNA expression loss in culture. MAb A103 allows the detection of melanoma-associated Melan A/MART-1 protein expression in routine archival tissue and thus enables the profiling of melanomas suited for immunotherapy approaches involving Melan A/MART-1 derived epitopes.

Antibodies, Monoclonal↗

PubMed2001

Cytokine profiles in paraffin-embedded biopsy samples of lepromatous leprosy patients: semi-quantitative measure of cytokine mRNA using RT-PCR.

A reproducible technique for fixation of tissue, RNA extraction and reverse transcription polymerase chain reaction (RT-PCR) analysis from paraffin-embedded leprosy biopsies, has been developed and used to study the mRNA profiles. This approach is valuable in retrospective analysis of gene expression, and the handling of infectious biopsy material is also minimized. Among the methods of RNA extraction compared, the most efficient method was found to be incubation of the tissue sections in digestion buffer followed by extraction with Trizol. The experimental conditions were optimized for first strand cDNA synthesis and PCR, and used to measure the quantity of cytokine transcripts in biopsy materials. Interleukin-10 (IL-10) mRNA was detectable in all cases examined, which correlates well with other earlier reports using frozen tissues. However, IL-5 transcripts were present in 60% of the biopsies, unlike the earlier reports which showed IL-5 mRNA in all LL cases. Transforming growth factor-beta (TGF-beta) mRNA was detected in 80% of the biopsies, and this confirmed earlier immuno-cytochemical data which showed TGF-beta protein in all cases. Tumor necrosis factor-alpha mRNA was found in about 60% of the biopsies; whereas interferon gamma mRNA was detected in 30% of the LL cases. In conclusion, the results obtained in our study confirm and extend earlier observations which examined cytokines in peripheral blood cells and dermal lesions of leprosy. The simplicity of this method would allow the examination of a large number of samples across the spectrum of leprosy.

Biopsy↗

PubMedDate not supplied

Cytokeratin positivity in paraffin-embedded malignant melanomas: comparative study of KL1, A4 and Lu5 antibodies.

The unclear role of cytokeratin (CK) in the progression and diagnostics of malignant melanomas stimulated us to compare the reactivity of three antibodies directed to CK in 109 paraffin-embedded melanomas. By far the majority of melanomas did not express cytokeratin even at the<1% level, only vimentin. In about 6% of melanomas it was possible to find CK expression ranging between 3 and 40% of melanoma cells. There was a correlation between CK expression and pT-stage. Cytokeratin-expressing tumours were found in the more advanced pT-stages. The independent prognostic values of none of the three CK antibodies investigated could be shown.

Adult↗

PubMedDate not supplied

Cellular immune response in atypical tuberculosis diagnosed by PCR in paraffin embedded material.

The aim was to evaluate the cellular immune response in atypical tuberculosis and granulomatous inflammation consistent with tuberculosis (TBC), negative histochemically for acid-fast bacilli and analysed by PCR for Mycobacterium tuberculosis (MT) detection in paraffin-embedded tissue. Thirty six samples of differently localized atypical tuberculous lesions and granulomatous tuberculoid lesions negative for acid fast bacilli and 4 positive cases on Ziehl-Nielsen stain were analysed by PCR for MT detection and were tested immunohistochemically (IHC) for the cellular immune response in the granulomas and perigranulomatous tissue. The samples selected were: 7 pulmonary and 33 extrapulmonary specimens, especially lymph nodes. Histologically, the atypical tuberculous lesions contained supurative necrosis, defective granulomas and cellular polymorphism. The epithelioid cells showed frequent mitoses. The immunoprofile of cells was polymorphous. L26 positive small lymphocytes were found in nodular lymphoid aggregates surrounding granulomas. A significantly increased number of positive UCHL1 cells were found in 33 out of the 40 analysed cases, with a larger percentage of CD4 positive T cells (81.8% of cases). CD44 was positive in multinucleated giant cells (17.5% of cases), epithelioid cells (60% of cases) and lymphocytes (30% of cases). CD68 was localized in multinucleated giant cells and epithelioid cells, in a 4%, respectively 62.5% of cases. The PCR was performed in all 40 cases; the tissue samples were heterogeneous (lung, lymph nodes, lever, nasopharynx, etc.) and needed a good quality extraction of DNA. Performing a control PCR for Beta Globin tested the extraction; a good result was obtained in 31 cases (77.5%); from these, 19 cases had amplification for IS 6110. The cellular immune response in the atypical tuberculous lesions was similar in cases with and without acid-fast bacilli, but positive for PCR. In the most cases with negative PCR reaction, it was due to a deficient fixation of the material. The T lymphocytes were numerous in all types of tuberculous granulomas, with the prominence of CD4 positive subtype. The immunoprofile of the epithelioid cells, positive for CD44 and CD68, presenting frequently mitoses suggests an activate state in a possible relationship to the T-cell-mediated immune response in tuberculosis.

