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Results for “Paraffin Embedding”

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At least 991 records · Page 55Linked to original sources

PubMed2006

Application of the polymerase chain reaction on formalin-fixed, paraffin-embedded tissue in the recognition of tuberculous osteomyelitis.

A retrospective series of 45 cases of chronic osteomyelitis collected over a period of 14 years was histologically classified into tuberculous osteomyelitis (25) and chronic non-granulomatous osteomyelitis (20). The tuberculous osteomyelitis group was divided into three subgroups: a) typical granulomas (13 cases); b) ill-defined granulomas (seven cases), and c) suspected granulomas (five cases). An in-house polymerase chain reaction amplifying the 245 bp nucleotide sequence, and capable of detecting 10 fg of DNA of Mycobacterium tuberculosis, was used on the DNA extracted from the paraffin blocks. The polymerase chain reaction was positive in 72% of cases (18) of tuberculous osteomyelitis, but when typical cases of tuberculous osteomyelitis with confirmed granulomas were considered (13), this increased to 84.6% (11). The chronic non-granulomatous osteomyelitis group gave positive polymerase chain reaction results in 20% of the cases (4). Our preliminary study on tuberculous osteomyelitis shows that the polymerase chain reaction can be a very useful diagnostic tool, since a good correlation was seen between typical granulomas and polymerase chain reaction with a sensitivity of 84.6% and a specificity of 80%. In addition, our study shows that tuberculous osteomyelitis can be diagnosed in formalin-fixed paraffin-embedded tissues in the absence of typical granulomas.

Chronic Disease↗

PubMed1997

The applicability of formalin-fixed and formalin fixed paraffin embedded tissues in forensic DNA analysis.

Historically, formalin fixed (FF) tissues could not be used as a source of DNA in forensic science due to the fact that the DNA was too degraded for DNA analysis. With the introduction of the polymerase chain reaction (PCR) technique to forensic science, the usefulness of DNA from this biological material has been re-evaluated. This study evaluates the potential use of DNA from FF and formalin fixed paraffin embedded (FFPE) tissues in 13 PCR systems; HLA DQ alpha, LDLR, GYPA, HBGG, D7S8, GC, D1S80, vWA31, THO1, F13A1, FES/FPS, TPOX, and CSF1PO. The first six, HLA DQ alpha, LDLR, GYPA, HBGG, D7S8, and GC are reverse dot blot systems, D1S80 is an amplified fragment length polymorphism (AmpFlp) system and the others are short tandem repeats (STRs). This study shows that FFPE tissue which has not been fixed in formalin for more than three days is a useful source of DNA for 12 of the 13 PCR systems. In contrast, FF tissue did not prove to be a reliable source of DNA for the PCR techniques examined here.

Chelating Agents↗

PubMed1992

Monoclonal and polyclonal antibodies for immunohistochemical detection of bovine parainfluenza type 3 virus in frozen and formalin-fixed paraffin-embedded tissues.

Accurate identification of bovine parainfluenza type 3 virus in bovine respiratory disease requires dependable, sensitive, and specific techniques for detection in affected animals. Immunohistochemical testing can be a rapid and reliable means of demonstration of virus in tissues from suspect cases; however, this procedure is dependent upon the quality of the antisera directed against the viral antigens. The production of rabbit polyclonal and murine monoclonal antibodies directed against bovine parainfluenza type 3 virus and techniques for their use in fresh-frozen and formalin-fixed paraffin-embedded tissues in immunofluorescence and immunoperoxidase-based immunohistochemical tests are described.

Animals↗

PubMed2000

Myogenin is a specific marker for rhabdomyosarcoma: an immunohistochemical study in paraffin-embedded tissues.

