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Results for “Paraffin Embedding”

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PubMed1997

Optimizing collagen antigen unmasking in paraffin-embedded tissues.

In order to optimize collagen antigen unmasking in paraffin-embedded tissue sections, the effects of various fixatives and duration of fixation in relation to enzyme pretreatment and microwave irradiation for collagen antigen unmasking were studied. A streptavidin-biotin-peroxidase complex method was used for the immunolocalization of type III and IV collagen antigens. Fixatives and fixation time had significant adverse effects on the immunoreactivity of the antigens. Enzyme pretreatment was found to be superior to microwave irradiation for collagen antigen unmasking. Fixation with paraformaldehyde required shorter enzyme pretreatment and yielded a more enhanced reaction than treatment with formalin and Bouin's fluid. The optimum conditions for type III and IV collagen unmasking were found to be fixation with 4% paraformaldehyde in 0.01 M phosphate-buffered saline, pH 7.4, for up to 3 weeks followed by enzyme pretreatment with 1 mg ml-1 pepsin in 0.01 N hydrochloric acid, pH 2.0, for 30 min (human tissues) or 60 min (rat tissues) at 37 degrees C. It is concluded that collagen antigen unmasking by enzyme pretreatment in tissue sections fixed for a long period of time can be successful if appropriate enzyme(s) and incubation time(s) are employed with regard to the antigen under study and fixative and fixation time used for tissue preparation.

Animals↗

PubMed1994

Detection of delta F508 cystic fibrosis mutation by polymerase chain reaction from old paraffin-embedded tissues: a retrospective autopsy study.

This study analyzes the usefulness of the polymerase chain reaction technique in the detection of delta F508 mutation in 11- to 25-yr-old formalin-fixed paraffin-embedded tissues obtained from the autopsy of 38 cystic fibrosis patients (nine with meconium ileus). Two different pairs of oligonucleotide primers were used: C16 B/C16 D that amplify 98 and/or 95 bp and FQ1/FQ2 that amplify 50 and/or 47 bp. After two independent rounds of polymerase chain reactions with the two sets of primers, amplification products were obtained in 67.5% of the cases when using C16 B/C16 D primers and in all cases when using FQ1/FQ2 primers. Fifty percent of the chromosomes analyzed in the 29 patients without meconium ileus had the delta F508 mutation, which was present in 13 heterozygous and 8 homozygous patients. The remaining eight cystic fibrosis patients did not carry that mutation. These results are similar to those reported in cystic fibrosis patients from Spain. In the meconium ileus group, we found a higher than expected proportion of delta F508 mutation; all patients showed the delta F508 mutation in at least one chromosome, seven patients (77.8%) being homozygous and two (22.2%) heterozygous. Present results indicate that delta F508 mutation can be detected by polymerase chain reaction in old paraffin-embedded tissues when appropriate primers are used.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1995

Detection of DNA mutations in acid formalin-fixed paraffin-embedded archival tumor specimens by polymerase chain reaction-single strand conformation polymorphism analysis.

A single strand conformation polymorphism analysis on polymerase chain reaction (PCR)-amplified genomic DNA was used for the detection of DNA mutations in acid formalin-fixed, paraffin-embedded tissues. Fifty non-small cell lung cancers were screened for mutations in the exon 5 of the p53 tumor suppressor gene. Structural abnormalities of the gene were found in nine (18%) of the tumors examined. The results show that paraffin-embedded tissues fixed in unbuffered formalin can be a good source of DNA for a mobility shift analysis by neutral polyacrylamide gel electrophoresis. The described procedure allows retrospective genetic studies on paraffin blocks available in all pathology departments.

Base Sequence↗

PubMed1993

Discrimination between immunoaccessory and phagocytic monocytes/macrophages of the skin in paraffin-embedded tissue by the monoclonal antibody Ki-M1P.

The variety of functions performed by monocytes and macrophages is reflected in their phenotypic diversity. Investigation of this complex system is facilitated by Ki-M1P, a new monoclonal antibody which recognizes a differentiation antigen on monocytes/macrophages in paraffin-embedded tissues. To test its usefulness as a pan-macrophage marker in the skin, we immunohistochemically analysed paraffin-embedded biopsy material from seven healthy individuals and 190 patients with a variety of dermatoses. Immunoreactivity was compared with results obtained with the antibodies KP-1, MAC-387, UCHL-1 and S-100. In normal skin, epidermal Langerhans cells were Ki-M1P-. Strong expression of this marker was detected on spindle-shaped as well as dendritic perivascular and intervascular macrophages. Pathological reaction forms such as giant cells and epithelioid cells in granulomatous dermatoses were also Ki-M1P+. The high specificity of Ki-M1P is reflected in the lack of reactivity with tumour cells in non-monocytic neoplasms and Langerhans cell histiocytosis. Thus, Ki-M1P is a useful marker for skin macrophages, discriminating between the immunoaccessory and the phagocytic compartments.