Antigens, CD↗

PubMed2005

Evaluation of seaFAST, a rapid fluorescent in situ hybridization test, for detection of Helicobacter pylori and resistance to clarithromycin in paraffin-embedded biopsy sections.

A commercially available rapid fluorescent in situ hybridization (FISH) test, (seaFAST H. pylori Combi-Kit; SeaPro Theranostics International, Lelystad, The Netherlands) was used to simultaneously detect the presence of Helicobacter pylori and determine clarithromycin susceptibility in paraffin-embedded biopsy sections. The FISH method was found to be 97% sensitive, 94% specific for the detection of H. pylori and comparable to agar dilution for the detection of resistance to clarithromycin.

Anti-Bacterial Agents↗

PubMed2003

Detection of Helicobacter pylori in paraffin-embedded and in shock-frozen gastric biopsy samples by fluorescent in situ hybridization.

We report on the successful application of fluorescent in situ hybridization for detection of Helicobacter pylori and determination of its clarithromycin susceptibility in formalin-fixed and paraffin-embedded gastric biopsy specimens that had been prepared for pathological examination. This method is useful when results from conventional culturing with antibiotic susceptibility testing are not available.

Anti-Bacterial Agents↗

PubMed1991

[DNA analysis of meningiomas using paraffin-embedded surgical specimens in connection with clinical recurrence].

Meningioma includes some clinically malignant cases which grow multifocally or recur rapidly. To develop methodology to distinguish clinically malignant cases, we examined the nuclear DNA of meningiomas by flow cytometry using paraffin-embedded specimens. 52 surgical specimens were studied from 52 cases of meningioma. Among these cases, 3 multiple meningiomas that recurred multifocally within 3 years were included. Malignancy was assessed by the proliferative index (%S + %G2/M) and DNA ploidy of the specimens. Six cases were histologically malignant, while aneuploidy was observed in only 2 (33.3%). No significant correlation was observed when analyzing the 23.9% aneuploidy rate among benign cases. Moreover, three cases of clinically malignant meningiomas were all diploid. In contrast, the proliferative index of 19.82 +/- 9.45% among histologically malignant cases was significantly higher as compared to that for benign cases (11.50 +/- 5.49%). The proliferative index was 15% or more (average 22.02 +/- 6.01%) for patients with clinically malignant meningioma. This was considerably higher than the corresponding value for clinically benign meningiomas. Our analysis indicated that the assessment of benignancy or malignancy of meningioma on the basis of DNA ploidy alone is difficult. The proliferative index so obtained relates significantly to prognosis, apparently providing a useful prognostic assessment.

Adult↗

PubMed1997

Application of fluorescence in situ hybridization to detect N-myc (MYCN) gene amplification on paraffin-embedded tissue sections of neuroblastomas.

Fluorescence in situ hybridization (FISH) was applied to neuroblastoma for detection of N-myc (MYCN) oncogene amplification, and the results were compared with Southern blot analysis (Southern). In nine neuroblastomas (formalin-fixed paraffin-embedded tissues were available in seven cases including two cases with touch preparations, and two cell lines), all five cases with N-myc amplification detected by Southern had cells with multiple N-myc signals by FISH, and three cases showed no N-myc amplification either by Southern or FISH procedure. One case, not examined by Southern, showed amplified signals of N-myc by FISH. These data indicate that FISH results for N-myc amplification have close correlation with Southern blot analysis. The chromosome 2-specific repetitive DNA probe was also applied for the analysis of ploidy by FISH. Six cases with N-myc amplification by Southern and/or FISH had diploid tumors and two cases without amplified N-myc showed aneuploidy. The remaining one case consisted of heterogeneous elements showing diploidy in undifferentiated tissue and both aneuploidy (ganglionic cells) and diploidy (Schwann cells) in differentiated area. We conclude that FISH is a practical, useful and reliable method over Southern especially for analysis of N-myc amplification in neuroblastoma, and simultaneous cohybridization with a specific chromosome probe is of great value in predicting the prognosis of patients.