Myogenin belongs to a group of myogenic regulatory proteins whose expression determines commitment and differentiation of primitive mesenchymal cells into skeletal muscle. The expression of myogenin has been demonstrated to be extremely specific for rhabdomyoblastic differentiation, which makes it a useful marker in the differential diagnosis of rhabdomyosarcomas (RMS) from other malignant small round cell tumors of childhood. Commercially available antibodies capable of detecting myogenin in routinely processed formalin-fixed paraffin-embedded (FFPE) tissue are now available. In this study, we evaluated myogenin expression using the monoclonal myf-4 antibody (Novocastra Labs) on FFPE in a large number of pediatric tumors in order to define the clinical utility of this marker. A total of 119 tumors were studied. These included 48 alveolar RMS (ARMS), 20 embryonal RMS (ERMS), one spindle cell RMS, 16 Ewing's sarcomas (ES), six nephroblastomas, two ectomesenchymomas, seven precursor hematopoietic neoplasms, five olfactory neuroblastomas, three neuroblastomas, six desmoplastic small round cell tumors, and five rhabdoid tumors. Distinct nuclear staining for myogenin was noted in all 69 RMS. Notably, the number of positive tumor cells differed between the ARMS and ERMS. In ARMS, the majority of tumor cells (75 to 100%) were positive, in contrast to ERMS, in which the positivity ranged from rare + to 25% in all but three tumors. Additionally, myogenin positivity was seen in two of two ectomesenchymomas and in two nephroblastomas with myogenous differentiation. All other tumors were clearly negative. Our results indicate that staining for myogenin is an extremely reliable and specific marker for rhabdomyoblastic differentiation. It gives consistent and easily interpretable results in routinely fixed tissues.

Biomarkers, Tumor↗

PubMed2001

API2-MALT1 fusion transcripts involved in mucosa-associated lymphoid tissue lymphoma: multiplex RT-PCR detection using formalin-fixed paraffin-embedded specimens.

Malignant lymphoma of mucosa-associated lymphoid tissue (MALT) type is a distinct clinicopathological disease entity in the category of extranodal marginal zone B-cell lymphoma. Recently, we and others have shown that the API2 gene on chromosome 11 and the MALT1 gene on chromosome 18 are fused as a result of t(11;18)(q21;q21) in MALT lymphomas. Here we report a detection assay that can be used for formalin-fixed, paraffin-embedded specimens. It consists of a multiplex one-tube reverse transcriptase-polymerase chain reaction (RT-PCR) followed by three parallel multiplex nested polymerase chain reactions. Eight variants of the fusion transcripts have been reported to date. When these variants were used as positive controls, all were successfully detected. The subsequent direct sequencing confirmed the results. Using this rapid and simple method, we could detect API2-MALT1 fusion transcripts in 5 of 15 (33%) archival cases of MALT lymphoma for a frequency comparable with those of RT-PCR assays using frozen materials. The lung was the preferential anatomical site of origin of MALT lymphomas harboring API2-MALT1 fusion. No fusion transcript was detected in any of 20 high-grade B-cell lymphomas. Our multiplex RT-PCR assay, which can be used for routinely-processed paraffin samples, should serve as a useful molecular tool for clarifying the clinicopathological significance of API2-MALT1 fusion in MALT lymphoma.

Adult↗

PubMed2006

Protocol modifications influence the result of EGF receptor immunodetection by EGFR pharmDx in paraffin-embedded cancer tissues.

EGF receptor (EGFR) became a useful target for several recently introduced therapies of various cancer types including colorectal, lung, head and neck cancers and glioblastoma. The successful clinical application of these novel molecularly targeted therapies requires the expression of their target, EGFR, determined by nucleic acid based or immunohistochemical techniques. However, until now, immunohistochemistry has not become a reliable diagnostic approach for this purpose. The golden standard for the determination of EGFR protein expression in paraffin-embedded cancer tissues is the EGFR pharmDxTM kit. Here we show that the recommended protocol may not be optimal for EGFR immunodetection. Microwave antigen retrieval and extended primary antibody incubation time converted four out of eight EGFR-negative tumors into EGFR-positive in a study of 50 lung adenocarcinoma cases. Accordingly, we recommend retesting cases negative for EGFR with EGFR pharmDxTM using protocol modifications optimizing antigen retrieval and the incubation periods.

Adenocarcinoma↗

PubMed1999

Application of the hybridization AT-tailing method for detection of human immunodeficiency virus RNA in cells and simian immunodeficiency virus RNA in formalin-fixed and paraffin-embedded tissues.

An in situ hybridization (ISH) AT-tailing method (HybrAT) was developed for the detection of viral genomes in infected cells and tissues. The method consists of hybridization with oligonucleotide probe which has a 3' end oligo d(A-T) tag, followed by elongation of the oligo d(A-T) by deltaTth DNA polymerase in the presence of the labeled nucleotide. The in situ HybrAT detected human immunodeficiency virus type 1 (HIV-1) in cells and simian immunodeficiency virus (SIV) in formalin-fixed and paraffin embedded sections with a sensitivity comparable to RNA ISH. The advantage of this method over other methods is discussed.