Antibodies, Monoclonal↗

PubMed1995

Evaluation of Ki-67 reactivity in neuroblastoma using paraffin embedded tissue.

AIMS: To examine the pattern of reactivity of Ki-67 in neuroblastoma and correlate this with a) clinical prognostic criteria and b) cell cycle statistics (using flow cytometry). METHODS: Four micron sections of paraffin embedded (PE) tissue from 55 patients (25 pre chemotherapy and 30 post) were placed on to aminosialinised slides, dewaxed and rehydrated. Slides were pretreated in a microwave oven, endogenous peroxidase activity blocked using 3% hydrogen peroxide and Ki-67 reactivity investigated using a streptavidin/biotin/peroxidase technique. DNA ploidy was also performed from an immediately adjacent section on the same block using a FACScan and Cellfit software. RESULTS: Ki-67 reactivity was well defined and highly reproducible. Eighteen out of 30 post chemotherapy samples were totally negative, despite evidence of proliferation on flow cytometry and all subsequently died of disease. As interpretation post chemotherapy was therefore deemed unreliable, this group was excluded from analysis. Reactivity in pretreatment samples ranged from 0% to 67%; staining was restricted to the nucleus with a distinct pattern noted in the nucleolus. Ki-67 positivity was lower in aneuploid compared with diploid tumours (mean 26% vs 36%, NS). Among diploid tumours, a lower percentage positivity was noted in those patients with better clinical prognostic parameters. Correlation however between Ki-67 and SG2M phases of cell cycle was poor (RS = 0.39, NS). CONCLUSION: Assessment of proliferation in neuroblastoma by Ki-67 reactivity in paraffin embedded tissue is reliable in pretreatment samples and can be incorporated into routine immunohistochemical evaluation. Larger multicentre studies are required to further evaluate Ki-67 reactivity as a prognostic indicator.

Biomarkers, Tumor↗

PubMed1990

Demonstration of Epstein-Barr viral DNA in formalin-fixed, paraffin-embedded samples of Hodgkin's disease.

The presence of Epstein-Barr virus (EBV) DNA in formalin-fixed, paraffin-embedded samples of Hodgkin's disease (HD) was investigated by Southern blot hybridization using a specific EBV Bam H1W fragment probe. DNA from 16 samples of HD in children and young adults was compared with that from ten samples in adults older than 45 years. A further eight cases of DNA extracted from fresh samples of HD were also studied, in order to compare the results obtained with fresh and fixed tissue samples. Hybridization was demonstrated in 15 of the 34 cases of HD studied in contrast to 3 or the 34 control specimens of non-Hodgkin's lymphoma, non-neoplastic lymph nodes, and carcinomas. No differences between the two age groups compared were found. The results of this study suggest that there is a definite association between EBV and HD, although the exact nature of this association remains to be established. It has also been shown that DNA of sufficient quality for the detection of EBV DNA can be extracted from formalin-fixed, paraffin-embedded material, and that comparable results can be obtained using DNA extracted from fresh and fixed tissue.

Adolescent↗

PubMed1992

Trisomy 12 in pediatric granulosa-stromal cell tumors. Demonstration by a modified method of fluorescence in situ hybridization on paraffin-embedded material.

The use of fluorescence in situ hybridization (FISH) to detect chromosomal abnormalities has many applications. Use of FISH on archival, paraffin-embedded material has been limited to microscopic sections. We have carried out FISH on preparations of disaggregated nuclei obtained from paraffin-embedded tissue to evaluate chromosome 12 copy number in granulosa-stromal cell neoplasms occurring in infants, children, and adolescents. Trisomy 12 was detected in the majority of cells from three of four juvenile granulosa cell tumors (three ovarian and one testicular) and one malignant granulosa cell tumor. Tetrasomy 12 was observed in a case of ovarian thecoma.

Cell Nucleus↗

PubMed2006

FISH analysis for the detection of lymphoma-associated chromosomal abnormalities in routine paraffin-embedded tissue.