Blotting, Southern↗

PubMed2005

Diagnosis of cat scratch disease with Bartonella henselae infection in formalin-fixed paraffin-embedded tissues by two different PCR assays.

Cat scratch disease (CSD) is commonly caused by Bartonella henselae infection. Clinical history and histologic findings are often insufficient to establish a definitive diagnosis of CSD. We retrospectively studied formalin-fixed, paraffin-embedded (FFPE) lymph nodes from 35 patients with histologically suspected CSD by 2 different PCR assays and immunohistochemistry (IHC). The first primer pair amplified a 163-bp fragment of the 16S rRNA gene in 19 of the 35 cases (54%). The second primer pair amplified a 191-bp fragment of the henselae citrate synthase (gltA) gene in 17 of the 35 cases (49%). IHC identified the organisms in 8 of 33 cases (24%). Fresh cultures of various Bartonella species showed a specific PCR product with an analytical sensitivity of 0.5 to 5 pg bacterial DNA. Bartonella species were identified by the unique size of the amplified PCR product. Twenty-two lymph nodes without morphologic evidence or a history of CSD were negative by PCR and immunostaining. Tissues from a patient with Legionella pneumophila were also negative by PCR and immunostaining for CSD supporting the specificity of the PCR reaction. The specific PCR products of the B. henselae were confirmed by sequencing. Human beta-actin for each case was amplified to check the integrity of the DNA. Our data indicate that detection of Bartonella DNA by PCR is useful to confirm the diagnosis of CSD.

Actins↗

PubMed2001

Evaluation of a group-specific 16S ribosomal DNA-based PCR for detection of Helicobacter bizzozeronii, Helicobacter felis, and Helicobacter salomonis in fresh and paraffin-embedded gastric biopsy specimens.

A new specific and sensitive 16S ribosomal DNA-based PCR assay was developed. The assay targets a 78-bp DNA fragment unique to Helicobacter bizzozeronii, Helicobacter felis, and Helicobacter salomonis and can be used with freshly frozen and formalin-fixed paraffin-embedded gastric biopsy specimens.

Animals↗

PubMed1998

A103: An anti-melan-a monoclonal antibody for the detection of malignant melanoma in paraffin-embedded tissues.

Melan-A is a previously defined, melanocyte differentiation antigen, and an anti-Melan-A murine monoclonal antibody, A103, was recently developed by our group. In this study, we evaluated A103 immunoreactivity on formalin-fixed, paraffin-embedded tissues, exploring the potential of A103 in the diagnosis of metastatic melanoma. Seventy-five metastatic melanomas, 10 primary melanomas, and 10 benign melanocytic nevi were tested. The reactivity of A103 was compared with HMB-4, an anti-gp100 antibody. Results showed that all nevi were A103 positive, and most primary melanomas were A103 and HMB45 positive. Of 75 metastatic melanomas, 61 (81%) were A103 positive, and 56 (75%) were HMB45 positive. Of 19 HMB45-negative lesions, 8 were A103 positive; of 14 A103-negative lesions, 3 were HMB45 positive. Eleven metastatic lesions, as well as 2 of 10 primary melanomas, were dual negative. These negative cases consisted mainly of the spindle cell and desmoplastic variants. Of the positive cases, A103 showed homogeneous staining in a significantly higher proportion of cases than HMB45 (72% versus 52%). In addition, focal staining with less than 5% reactive tumor cells was seen more frequently in HMB45 (12 of 56) than in A103 (5 of 61). These results indicated that A103 can be used as a first-line antibody in the diagnosis of metastatic melanoma. Our results also showed that A103 reacted with angiomyolipoma, which is known to be HMB45 positive. Of normal tissues, unexpected A103 reactivity was observed in the adrenal cortex, granulosa and theca cells of the ovary, and Leydig cells of the testis. This A103 immunoreactivity in benign and neoplastic tissues of nonmelanocytic origin, the basis of which is unclear, could also be of potential diagnostic value.