Animals↗

PubMed2006

Preliminary comparison of quantity, quality, and microarray performance of RNA extracted from formalin-fixed, paraffin-embedded, and unfixed frozen tissue samples.

Microarrays have been used to simultaneously monitor the expression of thousands of genes from biological samples, an approach that can potentially uncover previously unrecognized functions of genes. Microarray analyses can rarely be conducted retrospectively because of the requirement for RNA to be obtained from fresh or unfixed frozen tissues. Archived pathology specimens would need to be used for retrospective analyses, and these are typically preserved as formalin-fixed, paraffin-embedded (FFPE) tissue. Formalin-fixed tissues have been shown to yield compromised RNA compared with that obtained from frozen tissue. To begin to assess the performance of RNA extracted from FFPE samples on a microarray format, we compared RNA from a model system of pelleted lipopolysaccharide-stimulated human bone marrow stromal cells that were snap frozen with RNA from FFPE cells. RNA integrity and Affymetrix quality control parameters were assessed, and differentially regulated genes were analyzed with Ingenuity Pathway Analysis software. Results demonstrate that both snap-frozen and FFPE samples yielded intact RNA suitable for amplification prior to Affymetrix GeneChip analysis. Although some transcriptional information was lost with RNA extracted from the FFPE samples, Ingenuity Pathway Analysis revealed that the major pathways identified as affected by drug treatment were similar. Results show that FFPE samples are amenable to Affymetrix GeneChip analysis, expanding the possibility for expression profiling on archived tissue blocks in pathology laboratories.

Bone Marrow Cells↗

PubMed1977

Immunomorphologic methods in routine pathology. Application of immunofluorescence and the unlabeled antibody-enzyme (peroxidase-antiperoxidase) technique to formalin fixed paraffin embedded kidney biopsies.

Immunofluorescence and the unlabeled antibody enzyme (peroxidase - antiperoxidase) method were applied to formalin fixed conventionally paraffin-embedded kidney biopsy material, and optimal working conditions are reported. Both methods were suitable for the demonstration of immunoglobulin and complement deposits within glomerula. They were equally sensitive on dewaxed pronase-treated sections. Pronase treatment decreased nonspecific background fluorescence and increased the sensitivity of both immunomorphologic techniques, possibly by enhancing antigenicity.

Complement System Proteins↗

PubMed1995

A comparison of three avidin-biotin complex immunoenzyme systems for detection of African swine fever virus antigen in paraffin-embedded tissues.

The sensitivity and specificity of 3 avidin-biotin complex (ABC) immunostaining systems were compared on paraffin-embedded tissues from African swine fever virus (ASFV)-infected pigs. Results were also compared with immunofluorescent detection on cryosections of the same tissue for optimal detection of ASFV antigen. The ABC-alkaline phosphatase (ABC-AP) and ABC-peroxidase (ABC-PO) systems were at least as sensitive as direct fluorescent antibody (FA) and 10-fold more sensitive than the ABC-glucose oxidase system. Three ABC-AP and 2 ABC-PO chromagens with different counterstains were compared. In addition, 2 fixatives, 2 biotinylation procedures, 7 endogenous peroxidase blocking regimes, 6 tissue adhesives, and 3 mounting media were compared. The ABC-AP system with a red chromagen and hematoxylin counterstaining was preferred and most closely approximated routinely stained pathologic sections. Fixation in paraformalde-hydelysine-periodate fixative preserved ASFV antigen for research studies for at least 3 years. Formalin-fixed tissues retained some staining for up to 10 years.

African Swine Fever Virus↗

PubMed2004

Differentiation of bone and soft tissues in formalin-fixed, paraffin-embedded tissue by using methylene blue/acid fuchsin stain.

OBJECTIVE: To find a staining method for formalin-fixed, paraffin-embedded tissue that would distinguish bone from surrounding soft tissues, including muscle, periosteal tissue and bone marrow. STUDY DESIGN: A variety of stains were tested and compared with hematoxylin-eosin. The potential value of any given stain was evaluated based on its ability to stain bone and soft tissues different colors or shades that could be readily identified in photomicrographs. Stains were evaluated using both endochondral (tibia) and intramembranous bone (calvaria) samples. RESULTS: In contrast to standard hematoxylin-eosin stain, which stains both bone and soft tissues pink, the methylene blue/acid fuchsin stain demonstrates remarkable contrast between bone and other tissues. Methylene blue/acid fuchsin stained bone bright pink and the surrounding soft tissues blue-purple. CONCLUSION: In addition to the superior staining properties of methylene blue/acid fuchsin, other benefits of this stain include its stability, ease of use and low cost. This stain has many potential applications in the study of erosive bone disease in humans and also in animal models for research.