Over the last decade, fluorescence in situ hybridization (FISH) has become a firmly established technique in the diagnosis and assessment of lymphoid malignancies. However, this technique is not wide-ly used in the routine diagnostic evaluation of paraffin-embedded biopsies, most likely because of a perception that it is technically more demanding. There are also uncertainties regarding diagnostic thresholds and the way in which results should be interpreted. In this Review, we describe practical strategies for using FISH analysis to detect lymphoma-associated chromosomal abnormalities in routine paraffin-embedded lymphoma biopsies. Furthermore, we provide proposals on how FISH results should be interpreted (including how to calculate cutoff levels for FISH probes), recorded, and reported. An online appendix (available at http://jmd.amjpathol.org) details various simple, yet robust procedures for paraffin FISH analysis; it also provides additional information on the production of FISH probes, evaluating and reporting FISH results, sources for reagents and equipment, and troubleshooting. We hope that these suggestions will make FISH technology for the study of lymphoma biopsies more accessible to routine diagnostic and research laboratories.

Chromosome Aberrations↗

PubMed2005

beta-Tubulin mutations in ovarian cancer using single strand conformation analysis-risk of false positive results from paraffin embedded tissues.

Mutations in the beta-tubulin gene have been proposed as a resistance mechanism to paclitaxel. We therefore investigated the presence of mutations in the beta-tubulin M40 gene in 40 ovarian tumours (16 paraffin-embedded and 24 freshly frozen) selected for good or poor response to chemotherapy with paclitaxel or non-tubulin-affecting regimens. The presence of mutations was investigated using single strand conformation analysis followed by sequencing of the products with altered mobility. No sequence variants in the exons of the beta-tubulin M40 gene were detected. Non-reproducible shifts were identified, in eight out of 16 paraffin embedded samples. This may explain some of the previously published discrepancies. In conclusion, sequence variants in the beta-tubulin M40 gene are rare and are unlikely to be a clinically relevant explanation of resistance to paclitaxel.

Antineoplastic Agents, Phytogenic↗

PubMed1994

Detection of T-cell receptor beta chain mRNA in frozen and paraffin-embedded biopsy tissue using digoxigenin-labelled oligonucleotide probes in situ.

In situ hybridization techniques using a cocktail of digoxigenin-labelled T-cell receptor (TcR) constant (C) region beta oligonucleotide probes were used to detect TcR beta mRNA in frozen and paraffin-embedded tissue sections. The specificity of the C beta cocktail was confirmed by Northern blot analysis. The TcR C beta cocktail successfully hybridized to T cells in frozen and paraffin-embedded tissue obtained from patients with inflammatory arthropathies, B- and T-cell non-Hodgkin's lymphoma (NHL), and reactive tonsillitis, and showed staining patterns comparable to those obtained by conventional immunohistological detection of T cells. This is the first report of in situ studies using labelled TcR C beta oligonucleotide probes and may indicate the feasibility of investigating clonal T-cell populations using digoxigenin-labelled clonospecific probes in clinical samples in situ.

Biopsy↗

PubMed1996

[The relationship between placental pathology and certain pathogens detected in paraffin-embedded tissues in cases of intrauterine growth retardation].

OBJECTIVE: To study the relationship between placental pathology and certain pathogens detected in paraffin-embedded placental tissues in cases of intrauterine growth retardation (IUGR). METHODS: The PCR-DNA techniques were used for determination of pathogens, including toxoplasma, cytomegalovirus and herps simplex type II in paraffin-embedded placental tissues from 53 cases of IUGR. RESULTS: Among 53 cases of IUGR, the pathogens were detected in 19 cases (35.85%). The relationships of pathogens detected to placental pathology were analyzed. Pathogens were found in 10 of 18 placentas with villitis, 7 of 11 placentas with villous maturation retarded and 2 of 3 intervillous space infections respectively. The incidence of infection in the pathological placentas was 59.38%. CONCLUSIONS: The study suggested that the haematogenous infections could play important roles in unknown causes of IUGR.

Adult↗

PubMed1983

Selection of a fixative for identifying T cell subsets, B cells, and macrophages in paraffin-embedded mouse spleen.

Fixation techniques were investigated for their ability to preserve morphology, esterase activity and cell surface antigens in paraffin-embedded mouse lymphoid tissue. The avidin-biotin-peroxidase system was used to stain antigens Thy-1.2, Lyt-1, Lyt-2, RA32C2 and F4/80. Conventional fixatives were compared with fixatives containing periodate and lysine plus paraformaldehyde and/or glutaraldehyde. Conventional fixatives preserved esterase activity but not many cell surface antigens. Periodate-lysine fixatives allowed better preservation of membrane antigens, but esterase activity was often lost at antigen-preserving concentrations of paraformaldehyde or glutaraldehyde. However, a periodate-lysine fixative containing both paraformaldehyde and glutaraldehyde preserved esterase and showed good to excellent staining of Lyt-1, Thy-1.2, RA32C2, and F4/80. Lyt-2 could not be stained with any fixative, but was well preserved in frozen material post-fixed with periodate-lysine based fixatives. We conclude that with proper fixation immune cell surface markers can be identified in paraffin-embedded tissue.