Angiomyolipoma↗

PubMed2001

Optimized protocols for the detection of porcine circovirus 2 DNA from formalin-fixed paraffin-embedded tissues using nested polymerase chain reaction and comparison of nested PCR with in situ hybridization.

Optimized DNA extraction method and nested polymerase chain reaction (PCR) were developed for the detection of porcine circovirus 2 (PCV2) from formalin-fixed, paraffin-embedded tissues. Conventional PCR, nested PCR, and in situ hybridization methods were also compared for the detection of PCV2 in archival tissues. A method based on xylene deparaffinization followed by proteinase K digestion yielded DNA of sufficient quality for PCR analyses reliably and consistently. Twenty-six (70%) of the 37 tissues examined gave positive results with conventional PCR, whereas all the 37 tissues gave positive results using the nested PCR. A distinct positive signal for PCV2 was detected in spleen and lymph node from all the 37 pigs by in situ hybridization. The nested PCR and in situ hybridization could be applied successfully to archival tissues for the detection of porcine circovirus 2 DNA.

Animals↗

PubMedDate not supplied

The suitability of paraffin-embedded material to predict metastatic potential of oral squamous cell carcinoma.

BACKGROUND: The presence of regional metastasis in oral squamous cell carcinoma (OSCC) is an important prognostic factor. This study was undertaken to identify histological features and biological markers from paraffin-embedded primary OSCC that may predict the presence of regional metastases. MATERIALS AND METHODS: Fifty-three en-bloc primary OSCC resections were divided into two groups, 26 with lymph node metastases and 27 without metastases. The pattern of infiltration, presence of vascular or perineural infiltration and tumour necrosis were evaluated while expression of p53, p21 and Rb were assessed in the two groups. DNA ploidy status was also determined with a flow cytometer. RESULTS: The presence of DNA aneuploidy was found to be the only statistically significant predictor of regional metastases. Seventy-seven per cent of the primary OSCC with lymph node metastases showed DNA aneurploidy. CONCLUSION: DNA flow cytometry obtained from archival material could be used as a parameter to predict regional metastases.

Aneuploidy↗

PubMed2000

[Paraffin-embedded tissues as a source of control DNA].

During elucidation of crime cases, the need of a sample of DNA of the victim of the violent attack appears very often. Regarding to the obligatory archiving of histopathological preparations from sectioned persons, the paraffin-embedded tissues are easily achievable material suitable for the given purpose. Such a modification of an isolation method was elaborated which allows the yield of the sufficient amount of DNA from one histological section, and its usefulness was tested on tissues of different types and from different persons. The method was used successfully during solving of concrete cases.

DNA↗

PubMed1995

Interphase cytogenetic analysis of single cell suspensions prepared from previously formalin-fixed and paraffin-embedded tissues.

Fluorescence in situ hybridization (FISH) provides a rapid and accurate method for the detection of chromosomal aneuploidy. We have developed a technique for the use of FISH on single cell suspensions produced from either formalin-fixed or paraffin-embedded tissues. Preparation of such tissues involves sequential rehydration, enzymatic digestion to release single nuclei, and hybridization with a fluorescently labeled chromosome-specific centromeric probe. In a clinical setting formalin-fixed tissue from many tissue types is readily available for additional retrospective study. FISH on formalin-fixed tissues is especially beneficial in follow-up studies of cases involving termination after prenatal diagnosis or patients with a malignant disease where previous routine cytogenetics established the chromosomal aneuploidy. The use of this technique eliminates the biases of cytogenetic analysis due to clonal selection in tissue culture, the low number of cells analyzed, and the restriction to only dividing cell populations. We have demonstrated that this application of interphase cytogenetics to the study of various formalin-fixed tissues is amenable to the detection of chromosomal aneuploidies and has specific advantages over cytogenetic analysis.

Aneuploidy↗