Animals↗

PubMedDate not supplied

Immunohistochemical demonstration of the KI-67-antigen in paraffin-embedded tumor biopsies.

10 tumor specimens were processed according to a modification of the AMeX method, which allows for paraffin embedding plus immunostaining of the slices. We used this method to determine the growth fraction of tumors using the monoclonal antibody KI-67. Results were essentially the same as those obtained when studying frozen sections. The quality of the histological slices is equal to frozen sections, but greater areas of tumor can be examined. Furthermore, this method allows for compilation of a stock of tumor specimens which can be used for further immunohistochemical studies whenever needed.

Antibodies, Monoclonal↗

PubMed2000

Detection of hepatitis B virus surface antigen mutants in paraffin-embedded hepatocellular carcinoma tissues.

The antigenic "a" determinant of the human hepatitis B virus surface antigen (HBsAg) is highly conserved and involved in inducing neutralizing antibody. Mutations on this determinant have been associated with the hepatitis B virus (HBV) escape from vaccination but are also found in chronic HBV carriers, but their involvement in liver diseases including hepatocellular carcinoma (HCC) is unclear. To investigate the possible liver disease-associated role of HBsAg mutants, their incidence was analyzed in 11 paraffin embedded HCC tissues using an improved DNA extraction method. Mutations on the "a" determinant (Thr126Ser, Gly145Arg, a double mutant Thr126Ser/Gln129Asn, Met 133Leu and Thr140Ile) were identified in 5 samples while the wild type sequence was found in 2 others. Future characterization of these HCC-associated HBsAg mutants should provide new insights on their role in the pathogenesis of HCC.

Adult↗

PubMed2003

Molecular diagnosis of Epstein-Barr virus in paraffin-embedded tissues of tumors with abundant lymphoid infiltration.

To evaluate the significance of Epstein-Barr virus (EBV) infection in tumorogenesis, we examined ten archival samples of acinic cell carcinomas of salivary glands with lymphoid-rich stroma, four archival samples of lymphoepithelioma-like carcinomas (LELC) of the urinary bladder, ten samples of oncocytic papillary carcinoma of the thyroid (Warthin-like tumors) and one sample of lymphoepithelioma-like carcinoma of the cervix, together 25 paraffin-embedded tumor tissues. Polymerase chain reaction (PCR) and in situ hybridization (ISH) assays were used. The EBV genome was detected by PCR using primers targeting the IR region. ISH was performed using EBER oligonucleotide probes. Each examination was repeated two times. Positive PCR result was obtained in 12% of samples only. However, this result was not confirmed by the subsequent second PCR examination. ISH revealed negative signals in all samples. Our results demonstrate the importance of the diagnostic strategy based on combination at least two independent methods. PCR due to its sensitivity may produce false positive results depending on the degree of infiltration the tumor sample by EBV carrying lymphocytes.

DNA, Viral↗

PubMed2002

IgH PCR of zinc formalin-fixed, paraffin-embedded non-lymphomatous gastric samples produces artifactual "clonal" bands not observed in paired tissues unexposed to zinc formalin.

Helicobacter pylori (HP) causes dense gastritis that can be difficult to distinguish morphologically from MALT-type lymphoma (ML). Immunoglobulin heavy chain (IgH) gene analysis by polymerase chain reaction (PCR) is often used to resolve diagnosis. However, monoclonal bands have been reported in nonmalignant cases of gastritis. Retrospectively, 16 gastric ML with both formalin-fixed, paraffin-embedded (FF-PE) and ethanol-fixed samples (EF), and 9 cases of FF-PE HP-gastritis were analyzed by IgH PCR to document the presence of non-reproducible bands in HP-gastritis, but not ML samples. In duplicate analyses, 12 of 16 ML yielded identical monoclonal bands in FF-PE and EF samples whereas 3 of 9 FF-PE gastritis cases yielded different-sized (ie, non-reproducible) "clonal" bands. Sequencing of two PCR products from a gastritis case confirmed IgH gene sequences. To investigate whether FF-PE had a direct effect on producing these non-reproducible bands, 7 gastrectomy samples were prospectively divided into EF and FF-PE halves for IgH PCR. All 7 samples demonstrated polyclonal smears in EF portions while 4 of 7 FF-PE portions yielded either multiple distinct bands or non-reproducible bands. In conclusion, IgH PCR of FF-PE tissue can create artifactual "clonal" bands, which are the appropriate product size, contain IgH sequences, and, if not performed in duplicate, may confuse interpretation of B-cell clonality.