Animals↗

PubMed2000

Cytogenetical diagnosis in paraffin-embedded fetoplacental tissue using comparative genomic hybridization.

Comparative genomic hybridization (CGH) is a FISH-related technique used to assess global chromosomal aberrations in a variety of human tumours. Recently CGH has been applied to cytogenetic analysis of fresh frozen fetoplacental tissues. Here we report the application of CGH to paraffin-embedded placental samples. Ten samples from paraffin-embedded blocks of 6 control placentas and fetoplacental tissue from 10 aneuploidies, and 2 unbalanced aberrations were evaluated. Balanced karyotype profiles were obtained from samples of healthy placentas and all samples from the same placenta appeared to have similar confidence intervals. CGH analysis of four cases of trisomy 21, three cases of trisomy 18, one case of trisomy 13, one case of trisomy 15 and one case of trisomy 7 all showed overrepresentation of the respective trisomic chromosome. The CGH profile was also in accordance with the karyotyping of a case with isochromosome 21. The CGH profile of a case with der (2)t(2;6)(q37.3;q22.2) revealed partial trisomy for chromosome 6 between q21 and q27. CGH may be a useful adjunct in prenatal genetic diagnosis when retrospective diagnosis is needed from archival samples.

Chromosome Aberrations↗

PubMedDate not supplied

High-level detection of gene amplification and chromosome aneuploidy in extracted nuclei from paraffin-embedded tissue of human cancer using FISH: a new approach for retrospective studies.

A novel application of fluorescence in situ hybridization (FISH) to isolated nuclei is described. The method detects gene amplification and chromosome aneuploidy in extracted nuclei from paraffin-embedded tissue of human cancer with greater sensitivity and specificity than existing FISH methods. In this study, the method is applied to signal detection of the HER-2/neu (c-erbB-2) gene, whose amplification is one of the most common genetic alterations associated with human breast cancer. Nuclei were extracted and isolated from formalin fixed, paraffin embedded tissue of 43 different carcinomas (breast, ovary, endometrium, gastrointestinal stromal tumor and malignant mesothelioma). FISH was performed both on sections and extracted nuclei of each tissue using chromosome enumeration probes (CEP) for the centromeric regions of chromosomes 8 and 17, and a locus specific identifier (LSI) for the HER-2/neu oncogene. Differences between ploidy calculated in sections and extracted nuclei were seen in 3 breast carcinomas and 1 gastrointestinal stromal tumor (GIST). Furthermore, 1 breast cancer, previously considered to be borderline for HER-2/neu gene amplification turned out to be clearly amplified. Nuclei extraction and isolation bypass all the problems related to signal interpretation in tissue sections, and the adoption of this new technique, which improves the signal quality in several neoplastic samples, is suggested.

Aneuploidy↗

PubMed1989

Immunohistochemical localization and semi-quantification of atrial natriuretic polypeptide (ANP) in formalin-fixed-paraffin-embedded normal human hearts--comparative study with radioimmunoassay.

The presence of atrial natriuretic polypeptide (ANP) was immunohistochemically demonstrated in the formalin-fixed and paraffin-embedded tissues of 25 autopsied normal human hearts using monoclonal antibody. The ANP amounts were immunohistochemically semiquantified and compared with amounts measured by radioimmunoassay (RIA). The 25 autopsied hearts were divided into 5 groups according to the interval of formalin fixation or the length of time between death and fixation. Formalin-fixation intervals were one week in group 1A and 1B; 1 year in group 2; 4 to 5 years in group 3 and 10 to 12 years in group 4. The hearts of group 1A, 2, 3 and 4 were fixed within 5 hours after death. Those of group 1B were fixed 14 to 18 hours at 4 degrees C. After fixation, the left and right atrial appendages (LAA and RAA), the left and right atrial free walls (LA and RA), the left and right ventricular free walls (LV and RV) and the ventricular septum (VS) were transmurally dissected from each heart. They were embedded in paraffin, cut into 4 microns sections and immunohistochemically stained by the avidin-biotin-peroxidase complex (ABC) method using monoclonal antibody against alpha-human ANP. Under a light microscope, they were evaluated semiquantitatively according to the incidence of ANP-positive cells and the intensity of immunostaining. For every heart in group 1A, the tissue concentrations of ANP in the different parts were also measured separately by RIA before fixation. ANP-positive myocytes were noted in the atria of all hearts of all groups, but no in any ventricular myocytes. Both their incidence and grade in the atria were similar among groups 1A, 1B and 2. However, they were less in group 3, and least in group 4 among all groups. For all groups, they decreased in the following order: LAA greater than RAA not equal to LA greater than RA; the inner 1/3 greater than the middle 1/3 greater than the outer 1/3 of the atrial walls. The order in LAA, RAA, LA and RA.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMedDate not supplied