Base Sequence↗

PubMed1996

Ras oncogene mutations in thyroid tumors: polymerase chain reaction-restriction-fragment-length polymorphism analysis from paraffin-embedded tissues.

Ras mutations have been found in thyroid lesions. Different studies have shown different frequencies of mutations among benign and malignant lesions. The presence of point mutations in codons 12 and 13 of the c-K-ras, c-H-ras, and N-ras genes was studied in 58 thyroid lesions (10 nodular goiters, 10 follicular adenomas, and 15 papillary, 10 follicular, and 13 anaplastic carcinomas). DNA was extracted from formalin-fixed paraffin-embedded tissue, and target sequences were amplified in vitro by the polymerase chain reaction. Mutations were detected by the presence of restriction-fragment-length polymorphisms either occurring naturally or introduced artificially by the use of mutant primers. No characterization of the mutations was performed. Results were correlated with clinicopathologic features and patient follow-up. One goiter showed a mutation at codon 13, c-K-ras. All follicular adenomas, including three hyalinizing trabecular adenomas, were negative. Four papillary carcinomas presented mutations (one at codon 13, c-K-ras; three at codon 12, N-ras). Two follicular carcinomas showed mutations at codon 12, N-ras. Five anaplastic carcinomas showed mutations (two at codon 12 and two at codon 13, c-K-ras; one at codon 12, N-ras). In summary, the results confirm that ras oncogenes play a role in thyroid tumorigenesis, probably at an early step. Ras mutations appear not to be related to prognosis.

Adenoma↗

PubMed2006

Determination of sheep prion gene polymorphisms from paraffin-embedded tissue.

Amino acid polymorphisms of the prion protein (PrP) greatly influence the susceptibility of sheep to scrapie. Selective breeding to increase the prevalence of PrP gene alleles associated with scrapie resistance is a flock management practice that is important for scrapie control programs. Determination of sheep PrP alleles typically has required extraction of DNA from host tissues that are freshly derived or stored frozen. We describe application of a DNA extraction procedure for formalin-fixed, paraffin-embedded tissues (PET) for the purpose of PCR amplification and nucleotide sequencing of relevant codons (136-171) of the sheep PrP gene. Tissues derived from 96 sheep were studied. The DNA sequence identity was confirmed in 87 of 94 matched samples of PET and frozen tissue specimens. DNA from brainstem PET of 2 sheep, from which fresh tissue was not available, was amplified and sequenced after formalin fixation for 7-70 days. This method will allow retrospective analysis of PrP genetics of sheep subsequent to postmortem diagnosis of scrapie when nonfixed tissue is unavailable for DNA extraction; however, it is not recommended that submission of fixed tissue supplant collection of fresh tissues for the purpose of determining PrP gene polymorphisms.

Animals↗

PubMed1993

Frequent detection of human herpesvirus-6 sequences by polymerase chain reaction in paraffin-embedded lymph nodes from patients with angioimmunoblastic lymphadenopathy and angioimmunoblastic lymphadenopathy-like lymphoma.

In search of a possible involvement of viral agents in angioimmunoblastic lymphadenopathy with dysproteinaemia (AILD) and AILD-like lymphoma (AILD-L), we studied the presence of human herpesvirus-6 (HHV-6) in the lymph node biopsies of 12 cases by polymerase chain reaction (PCR). Given the rarity of this lymphoproliferative disorder, we investigated archival specimens, consisting of formalin-fixed and paraffin-embedded tissues, obtained from patients with a clinical and histologic diagnosis of AILD and AILD-L. HHV-6 sequences were detected in the lymph node biopsies of 7 out of the 12 AILD and AILD-L cases examined. HHV-6 sequences were identified also in the involved liver and spleen tissues of one patient and in the PBMCs of two patients, all carrying viral sequences in the affected lymph nodes. We also used PCR to characterize the HHV-6 genomes, showing that two different viral strains are represented in the pathologic tissues. This study provides evidence of the presence of HHV-6 specific sequences in an unexpectedly high proportion of our series of AILD and AILD-L cases, suggesting a possible involvement of this lymphotropic virus in the pathogenesis of at least some cases of the disease.

Base Sequence↗