[Prognostic factors in neuroblastoma. Usefulness of DNA analysis using flow cytometry on paraffin-embedded material].

Nuclear DNA content was determined on paraffin blocks of 28 neuroblastic tumors retrieved from Surgical Pathology files. In 26 cases cytofluorimetric analysis for DNA content was satisfactory. 8 tumor were diploid, 12 were aneuploid and 6 tetraploid. All but one of the children with diploid neuroblastoma (NB) died after a survival ranging from 14 to 32 months. The only surviving child had a Stage I thoracic ganglioneuroblastoma (GNB) and was alive after 27 months. Conversely 11 of 12 patients with aneuploid tumors were alive with prolonged follow-up. Two children are alive after a period exceeding 146 months. The only exception in this group of aneuploid tumors was a child with Stage IV neuroblastoma of the adrenal gland with pleuro pulmonary metastasis who died after 6 months. In the group with tetraploid tumors 3 patients died (2 children with Stage III and IV died early while the third, with thoracic Stage II GNB, survived for 72 months). Two patients with tetraploid GNB Stage II and III are alive 65 and 72 months after diagnosis respectively. Another child with Stage IV adrenal NB is alive after 14 months but with metastatic spread to bones, bone marrow and lymph nodes. Cox analysis of the cases demonstrated ploidy as an independent prognostic factor. These results are in agreement with other molecular analysis linking near-ploidy of Neuroblastic tumors to poor prognosis. Ploidy, as detected by flow cytometry for nuclear DNA content, may represent an important prognostic factor in Neuroblastic tumors. The advantage of flow cytometry over other techniques of molecular analysis is represented by the simple methodology suitable for fixed and paraffin embedded tissue, even on retrieval material.

Adrenal Gland Neoplasms↗

PubMed1987

Flow cytometric analysis of relative mean DNA content of urogenital cancer cells in fresh and paraffin-embedded materials.

The relative mean DNA content calculation was performed by flow cytometry on single cell suspensions prepared from fresh and paraffin-embedded specimens of 10 patients with surgically resected urogenital cancer. Samples were processed by a modified method of Hedley et al. including two hours of pepsinizing time, ribonuclease digestion, and propidium iodide staining. The mean DNA content which is a quantitative description of flow cytometric characteristics was significantly correlated between the fresh and paraffin-embedded materials (n = 10, r = 0.869, p less than 0.01). This method allows for the objective, retrospective analysis of DNA content in relation to diagnosis and prognosis of urogenital cancer.

DNA, Neoplasm↗

PubMed1998

Extraction and analysis of diagnostically useful proteins from formalin-fixed, paraffin-embedded tissue sections.

We describe and discuss a method of protein extraction for Western blot analysis from formalin-fixed, paraffin-embedded tissue sections. From 5-mm2 50-micron-thick tissue sections, an abundance of proteins could be extracted by incubating the sections in lysis buffer containing 2% sodium dodecyl sulfate (SDS) at 100C for 20 min followed by incubation at 60C for 2 hr. Extracts yielded discernible protein bands ranging from 10 kD to 120 kD as identified by SDS-polyacrylamide gel electrophoresis (PAGE). Western blot analysis successfully detected membrane-bound protein such as E-cadherin, cytosolic protein such as beta-catenin, and nuclear proteins including proliferating cell nuclear antigen (PCNA), mutant-type p53, cyclin D1, cyclin E, and cyclin-dependent kinases (CDKs). With this technique, we could examine cyclin D1 and CDK2 expression in small adenomas compared with cancer tissues and normal mucosa. The simple method of protein extraction described here should make it possible to use large-scale archives of formalin-fixed, paraffin-embedded samples for Western blot analysis, and its application could lead to detailed analysis of protein expression. This new technique should yield valuable information for molecular biology.

Adenoma↗